Joint annual meeting of the Australian Physiological Society and

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AuPS/ASB Meeting

Canberra 2005

Joint annual meeting of the Australian Physiological Society and Australian Society for Biophysics Canberra 2005

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AuPS/ASB Meeting

Canberra 2005

AuPS Sponsors

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AuPS/ASB Meeting

Canberra 2005

Meeting Sponsors

School of Biochemistry & Molecular Biology

Research School of Physical Science and Engineering

John Curtain School of Medical Research

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PDF Copy of Web-based Programme of AuPS/ASB Canberra 2005 Meeting With appreciation to AuPS Editor and Web-Master, Dr Dave Davey Abstracts and Author Index follows

AuPS/ASB Canberra 2005 Meeting Programme In the programme below, each presentation has, beside the time, the page number in volume 36 of the Proceedings. These numbers are links to the HTML versions of the pages. Each HTML page has a link to a corresponding PDF print version. A PDF file of the abstracts for each platform and poster session can be accessed by click on the

symbol in the upper right of the programme block.

Links to the abstracts can also be found in the author index.

Tuesday 27 September 2005 0800 Ion Channel Modelling Workshop Computer Laboratory, Copland G18 Australian National University 1730 Close Huron and Michigan Rooms Meeting Opening 4

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Opening remarks Obituary Peter Gage 1830 Plenary Lecture - Francisco Bezanilla Chair: David Adams 1P How does the membrane electric field gate an ion channel open and close? Francisco Bezanilla, Departments of Physiology and Anesthesiology. University of California at Los Angeles, Los Angeles, CA, U.S.A. 1930 Close 1930 Mixer

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Wednesday 28 September 2005 Superior

Huron

Symposium 1: Ion Channel Gating

Symposium 2: Functional Roles of Potassium Channels in the Vasculature

Chair: Shin-Ho Chung

Chair: Mike Hill

0845 2P The domains in the Na channel have specific functions Francisco Bezanilla and Baron Chanda, Departments of Physiology and Anesthesiology. University of California at Los Angeles, Los Angeles, CA, U.S.A. 0915 3P Modulation of potassium channel conformation and function by permeating ions Stephen J. Korn, Department of Physiology and Neurobiology University of Connecticut Storrs, Connecticut 06269 USA. 0945 4P The gating of mechanosensitive ion channels Boris Martinac, School of Biomedical Sciences, The University of Queensland, Brisbane, QLD 4072. 1005 5P The mechanism of fast gating in ClC chloride channels B. Corry1, D. Bisset2 and S.H. Chung2, 1Chemistry. School of Biomedical, Biomolecular and Chemical Science, The University of Western Australia, Crawley, WA 6009, Australia and 2Research School of Physical Sciences and Engineering, The Australian National University Canberra, ACT 0200, Australia. 1025 6P Conformational changes associated with glycine receptor activation J.W. Lynch and R.L. Hawthorne, School of Biomedical Sciences, University of Queensland, Brisbane, QLD 4072, Australia. 1045 Close

0845 Introduction and overview: Mike Hill 0850 7P Endothelium-dependent vasodilatation: Fundamental role of SKCa and IKCa potassium channels C.J. Garland, Vascular Pharmacology Group, Department of Pharmacy & Pharmacology, University of Bath, Bath BA2 7AY, U.K. 0925 8P Functional effects of vascular KIR channels C.G. Sobey, Department of Pharmacology, The University of Melbourne, Parkville, Victoria 3010, Australia. (Introduced by M. Hill) 0935 9P Kv as a target for nitroxyl anion (NO−)-mediated vasodilatation B.K. Kemp-Harper and J.L. Favaloro, Department of Pharmacology, Monash University, VIC 3800, Australia. (Introduced by M. Hill) 1005 10P The smooth muscle BKCa potassium channel and its interaction with arteriolar myogenic tone T.V. Murphy1, Y.T. Hwang1, H. Ding2, N. Kotecha2 and M.A. Hill1, 1Physiology and Pharmacology, School of Medical Sciences, University of New South Wales, NSW 2052, Australia and 2Division of Biosciences, School of Medical Sciences, RMIT University, VIC 3083, Australia. 1025 11P Potassium channels in vascular dysfunction C.R. Triggle1, A. Ellis2, L. Ceroni3, W. Wiehler3 and H. Ding1, 1School of Medical Sciences, RMIT University, Melbourne, VIC, Australia, 2Division of Chinese Medicine, School of Health Sciences, RMIT University, Melbourne, VIC, Australia and 3Smooth Muscle Research Group, University of Calgary, Canada. (Introduced by Michael Hill) 1045 Close

1045-1115 Morning Tea

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Free Communications 1: Exercise Physiology

Free Communications 2: Ion channels

Chair: David Allen

Chair: Peter Barry

1115 12P Sudomotor responses during isometric exercise appear to be intensity- and muscle mass-dependent C.J. Gordon, C.D. Haley, J.N. Caldwell and N.A.S. Taylor, Department of Biomedical Science, University of Wollongong, Wollongong, NSW 2522, Australia. 1130 13P Hydration indices in exertional heat stress A.T. Garrett1,2, N.G. Goossens1, N.J. Rehrer1, M.J. Patterson3 and J.D. Cotter1, 1School of Physical Education, University of Otago, Dunedin, New Zealand, 2 College of Education, University of Canterbury, Christchurch, New Zealand, and 3Defence Science and Technology Organisation, Melbourne, Australia. 1145 14P Plasma ammonia responses during heavy-intensity constant-load cycling in young and older individuals S. Sabapathy, D.A. Schneider and N.R. Morris, School of Physiotherapy and Exercise Science, and Heart Foundation Research Centre, Gold Coast campus, Griffith University, Southport, QLD 4215, Australia. 1200 15P Abnormal muscle Na+,K+-pumps, plasma K+, and exercise limitation in renal failure patients A.C. Petersen1, M.J. Leikis2, K.T. Murphy1, J.A. Leppik1, X. Gong1, A.B. Kent2, L.P. McMahon2 and M.J. McKenna1, 1 Muscle, Ions and Exercise Group, Centre for Ageing, Rehabilitation, Exercise and Sport, School of Human Movement, Recreation and Performance, Victoria University, Melbourne, VIC 8001, Australia and 2 Department of Nephrology, Royal Melbourne Hospital, Department of Medicine, University of Melbourne, Melbourne, VIC 3052, Australia. 1215 16P The effect of eccentric exercise on plasma K+ regulation and skeletal muscle Na+,K+-ATPase activity and content J.A. Bennie1, C.A. Goodman1, M.J. Leikis2 and M.J. McKenna1, 1Muscle, Ions and Exercise Group, School of Human Movement, Recreation and Performance, Centre for Ageing, Rehabilitation, Exercise and Sport, Victoria University, PO Box 14428, Melbourne, VIC 8001, Australia and 2Department of Nephrology, Royal Melbourne Hospital, Department of Medicine, University of Melbourne, Melbourne, VIC, Australia.

1115 18P Investigating the mechanism of proton transfer through the bacterial ClC transporter M. O’Mara, J. Yin, M. Hoyles and S.H. Chung, Research School of Physical Sciences and Engineering, Australian National University, Canberra, ACT 0200, Australia. 1130 19P An electrostatic basis for valence selectivity in cationic channels T. Vora1, B. Corry2, and S.H. Chung1, 1The Department of Theoretical Physics, RSPhysSE, The Australian National University, Canberra, ACT 0200, Australia and 2Chemistry, School of Biomedical and Chemical Science, The University of Western Australia, Crawley, WA 6009, Australia. 1145 20P A current source and a cation conductance are components of an electrical circuit connected across the plasma membrane of the malaria parasite Plasmodium falciparum R.J. W. Allen, K.J. Saliba and K. Kirk, School of Biochemistry and Molecular Biology, Linnaeus Way, Australian National University, Canberra, ACT 0200, Australia. 1200 21P Role of protein flexibility in gramicidin A channel permeability Turgut Bastug, School of Physics, University of Sydney, NSW 2006, Australia. 1215 22P Ca2+ influx through store-operated Ca2+ channel in mouse sinoatrial node Y.K. Ju1, H. Chaulet2, R.M. Graham2 and D.G. Allen1, 1School of Medical Sciences, University of Sydney, NSW 2006, Auatralia and 2 Victor Chang Cardiac Research Institute, NSW 2010, Australia. 1230 23P A hydrogen peroxide insult causes sustained alteration in the sensitivity of the L-type Ca2+ channel to β-adrenergic receptor stimulation in ventricular myocytes L.C. Hool, H.M. Viola and P.G. Arthur, School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, Crawley, WA 6009, Australia.

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1230 17P N-acetylcysteine infusion enhances skeletal muscle Na+,K+-ATPase activity and plasma K+ regulation, and delays fatigue, during prolonged submaximal exercise in well-trained individuals C.A. Goodman1, I. Medved1, M.J. Brown2, A.R. Bjorksten3, K.T Murphy1, A.C Petersen1, S. Sostaric1, X. Gong1 and M.J. McKenna1, 1Muscle, Ions & Exercise Group, Centre for Ageing, Rehabilitation, Exercise and Sport, School of Human Movement, Recreation and Performance, Victoria University, PO Box 14428, Melbourne, VIC 8001, Australia, 2Department of Anaesthesia, Austin Health, Heidelberg, VIC, Australia and 3Department of Anaesthesia and Pain Management, Royal Melbourne Hospital, Melbourne, VIC, Australia. 1245-1345 Lunch

Superior Plenary Lecture - Nigel Unwin Chair: Louise Tierney 1345 24P Structure and gating mechanism of the nicotinic acetylcholine receptor N. Unwin, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. Superior

Huron

Free Communications 3: Ligand-Gated Ion Channels

Free Communications 4: Skeletal Muscle Regulation: From Molecular Mechanism to Physiology

Chair: Louise Tierney 1445 25P Analysis of a GABAAγ2 (R43Q) knock-in mouse model of familial epilepsy S. Petrou, H. Tan, P. Davies and S. Murphy, Howard Florey Institute, The University of Melbourne, VIC 3010, AUSTRALIA.

1505 26P The receptor-associated protein, rapsyn, and regulation of postsynaptic acetylcholine receptor packing density and turnover at

Chair: Brett Cromer, David Allen 1445 30P Calcium-phosphate precipitation in the sarcoplasmic reticulum reduces action potential-mediated Ca2+ release in mammalian skeletal muscle T.L. Dutka, L. Cole and G.D. Lamb, Department of Zoology, La Trobe University, Victoria 3086, Australia. 1500 31P Digoxin effects on muscle strength, fatigue and K+ fluxes during exercise in healthy young adults M.J. McKenna1, S. Sostaric1, M.J. 8

the neuromuscular synapse W.D. Phillips and O.L. Gervásio, Brown2, C.A. Goodman1, X. Gong1, A.C. Petersen1, J. Aw3, J. Leppik1, C.H. Steward1, S.F. Fraser4, R.J. Snow5 and H. Krum3, 1Muscle, Ions Department of Physiology, Institute for Biomedical Research, and Exercise Group, School of Human Movement, Recreation and University of Sydney, NSW 2006, Australia. Performance, Centre for Ageing, Rehabilitation, Exercise and Sport, 1525 27P Functional consequences of clustering GABAA receptors M.L. Tierney, Victoria University, PO Box 14428, Melbourne, VIC 8001, Australia, T. Luu, A.E. Everitt, P.W. Gage, Membrane Physiology & Biophysics 2 Department of Anaesthesia, Austin Health, Heidelberg, VIC, Group, The John Curtin School of Medical Research, The Australian Australia, 3Department of Epidemiology and Preventive Medicine, National University, Canberra 0200, Australia. Monash University, Alfred Hospital, Melbourne, VIC, Australia, 4School 1545 28P The charge of the P-loop glutamate controls cation-anion selectivity in of Medical Sciences, RMIT University, Bundoora, VIC, Australia and CNG channels W. Qu1, A.J. Moorhouse1, M. Chandra1, K.D. Pierce2, 5 School of Exercise Science and Nutrition, Deakin University, 1 1 1 T.M. Lewis and P.H. Barry , Dept of Physiology and Pharmacology, Burwood, VIC, Australia. School of Medical Sciences, The University of New South Wales, 1515 32P The peak tetanic force-[K+]o relationship in mouse fast- and slowNSW 2052, Australia and 2Neurobiology Research Program, The twitch muscle: modulation with [Na+]o or [Ca2+]o S.P. Cairns, Division Garvan Institute of Medical Research, 384 Victoria Street, of Sport & Recreation, Auckland University of Technology, Auckland Darlinghurst, NSW 2010, Australia. 1020, New Zealand. 1600 29P Structure and dynamics of the periplasmic loop of the MscL 1530 33P The effect of dithiothreitol (DTT) application on isolated mouse muscle mechanosensitive channel studied by electron paramagnetic 1,2,3 2 1,3 fatigued at 37°C T.R. Moopanar and D.G. Allen, School of Medical resonance spectroscopy G. Meyer , E. Perozo and B. Martinac , 1 Sciences, University of Sydney F13, NSW 2006, Australia. School of Medicine and Pharmacology, University of Western Australia, Crawley, WA 6009, Australia and 2Department of Molecular 1545 34P Cytoplasmic ATP-sensing CBS domains regulate gating of skeletal muscle ClC-1 chloride channels B. Bennetts1, G.Y. Rychkov2, H-L. Physiology and Biological Physics, University of Virginia, 3 Charlottesville, VA 22906, USA. (Present address: School of Ng1, C.J. Morton1, D. Stapleton3, M.W. Paarker1 and B.A. Cromer1, 1 Biomedical Sciences, University of Queensland, Brisbane, QLD 4072, St. Vincent’s Institute, Fitzroy, VIC 3065, Australia,2 The University of Australia). Adelaide, Adelaide, SA 5005, Australia and 3Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC 3010, Australia. 1600 35P Modelling diffusive O2 supply to isolated muscle preparations C.J. Barclay, Muscle Energetics Laboratory, School of Physiotherapy & Exercise Science, Griffith University Gold Coast, PMB50 Gold Coast Mail Centre, Gold Coast, Queensland 9726, Australia.

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Superior 1615 - Poster Session, Afternoon Tea and Drinks 1 2

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Ion channel structure and function 36P Effects of gadolinium and static magnetic fields on MscL channel activity E. Petrov, Z.-W. Liu and B. Martinac, School of Biomedical Sciences, University of Queensland, St Lucia, 4072 Australia. 37P Conformational changes involved in MscL channel gating measured using FRET spectroscopy B. Corry1, P. Rigby2 and B. Martinac3, 1Chemistry, School of Biomedical, Biomolecular and Chemical Science, 2Biomedical Imaging and Analysis Facility and 3School of Medicine and Pharmacology, The University of Western Australia, Crawley, WA 6009, Australia. 38P C-terminal charged cluster of the mechanosensitive channel MscL, RKKEE, functions as a pH sensor Anna Kloda and Boris Martinac, School of Biomedical Sciences, University of Queensland, Brisbane, Queensland 4072 Australia. 39P The effects of eriochrome cyanine R on the mechanosensitive channels of E. coli T. Nguyen1,2, B. Clare2, L. Hool2 and B. Martinac3, 1School of Medicine and Pharmacology, University of Western Australia, WA 6009, Australia2, School of Biomedical, Biomolecular and Chemical Sciences, University of Western Australia, WA 6009, Australia and 3School of Biomedical Sciences, University of Queensland, QLD 4072, Australia. 40P Mutations within the selectivity filter of the NMDA receptor channel influence voltage-dependent block by extracellular 5-Hydroxytryptamine Anna Kloda and David Adams, School of Biomedical Sciences, University of Queensland, Brisbane QLD 4072, Australia. 41P Ion selectivity of glycine receptors with mutations of charged residues in the intracellular portals T.M. Lewis1, Sugiharto1, J.A. Peters2, J.J. Lambert2, P.H. Barry1 and A.J. Moorhouse1, 1School of Medical Sciences, The University of New South Wales, NSW 2052, Australia and 2Department of Pharmacology and Neuroscience, The University of Dundee, Dundee DD1 9SY, United Kingdom. 42P A molecular determinant of tropisetron inhibition of the glycine receptor Cl- channel Z. Yang, A.D. Ney and J.W. Lynch, School of Biomedical Sciences, University of QLD, Brisbane QLD 4072, Australia. 43P Subunit-specific inhibition of recombinantly expressed glycine receptors by ginkgolides and bilobalide R.L. Hawthorne and J.W. Lynch, School of Biomedical Sciences, University of Queensland, Brisbane QLD 4072, Australia. 44P Crosslinking of α1β1 GABAA receptor subunits via cysteines introduced into the transmembrane domain T.I. Webb, Z. Yang and J.W. Lynch, School of Biomedical Sciences, University of QLD, Brisbane QLD 4027, Australia. 45P Etomidate alters the single-channel properties of GABAA receptors in newborn rat hippocampal neurons V.A.L. Seymour, P.W. Gage and M.L. Tierney, The John Curtin School of Medical Research, The Australian National University, Canberra, ACT 2601, Australia. 46P GABAA αβ receptors open spontaneously when the conserved M2 leucine 9′ residue is mutated to a threonine T.L. Luu, M.L. Tierney and P.W. Gage, Division of Molecular Bioscience, The John Curtin School of Medical Research, The Australian National University, ACT 2601, Australia. 47P GABARAP influences the conductance of recombinant GABAA channels A.B. Everitt, M.L. Tierney and P.W. Gage, The John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia. 48P C-Terminal peptide of M protein from dengue virus (DVM-C) forms ion channels A. Premkumar, C.R. Horan and P.W. Gage, Division of Molecular Biosciences, John Curtin School of Medical Research, Australian National University, PO Box 334, Canberra City, ACT 2601, Australia. 49P The role of the M1-P1 loop in acid sensitive two-pore domain potassium (TASK) channel regulation Catherine E. Clarke1,2, Alistair Mathie3 and Jamie I. Vandenberg1,2, 1St Vincent's Hospital Clinical School, University of New South Wales, Victoria Road, Darlinghurst NSW 2010, Australia, 2Electrophysiology and Biophysics Unit, Victor Chang Cardiac Research Institute, 384 Victoria Street, Darlinghurst, NSW 2010, Australia and 3Department of Biological 10

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Sciences, Imperial College London, Exhibition Road, London SW7 2AZ, UK. 50P Structural studies of chloride intracellular ion channel proteins A.V. Mynott1, D.R. Littler1, P.M.G. Curmi1, L.J. Brown2 and S.N. Breit3, 1School of Physics, University of New South Wales, NSW 2052, Australia, 2Department of Chemistry and Biomolecular Sciences, Macquarie University, NSW 2109, Australia and 3St Vincent's Hospital, Sydney, NSW 2010, Australia. Biomolecular structure and dynamics 51P Photochemical behaviour and Na+,K+-ATPase sensitivity of voltage-sensitive styrylpyridinium fluorescent membrane probes S. Amoroso1, V.V. Agon1, T. Starke-Peterkovic1, M.D. McLeod1, H.-J. Apell2, P. Sebban3 and R.J. Clarke1, 1School of Chemistry, University of Sydney, NSW 2006, Australia, 2Faculty of Biology, University of Constance, D-78434 Constance, Germany and 3Laboratory of Physical Chemistry, UMR 8000, University of Paris XI, Orsay 91405, France. 52P Applications of styrylpyridinium dyes in elucidating ion-transport mechanisms in plant cells S. Amoroso1, R.J. Clarke2, A. Larkum1 and R. Quinnell1, 1School of Biological Sciences, University of Sydney, NSW 2006, Australia and 2School of Chemistry, University of Sydney, NSW 2006, Australia. 53P The crystal structure of Pichia pastoris lysyl oxidase at 1.23Å reveals a lysine-lysine covalent cross-link, dehydrolysinonorleucine A.P. Duff1, A.E. Cohen2, P.J. Ellis2, D.B. Langley1, D.M. Dooley3, H.C. Freeman1 and J.M. Guss1, 1School of MMB, University of Sydney, NSW 2006, Australia, 2Stanford Synchrotron Radiation Laboratory, CA, USA and 3Chemistry and Biochemistry, Montana State University, Bozeman MT, USA. 54P Molecular dynamics study of conformational changes in human serum albumin by binding of fatty acids S. Fujiwara and T. Amisaki, Department of Biological Regulation, Faculty of Medicine, Tottori University, 86 Nishi-machi, Yonago, 683-8503, Japan. 55P NMR probes of red cell deformation P.W. Kuchel, B.E. Chapman, D.J. Philp and W.A. Bubb, School of Molecular and Microbial Biosciences, University of Sydney, NSW 2006, Australia. 56P Current-voltage analysis of response to salt stress by salt-tolerant and salt-sensitive charophyte cells Mary J. Beilby and Virginia A. Shepherd, Biophysics, School of Physics, University of NSW, NSW 2052, Australia. 57P Oxygen evolution in chimeric spinach photosystem II with cyanobacteria manganese stabilising protein Adele Williamson, Warwick Hillier, Reza Razeghifard and Tom Wydrzynski, Photobioenergetics Group, Research School of Biological Sciences, The Australian National University, Canberra, ACT 0200, Australia. 58P The role of an oil droplet lens in vision enhancement L. Fischer1, M. Vorobyev2, A. Zvyagin1 and T. Plakhotnik1, 1Department of Physics, University of Queensland, QLD 4072, Australia and 2Vision Touch and Hearing Research Centre, University of Queensland, QLD 4072, Australia. 59P Circular dichroic spectra of the N-terminal region of cardiac myosin binding protein – C C.E. Oakley1, L.J. Brown2 and B.D. Hambly1, 1Department of Pathology, University of Sydney, NSW 2006, Australia and 2Department of Chemistry and Biomolecular Sciences, Macquarie University, NSW 2109, Australia. 60P Changes in mechanical properties of live cell wall during turgor regulation monitored by atomic force microscopy E.M. Mahomudally1, M.J. Beilby2, V. Shepherd2 and A.R. Moon1, 1Department of Applied Physics, University of Technology, Sydney, NSW 2007, Australia and 2Biophysics Department, School of Physics, University of New South Wales, Kensington, NSW 2052, Australia. (Introduced by M.J. Beilby) Cardiac muscle and Skeletal muscle 61P Effect of temperature on stretch-induced cardiac action potential shortening in the rat heart: involvement of TREK-1 D.R. Kelly, L. Mackenzie and D.A. Saint, Department of Physiology, University of Adelaide, SA 5005, Australia. 62P Immunohistochemical identification of stretch-sensitive two-pore-potassium (TREK) channels in the human heart S.Y. Yuan, H.P. Zhu and D. Saint, Department of Physiology, School of Molecular & Biomedical Science, University of Adelaide, Adelaide, SA 5005, Australia. 63P More than one type of stretch activated channel contributes to the action potential duration in guinea pig L. Mackenzie, D.R. Kelly and D.A. Saint, 11

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Department of Physiology, School of Molecular and Biomedical Science, The University of Adelaide, SA 5000, Australia. 64P Fatty acid composition of red blood cell membranes as a marker of human heart membrane phospholipid fatty acids Mandy L Theiss1, Salvatore Pepe2 and Peter L. McLennan1, 1Smart Foods Centre, Department of Biomedical Science, University of Wollongong, Wollongong, NSW 2522, Australia and 2Cardiac Surgical Research Lab, Alfred Hospital & Baker Medical Research Institute, Prahran, VIC 3181, Australia. 65P Confocal Ca2+ imaging of mouse sinoatrial node Y.K. Ju1, D.G. Allen1 and M.B. Cannell2, 1School of Medical Sciences, University of Sydney, NSW 2006, Australia and 2The Faculty of medical and Health Sciences, University of Auckland, Auckland, New Zealand. 66P Reactive oxygen species generated from the mitochondria and not NAD(P)H-oxidase regulate L-type Ca2+ channel function during acute hypoxia in ventricular myocytes L.C. Hool, H.M. Viola, C.A. Di Maria and P.G. Arthur, School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, WA 6009, Australia. 67P Eccentric damage is accentuated in aged dystrophin-deficient EDL muscles from dystrophic mice (MDX) S. Chan and S.I. Head, School of Medical Sciences, UNSW, NSW 2052, Australia. 68P Phosphorylation of CSQ affects Ca2+ binding and interactions with anchoring protein junctin N.A. Beard1, S. Cheung1, L. Wei1, M. Varsànyi2 and A.F. Dulhunty1, 1John Curtin School of Medical Research, ANU, Canberra, ACT 0200, Australia and 2Institut für Physiologische Chemie, Ruhr Universität, Bochum, Germany. 69P A calsequestrin polymer is necessary for the Ca2+ binding protein to regulate RyR channels L. Wei, N.A. Beard and A.F. Dulhunty, John Curtin School of Medical Research, Australian National University, Canberra, ACT 0200, Australia. 70P Digoxin and exercise effects on Na+,K+-pump activity, content, isoform gene and protein expression in human skeletal muscle X. Gong1, A. Petersen1, S. Sostaric1, C. Goodman1, D. Cameron-Smith2, R. Snow2, K. Murphy1, K. Carey2, J. Aw3, H. Krum3 and M. McKenna1, 1School of Human Movement, Recreation and Performance, Centre for Ageing, Rehabilitation, Exercise and Sport, Victoria University, Melbourne, VIC 8001, Australia, 2School of Exercise Science and Nutrition, Deakin University, Melbourne, VIC 3125, Australia and 3Department of Epidemiology and Preventive Medicine, Monash University, Alfred Hospital, Melbourne, VIC, Australia. Nervous system 71P Reduced long-term depression is recovered in aging mdx cerebellar Purkinje cells J.L. Anderson, S.I. Head and J.W. Morley, School of Medical Sciences, UNSW, NSW 2052, Australia. 72P Differential action of ω-conotoxins CVID and CVIB on voltage-gated calcium channels in rat sensory neurons L.M. Motin, R.J. Lewis and D.J. Adams, School of Biomedical Sciences, University of Queensland, Brisbane, QLD 4072, Australia. 73P Evidence from collision experiments that onset chopper neurons in the guinea pig cochlear nucleus receive excitatory input from centrifugal collaterals D. Robertson and W.H.A.M. Mulders, The Auditory Laboratory, Discipline of Physiology, School of Biomedical Biomolecular and Chemical Sciences, The University of Western Australia, Crawley, Western Australia 6009, Australia. 74P P2Y receptor activation inhibits the formation and proliferation of primary mouse sub-ventricular-derived neurospheres M.R. Stafford, P.F. Bartlett and D.J. Adams, School of Biomedical Sciences and The Queensland Brain Institute, University of Queensland, Brisbane QLD 4072 Australia. Membrane transport and its regulation 75P Protein kinase A inhibits cell growth induced by overexpression of IK channels C.J. Fowler, K. Ngui, B. Hunne, D. Poole, J.B. Furness and C.B. Neylon, Department of Anatomy and Cell Biology, The University of Melbourne, VIC 3010, Australia. 76P Post-transcriptional regulation of CFTR protein expression by 5′untranslated region encoded regulatory elements S-J. Conroy, W.L Davies and A.E.O. Trezise, School of Biomedical Science, The University of Queensland, QLD 4072, Australia. 77P Nedd4-2, CLC-5 and albumin endocytosis in the proximal tubule: a role for SGK-1? D.H. Hryciw1, J. Ekberg1, A. Lee1, I.L. Lensink2, S. Kumar2, W.B. 12

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Guggino3, D.I. Cook4, C.A. Pollock5 and P. Poronnik1, 1School of Biomedical Sciences, University of QLD, Brisbane, QLD 4072, Australia, 2Hanson Institute, IMVS, Adelaide, SA 5000, Australia, 3Department of Physiology, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA, 4Department of Physiology, University of Sydney, NSW 2006 Australia, and 5Kolling Institute, RNSH, University of Sydney, NSW 2065, Australia. 78P Na+ H+ exchanger regulatory factor 2 (NHERF-2) is a scaffold for the plasma membrane Ca2+ ATPase (PMCA) W.A. Kruger1, G.R. Monteith2, L. Tongpao1 and P. Poronnik1, 1School of Biomedical Sciences, The University of Queensland, St Lucia, QLD 4072, Australia and 2School of Pharmacy, The University of Queensland, St Lucia, QLD 4072, Australia. 79P NHERF1 - a novel scaffold protein for the astroglial glutamate transporter GLAST A. Rayfield1, A. Lee1, D. Pow2, D. Hryciw1, T.A. Ma1, S. Broer3, C. Yun4 and P. Poronnik1, 1School of Biomedical Sciences, University of Queensland, Brisbane, QLD 4072, Australia,2 Department of Anatomy, University of Newcastle, NSW 2308, Australia,3 Division of Biochemistry and Molecular Biology, Faculty of Science, Australian National University, Canberra, ACT 0200, Australia and 4Department of Medicine, Emory University, Atlanta, Georgia 30322, USA. 80P Molecular cloning and characterisation of the mouse ‘system IMINO’ transporter S. Kowalczuk1, A. Bröer1, M. Munzinger1, N. Tietze1, K. Klingel2 and S. Bröer1, 1School of Biochemistry & Molecular Biology, Australian National University, Canberra, ACT 0200, Australia and 2Department of Molecular Pathology, University of Tübingen, 72076 Tübingen, Germany. 81P Increased acetaminophen hepatotoxicity in the NaS1 sulphate transporter null mouse S. Lee, P.A. Dawson and D. Markovich, School of Biomedical Sciences, University of Queensland, Brisbane, QLD 4072, Australia. Nutrition 82P The influence of dietary fish oil and exercise upon oxidative status biomarkers in a rat model R. Henry, A.J. Owen and P.L. McLennan, Smart Foods Centre, Department of Biomedical Science, University of Wollongong, NSW 2522, Australia. Education 83P Simulation of visual processing in retinal ganglion cells M. Watson,1 G. Holmes,1 T. Byrne2 and S. Cornford,1 1Department of Biological & Physical Sciences, Faculty of Sciences, 2Faculty of Engineering and Surveying, University of Southern Queensland, Toowoomba, QLD 4350, Australia.

Superior AuPS Invited Lecture - Angela Dulhunty Chair: David Adams 1800 84P Excitation-contraction coupling from 1969 to 2005 A.F. Dulhunty, Division of Molecular Bioscience, John Curtin School of Medical Research, Building 54, Off Mills Rd, Australian National University, ACT 0200, Australia. 1900- Student Function

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Thursday 29 September 2005 Superior

Huron

Symposium 3: Physiology Teaching in the 21st Century: Trends, Challenges and Innovations

Symposium 4: Membrane Protein Structure and Interactions

Chair: Kay Colthorpe and Hardy Ernst

Chair: Frances Separovic

0845 85P Challenges facing physiology educators in the 21st century Ann Sefton, Faculty of Medicine, University of Sydney, NSW 2006, Australia. 0915 86P Research led teaching and learning in physiology R.E. Kemm, Department of Physiology, University of Melbourne, Parkville, VIC 3010, Australia. 0945 87P Problem-based learning (PBL): A novel and effective approach for teaching research skills by addressing contemporary research problems in physiology J. Schwartz1 and P. Buckley1,2, 1Discipline of Physiology, School of Molecular and Biomedical Science, Univeristy of Adelaide, Adelaide SA, Australia and 2School of Pharmacy & Medical Sciences, University of South Australia, Adelaide SA, Australia. (Introduced by Michael Roberts) 1015 88P Improving learning outcomes for students in Clinical Physiology C.R Dallemagne, School of Life Sciences, Queensland University of Technology, GPO Box 2434, Brisbane, Queensland 4001, Australia. 1045 Close

0845 89P Seeing spots: miscibility transitions in lipid/cholesterol membranes S.L. Keller and S.L. Veatch, Departments of Chemistry and Physics, University of Washington, Seattle, WA 98195-1700, U.S.A. (Introduced by Frances Separovic) 0925 90P Role of the plasma membrane in amyloid formation and toxicity M.I. Aguilar1, X. Hou1, S. Subasinghe1, A. Mechler2, K. Hall1, L. Martin2 and D.H. Small1, 1Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC 3800, Australia and 2School of Chemistry, Monash University, Clayton, VIC 3800, Australia. 0950 91P Are chloride intracellular ion channel proteins (CLICs) really channels? Exploring their membrane structure L.J. Brown1, D.R. Littler2, A. Mynott2, S.J. Harrop2, S.N. Breit3, M. Mazzanti4 and P.M.G. Curmi2, 1Department of Chemistry and Biomolecular Sciences, Macquarie University, NSW 2109, Australia, 2School of Physics, University of New South Wales, NSW 2052, Australia, 3Centre for Immunology, St Vincent’s Hospital, Sydney NSW 2010, and 4 University or Rome, La Sapienza, Roma, Italy. 1015 92P Structure and dynamics of cellular components using fluorescence and X-ray diffraction techniques Leann Tilley and Nick Klonis, Department of Biochemistry, La Trobe University, Bundoora, VIC 3083, Australia. 1045 Close

1045-1105 Morning Tea

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Superior

Huron

Symposium 5: Regulation of Membrane Transport

Symposium 6: Membrane Associated Proteins that Regulate Muscle Contraction

Chair: David Adams

Chair: Angela Dulhunty

1105 93P The canonical transient receptor potential channel 1 is an essential structural component of the mechanosensitive calcium permeable channel in vertebrate cells O.P. Hamill1, R. Maroto1, A. Kurosky2, T.G. Wood2, A. Raso3 and B. Martinac3, 1Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, Texas, U.S.A., 2Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas, U.S.A. and 3 Department of Pharmacology, University of Western Australia, Crawley, WA, Australia. 1135 94P Sensing pressure with K2P channels Eric Honore, Institut de Pharmacologie Moléculaire et Cellulaire, CNRS-UMR 6097, Institut Paul Hamel, 660 Route des Lucioles, Sophia Antipolis, 06560 Valbonne, France. 1205 95P Varieties of mechanotransduction: the cytoskeletal stress fibre as a force transmitter and a mechanosensor Masahiro Sokabe1, Kimihide Hayakawa2 and Hitoshi Tatsumi1, 1Department of Physiology, Nagoya University Graduate School of Medicine, Nagoya, 464-8558, Japan and 2ICORP/SORST Cell Mechanosensing, JST, Nagoya, 464-8550, Japan. 1235 96P Role of tryptophan residues in ion channel function Amitabha Chattopadhyay, Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India. 1305 Close

1105 97P From DHPR to RyR and back again: What lies along the way? Kurt Beam, Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado, U.S.A. 1150 98P Regulation of ryanodine receptors from skeletal and cardiac muscle by components of the cytoplasm and lumen D.R. Laver, School of Biomedical Sciences, University of Newcastle, and Hunter Medical Research Institute, Callaghan, NSW 2308, Australia. 1215 99P Structural and functional characterisation of the interaction of the dihydropyridine receptor II-III loop with the ryanodine receptor M.G. Casarotto, Y. Cui, Y. Karunasekara, P.J. Harvey, N. Norris, P.G. Board and A.F Dulhunty, Division of Molecular Bioscience, The John Curtin School of Medical Research, The Australian National University, ACT 0200, Australia. 1240 100P ClC-1 chloride channel - matching its properties to a role in skeletal muscle G. Rychkov, School of Molecular and Biomedical Science, The University of Adelaide, ADELAIDE, 5005. 1305 Close

1305-1350 Lunch

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Superior Plenary Lecture D. Peter Tieleman Chair: Boris Martinac 1350 101P Pores, channels and transporters: computational studies of membrane transport Peter Tieleman, Department of Biological Sciences, University of Calgary, 2500 Unniveristy Drive NW, Calgary AB, T2N 1N4, Canada. Free Communications 5: Cardiac muscle

Free Communications 6: Cellular signaling

Chair: Lea Delbridge

Chair: Craig Neylon

1450 102P Does lignocaine increase the chance of survival from massive heart attack? S.M. Weiss and P.W. Gage, John Curtin School of Medical Research, Australian National University, ACT 0200, Australia. 1505 103P Acute application of n-3 polyunsaturated fatty acids modify calcium sparks in permeabilised rat cardiac myocytes Bonny Honen, Rebecca Dalton, Dirk van Helden and Derek Laver, School of Biomedical Sciences, University of Newcastle and Hunter Medical Research Institute, Callaghan, NSW 2308, Australia. 1520 104P Abnormal calcium transients and calcium handling protein expression in cardiomyocytes from mdx (dystrophic) mice I.A. Williams and D.G. Allen, Institute for Biomedical Research, School of Medical Sciences, University of Sydney F13, NSW 2006, Australia. 1535 105P Mechanisms underlying the stretch-dependent slow inotropic response in isolated mouse myocardium M.L. Ward1 and D.G. Allen2, 1Department of Physiology, University of Auckland, New Zealand and 2School of Medical Sciences, University of Sydney F13, NSW 2006, Australia. 1550 106P Salutary effects of pyruvate are more evident in female than male glut4-deficient mouse hearts C.E. Huggins1, J. Favaloro2, J. Proietto2, S. Pepe3 and L.M.D. Delbridge1, 1Department of Physiology, University of Melbourne, VIC 3010, Australia,

1450 108P Analyses of the actin cytoskeleton using fluorescence resonance energy transfer (FRET) C.G. dos Remedios1, D. Chhabra1, I. Dedova1, D. Safer2 and E DeLaCruz3, 1Institute for Biomedical Research F13, University of Sydney, NSW 2006, Australia, 2 Department of Physiology, University of Pennsylvania, School of Medicine, Philadelphia, PA, 19104, USA, and 3Department of Molecular Biophysics & Biochemistry, Yale University, 266 Whitney Ave, PO Box 208114, New Haven, CT, 06520-8114, USA. 1505 109P Profilin binding to sub-micellar concentration of polyphosphoinositides PI(4,5)P2 and PI(3,4,5)P3 P.D.J. Moens, School of Biological, Biomedical and Molecular Sciences, The University of New England, Armidale, NSW 2351, Australia. 1520 110P Phospholipase Cγ is essential for activation of store-operated Ca2+ channels in liver cells T. Litjens1, T. Nguyen1, E. Aromataris1, M. Roberts1, G. Barritt2 and G. Rychkov1, 1School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, SA 5005 and 2School of Medicine, Flinders University of South Australia, G.P.O. Box 2100, Adelaide, SA 5001, Australia. 1535 111P Distinct characteristics of exocytosis in mouse pancreatic acinar cells Peter Thorn1, Olga Larina1 and Ian Parker2, 1Department of Pharmacology, University of Cambridge, Tennis Court Road,

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2

Department of Medicine, University of Melbourne, Austin and Repatriation Medical Centre, Heidelberg, VIC 3084, Australia and 3 Alfred Hospital and Baker Heart research Institue, Prahran, VIC 3181, Australia. 1605 107P The angiotensin type 2 receptor prevents cell death in neonatal cardiomyocytes of the hypertrophic heart rat E.R. Porrello1,2, A. D'Amore2, C.L. Curl1, S.B. Harrap1, W.G. Thomas2 and L.M.D. Delbridge1, 1Department of Physiology, The University of Melbourne, VIC 3010, Australia and 2Baker Heart Research Institute, Prahran, VIC 3004, Australia.

Cambridge, CB2 1PD and 2Department of Neurobiology and Behavior, University of California Irvine, CA 92697, USA. 1550 112P Synchronization of Ca2+ oscillations through interaction of intracellular Ca2+ stores and L-type Ca2+ channels M.S. Imtiaz, J. Zhao, K. Hosaka and D.F. van Helden, The Neuroscience Group, School of Biomedical Sciences, Faculty of Health and Medical Sciences, The University of Newcastle, NSW 2308, Australia. 1605 113P Characterization of the of the electrical activity underlying spontaneous contractions in the mouse ureteropelvic junction R.J. Lang, B. Zoltkowoski, J. Hammer, W. Meeker, I. Wendt and H. Parkington, Department of Physiology, Monash University, Clayton, Vic 3800, Australia.

1620-1635 Afternoon Tea Free Communications 7: Muscle physiology

Free Communications 8: Biophysics

Chair: Derek Laver

Chair: Paul Smith

1635 114P Active metabolism of mouse papillary muscle C. Widén and C.J. Barclay, Muscle Energetics Laboratory, Heart Foundation Research Centre, School of Physiotherapy & Exercise Science, Griffith University, PMB50 Gold Coast Mail Centre, Gold Coast, QLD 9726, Australia. 1650 115P Functional and electrophoretic identification of two Troponin C isoforms in toad skeletal muscle fibres B. O'Connell, R. Blazev and G.M.M. Stephenson, School of Biomedical Sciences, Victoria University, Melbourne, VIC 3011, Australia. 1705 116P X-ray diffraction analysis of the effects of myosin chain-2 phosphorylation on the structure of fast skeletal muscle fibres Joseph F.Y. Hoh1, Maki Yamaguchi2, Masako Kimura2, Shigeru Takemori2 and Naoto Yagi3, 1Department of Physiology and Institute for Biomedical Research, The University of Sydney, NSW 2006, Australia, 2Department of Physiology, The Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan and 3Japan Synchrotron Radiation Research Institute (JASRI), Sayo-gun 6795198, Japan.

1635 Introduction by Boris Martinac 1645 ASB Robertson Lecture (TBA) 1715 120P Bicarbonate is not a physiological substrate of Photosystem II I.L. McConnell, W. Hillier and T. Wydrzynski, Photobioenergetics Group, The Research School of Biological Sciences, The Australian National University, Canberra, ACT 0200, Australia. 1730 121P Spectra of reef fish – a physics approach to colourful patterns Misha Vorobyev and Justin Marshall, Vision Touch and Hearing Research Centre, School of Biomedical Sciences, University of Queensland, QLD 4072, Australia. 1745 122P Resonances of the human vocal tract and some of their uses John Smith and Joe Wolfe, School of Physics, University of New South Wales, NSW 2052, Australia. 1800 Close

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1720 117P Calpain-1 and calpain-3 are not autolysed with exhaustive exercise in humans R.M. Murphy1, R.J. Snow2 and G.D. Lamb1, 1Department of Zoology, La Trobe University, VIC 3086, Australia and 2School of Exercise and Nutrition Sciences, Deakin University, Burwood, VIC 3125, Australia. 1735 118P Increased fatigue resistance in EDL muscle of the obese mouse is associated with an increase in the proportion of hybrid IIB+IID fibres R. Blazev1, J.G. Kemp1,2, D.G. Stephenson3 and G.M.M. Stephenson1, 1School of Biomedical Sciences, Victoria University, VIC 3011, Australia, 2School of Exercise Science, Australian Catholic University, VIC 3065, Australia and 3Department of Zoology, La Trobe University, VIC 3083, Australia. 1745 119P Insulin-like growth factor-I gene transfer by electroporation enhances skeletal muscle regeneration and function after injury J.D. Schertzer and G.S. Lynch, Basic and Clinical Myology Laboratory, Department of Physiology, The University of Melbourne, Victoria 3010, Australia. 1900 - 2200 Conference Dinner - Australian National Museum

Friday 30 September 2005 Superior

Huron

Free communications 9: Education

Free communications 10: Skeletal Muscle 1

Chair: Ann Sefton

Chair: Robyn Murphy

0845 123P Enhancing the first-year experiences of undergraduate students enrolled in large classes Roger W. Moni, Karen B. Moni, Lesley Lluka and Philip Poronnik, School of Biomedical Sciences The University of Queensland, St. Lucia, 4072, Brisbane, Australia. 0900 124P Creating an effective learning community in a large-class service teaching physiology course H. Ernst and K. Colthorpe, School of Biomedical Sciences, The University of Queensland, St Lucia, QLD 4072, Australia. 0915 125P The place of physiology in an integrated medical curriculum T.O. Neild, Department of Human Physiology, Flinders University, GPO

0845 129P Low dose formoterol treatment reverses sarcopenia and improves muscle function in fast- but not slow-twitch skeletal muscles of aged rats G.S. Lynch and J.G. Ryall, Basic and Clinical Myology Laboratory, Department of Physiology, The University of Melbourne, Victoria 3010, Australia. 0900 130P β-adrenergic signalling in skeletal muscle regeneration after myotoxic injury F. Beitzel1, M.N. Sillence2 and G.S. Lynch1, 1Basic and Clinical Myology Laboratory, Department of Physiology, The University of Melbourne, VIC 3010, Australia and 2School of Agriculture, Charles Sturt University, Wagga Wagga, NSW 2678,

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Box 2100, Adelaide SA, 5001. Australia. 0930 126P Using a student-centred approach to enhance understanding of the physiology of metabolism and energy balance K.L. Colthorpe and H.G.G. Ernst, School of Biomedical Sciences, University of Queensland, St Lucia, Queensland 4072, Australia. 0945 127P Student perceptions and use of pre-specified criteria in constructing complex concept maps in physiology Roger W. Moni, Eileen Beswick, Alex Forrest and Karen B. Moni, School of Biomedical Sciences The University of Queensland, St. Lucia, QLD 4072, Australia. 1000 128P The opinion editorial – a novel assessment task in final year physiology Deanne Hryciw, Philip Poronnik and Roger W. Moni, School of Biomedical Sciences The University of Queensland, St. Lucia, QLD 4072, Australia.

Australia. 0915 131P Streptomycin reduces stretch-induced membrane permeability in isolated muscles from mdx (dystrophic) mice N.P. Whitehead, M. Streamer and D.G. Allen, School of Medical Sciences, University of Sydney (F13), NSW 2006, Australia. 0930 132P Muscle weakness in a mouse model of nemaline myopathy can be reversed with exercise and reveals a novel myofibre repair mechanism A.J. Kee, J.E. Joya, V. Nair-Shalliker, M.-A. Nguyen, M. Ghoddusi and E.C. Hardeman, Muscle Development Unit, Children’s Medical Research Institute, Westmead, NSW 2145, Australia. 0945 133P Contraction-mediated damage in mdx dystrophic mouse tibialis anterior muscles is not affected by the membrane sealant poloxamer D.R. Plant, J.G. Ryall and G.S. Lynch, Basic and Clinical Myology Laboratory, Department of Physiology, The University of Melbourne, VIC 3010, Australia. 1000 134P Aberrant splicing of ryanodine receptor reduces Ca2+ release via an inter-domain interaction in myotonic dystrophy type 1 T. Kimura1,2, M. Nakamori2, J.D. Lueck3, P. Pouliquin1, R.T. Dirksen3, M.P. Takahashi2, S. Sakoda2 and A.F. Dulhunty1, 1Muscle Research, John Curtin School of Medical Research, Australian National University, Canberra ACT, Australia, 2Clinical Neuroscience (Neurology), Graduate School of Medicine, Osaka University, Suita, Osaka, Japan and 3Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY, USA.

1015-1045 Morning Tea Free communications 11: Skeletal muscle 2

Free communications 12: Systems physiology

Chair: Gordon Lynch

Chair: Rick Lang

1045 135P The effect of altering the rest period during interval training on adaptations to muscle metabolism, ion regulation and exercise performance J. Edge, D. Bishop and C. Goodman, School of Human Movement and Exercise Science, The University of Western Australia, 35 Stirling Highway, Crawley WA 6009, Australia. 1100 136P Long lasting muscle fatigue: partial disruption of EC-coupling by the

1045 140P Novel nifedipine-insensitive high voltage activated calcium channels play a role in vascular tone of cerebral arteries M.F. NavarroGonzalez and C.E. Hill, Division of Neuroscience, John Curtin School of Medical Research, Australian National University, Canberra, ACT 2601, Australia. 1100 141P Accentuation during diabetes of differential connexin expression 19

between the preglomerular and postglomerular renal vasculature elevated cytosolic calcium during contractions E. Verburg, T.L. Dutka J.H. Zhang and C.E. Hill, Division of Neuroscience, JCSMR, ANU, and G.D. Lamb, Department of Zoology, La Trobe University Acton, ACT 2602, Australia. Bundoora Campus, Melbourne, VIC 3086, Australia. 1115 142P High-amplitude oscillations in human skin blood flow are distinct from 1115 137P The role of reactive oxygen species on stretch-induced muscle damage in dystrophic mice D.G. Allen1 and E.Y. Yeung2, 1School of known cardiac or respiratory influences C.D. Haley, C.J. Gordon, N.A. S. Taylor and A.B. Jenkins, Department of Biomedical Science, Medical Sciences, University of Sydney F13, NSW 2006, Australia University of Wollongong, Wollongong, NSW 2522, Australia. and 2Department of Rehhabilitation Sciences, Hong Kong Polytechnic University, Hong Kong. 1130 143P Activation of at least three classes of ion channels by β-adrenoceptor 1130 138P Effects of raising the temperature from 25°C to 37°C on twitch activation in pregnant uterine smooth muscle H.C. Parkington, M.A. responses in fast-twitch mechanically skinned muscle fibres of the Tonta, S. Simon, S.A. Cohen, A. Satragno, R.J. Lang and H.A. rat C. van der Poel, J. Edwards and D.G. Stephenson, Department Coleman, Department of Physiology, Monash University, Vic 3800, Australia. of Zoology, La Trobe University, VIC 3086, Australia. 1145 144P Expression of a constitutively active K+ channel prevents cell division 1145 139P Exposure of mammalian skeletal muscle to sub-physiological temperatures reduces its ability to function at physiological in the mouse preimplantation embryo M.L. Day1, C.G. Bailey2, J.E. temperatures J. Edwards, C. van der Poel and D.G. Stephenson, Rasko2, M.H. Johnson3 and D.I. Cook1, 1Physiology, School of Department of Zoology, La Trobe University, VIC 3086, Australia. Medical Sciences, University of Sydney, NSW 2006, Australia, 2 Gene Therapy, Centenary Institute of Cancer Medicine & Cell Biology, University of Sydney, NSW 2042, Australia and 3 Department of Anatomy, Downing St., University of Cambridge, CB2 3DY, UK. 1200-1245 Lunch - Joint meeting AuPS/ASB - Student Prizes - Chair: Peter Barry Symposium 7: Function and Regulation of Ion Transport Membrane Proteins Chair: Ron Clarke

Symposium 8: Epithelial Transport of Ions and Metabolites Chair: Stefan Bröer, David Cook

1245 150P A systems biology approach to understanding the role of peptide 1245 145P The transition between gating states in inward rectifier K+ channels J. transporters in biology H. Daniel, Meissner, B. Spanier, D. Weitz and Gulbis, Structural Biology Division, The Walter and Eliza Hall I. Frey, Molecular Nutrition Unit, Technical University of Munich, Am Institute, 1G Royal Parade, Parkville, VIC 3052, Australia. forum 5, D-85350 Freising-Weihenstephan, Germany. 1305 146P Channelrhodopsin 1,2, a new class of ion channels: functional 1315 151P Na+-H+ exchange regulatory factors NHERF-1 and NHERF-2: roles description and cellular applications Georg Nagel1, Peter in albumin endocytosis in the proximal tubule P. Poronnik1, C. Hegemann2, Suneel Kateriya2 and Ernst Bamberg1, 1Max-PlanckFerguson2, R. Parton2, C.H. Yun3 and D.H. Hryciw1, 1School of Institut für Biophysik, Frankfurt, Germany and 2Institute of Biology, Biomedical Science, The University of Queensland, Brisbane, QLD Humboldt University, Berlin, Germany. 4072, Australia, 2Institute of Molecular Biosciences, The University of 1325 147P Membrane lipid composition and its effect on Na+,K+-ATPase Queensland, Brisbane, QLD 4072, Australia and 3Department of 20

molecular activity: insights from mammals, birds and ectotherms N. Turner1,2,4, P.L. Else1,2, B.J. Wu1,2 and A.J. Hulbert1,3, 1Metabolic Research Centre, 2Department of Biomedical Science and 3School of Biological Sciences, University of Wollongong, Wollongong NSW 2522, Australia (4Present address: Diabetes and Obesity Program, Garvan Institute of Medical Research, 384 Victoria St, Darlinghurst, NSW 2010, Australia. 1355 148P Testing the membrane pacemaker model of metabolism Paul L. Else1, Nigel Turner1, Todd W. Mitchell1, Ben J. Wu1 and Anthony J. Hulbert2, 1Metabolic Research Centre, Department of Biomedical Science University of Wollongong, Wollongong, NSW 2522, Australia and 2Metabolic Research Centre, School of Biological Sciences, University of Wollongong, Wollongong, NSW 2522, Australia. (Introduced by R. Clarke) 1420 149P Regulation of the Na,K-ATPase Helge Rasmussen, Department of Cardiology, Royal North Shore Hospital, and Department of Medicine, University of Sydney, NSW, Australia. 1445 Close

Medicine, Division of Digestive Diseases, Emory University, Atlanta, USA. 1335 152P Sulphate ions in mammalian physiology: lessons from sulphate transporter knock-out mice P.A. Dawson1, B. Gardiner2, S. Lee1, M.C. Ku1, S.M. Grimmond2 and D. Markovich1, 1School of Biomedical Sciences, and 2Institute of Molecular Bioscience, University of Queensland, Brisbane, QLD 4072, Australia. 1355 153P Regulation of epithelial Na+ channels A. Dinudom, I.-H. Lee and D.I. Cook, School of Medical Sciences, University of Sydney, NSW 2006, Asutralia. 1420 154P Disorders of neutral amino acid resorption in epithelial cells S. Bröer, School of Biochemistry & Molecular Biology, Australian National University, Canberra, ACT 0200, Australia. 1445 Close

1445 AuPS Annual General Meeting 1545 Close

1445 ASB General Meeting 1545 Close End of Conference

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Abstracts and Author Index follows

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How does the membrane electric field gate an ion channel open and close? Francisco Bezanilla, Departments of Physiology and Anesthesiology. University of California at Los Angeles, Los Angeles, CA, U.S.A. Voltage dependent sodium and potassium channels are responsible for the generation and propagation of the nerve impulse. Voltage dependence is achieved by the translocation of about 12 e across the membrane field. The movement of this charge generates a transient current called gating current that precedes channel opening. Most of the gating charge is located in the first most extracellular arginines of the fourth transmembrane helix (S4 segment) found in voltage-gated channels. Accessibility studies with protons in mutants that replace the arginines by histidine, reveal that those residues are alternatively exposed to the inside at hyperpolarized potentials and outside at depolarized potentials because the histidine-replaced voltage sensor can act as a proton transporter. The most extracellular site generates a proton pore in the closed state, indicating that the electric field becomes very intense in a narrow region of the channel. A voltage sensing probe attached in this region confirms that the field is at least three times as large as the field in the bilayer. Resonance energy transfer experiments to several sites in S4 using the pore or the bilayer as a reference show that the S4 does not translocate across the membrane. The large number of charges (12 e) must then move in a small region where all the electric field is concentrated. In the hyperpolarized state, a water crevice penetrates from the intracellular medium all the way to the most extracellular charge exposing all the gating charges to the inside. We propose that, when the membrane is depolarized, the S4 changes its tilt and the charges change their exposure to an extracellular water-filled crevice while the intracellular crevice is obliterated. Thus in the process, the charges have been translocated without a significant translocation of the S4 segment in much the same way that a transporter operates. The change in tilt of the S4 segment induced by depolarization couples its motion to the S5 segment that allows the conformational change of S6 that opens the conduction pathway. A molecular model of the open and closed states based on multiple experimental results and the crystal structure of KvaP is able to reproduce quantitatively the movement of 12 e with less than 2 Å of S4 translocation. Supported by NIH grant GM30376.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/1P

AuPS/ASB Meeting - Canberra 2005 Symposium 1: Ion Channel Gating Wednesday September 28 2005 Chair: Shin-Ho Chung

The domains in the Na channel have specific functions Francisco Bezanilla and Baron Chanda, Departments of Physiology and Anesthesiology. University of California at Los Angeles, Los Angeles, CA, U.S.A. The classical voltage-dependent sodium channel is responsible for the upstroke of the action potential. Its pore forming subunit is a single polypeptide that has four homologous domains. Although each one of these domains is similar to a single subunit of the voltage gated potassium channel, the structures differ among each other. For example, the number of basic residues in S4 segments, responsible for voltage-sensing, is different in each domain. We have probed the function of each domain in the overall operation of the channel using fluorescent tags because they can detect local conformational changes. The results indicate that the S4 segments of domains I, II and III have faster kinetics than the S4 segment of domain IV. The kinetics and voltage dependence as reported by the fluorescent probe in the first three domains agree well with that of the fast component of the gating currents. On the other hand, the kinetics of the fluorescence probe attached in S4 of domain IV matches the kinetics of the slow component of the gating current. In addition, the turn-on of the fluorescence of domain IV exhibits a lag that is not observed in the fluorescence of the first three domains, indicating that the movement of S4-DIV does not start until one of the other three S4’s has moved. The kinetics of S4-DIV (or the slow component of the gating current) is too slow to account for the activation of the conductance but is faster that the time course of inactivation. In fact, the ionic current develops before the fluorescence change in S4-DIV. Taken together, these results indicate that the first three domains are responsible for the activation of the conductance and suggest that S4-DIV does not participate in channel opening. Other correlations of the fluorescence of S4-DIV with the kinetics and steady-state properties of inactivation indicate that the function of S4-DIV is related to the voltage dependence of inactivation that is ultimately produced by the IFM motif that blocks ion conduction. The local detection of conformation by the fluorescent probe can also be used to test possible interactions between domains during the operation of the channel. Thus, by introducing a mutation in one domain that affects the voltage dependence of the movement of that domain, one can ask whether that mutation has an effect in the movement of another domain. We have found that in fact all domains interact with each other with positive cooperativity. It can be demonstrated that positive cooperativity among voltage sensors increases the overall kinetics of channel opening. These results provide at least a partial explanation as to why sodium channels are so much faster than potassium channels, a requirement to generate the action potential. Supported by NIH grant GM30376 and American Heart Association.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/2P

Modulation of potassium channel conformation and function by permeating ions Stephen J. Korn, Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut 06269, USA. The Kv2.1 potassium channel is a very slowly inactivating delayed rectifier, with widespread distribution in brain, peripheral neurons and excitable cells such as heart and pancreas. In hippocampal neurons, where its function has been best studied, it appears to be profoundly important under conditions of high frequency firing or elevated extracellular [K+]. For example, with physiologically relevant elevation of extracellular [K+], action potential duration is increased ∼10 fold in the absence of Kv2.1 function, yet is unaffected in the presence of normal Kv2.1 function (Du et al., 2000). In contrast, Kv2.1 appears to have little importance to the integrity of single action potentials under physiological conditions. We have studied the molecular mechanism by which this seemingly standard delayed rectifier performs this very specific function. Kv2.1 channels open into one of two conformations. This conformational difference influences activation rate, inactivation rate, current magnitude and channel pharmacology. All of the functional effects of this difference in conformation are related to the orientation of a single outer vestibule lysine. In one conformation, currents are bigger and activate faster, whereas in the other, currents are smaller and activate more slowly. Which conformation the channel opens into appears to be determined by the occupancy of a particular K+ binding site in the channel’s selectivity filter. Thus, with elevation of [K+], occupancy of this site is greater, and channels open into a conformation that produces a larger current. In addition, two outer vestibule lysines dramatically reduce current magnitude variation associated with changes in K+ driving force that accompany changes in extracellular [K+]. Together, these two mechanisms produce the unique phenotype of the Kv2.1 channel. Upon elevation of external [K+], current density through Kv2.1 is increased at all membrane potentials, whereas in all other K+ channels, it is reduced. We propose that this increase in outward current density acts to maintain action potential integrity in the face of elevated extracellular [K+], which occurs during high frequency firing. From a biophysical perspective, the Kv2.1 channel displays another apparently unique mechanism. One of the fundamental mysteries in ion channel biophysics, which has potentially significant clinical importance, is how single channel conductance is controlled. Recent experimental and theoretical studies suggest that, in many K+ channels, single channel conductance is determined by inner vestibule characteristics. In contrast, our data demonstrate that single channel conductance in Kv2.1 can be modulated by reorientation of the outer vestibule. Thus, it may be that targets of conductance modulation will be different in different K+ channels. Du, J., L.L. Haak, E. Phillips-Tansey, J.T. Russell & C.J. McBain (2000) Journal of Physiology 522: 19-31.

Proceedings of the Australian Physiological Society

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The gating of mechanosensitive ion channels Boris Martinac, School of Biomedical Sciences, The University of Queensland, Brisbane, QLD 4072, Australia. Mechanosensitive (MS) ion channels are a special type of integral membrane proteins activated by membrane deformation caused by mechanical stimuli experienced by living cells. They convert mechanical stimuli into electrical and/or chemical intracellular signals. There is a great diversity of these channels in terms of ionic conductance, selectivity or voltage dependence. These channels have been found in all types of prokaryotic and eukaryotic cells. In animals and humans they play a role in hearing, touch, proprioception or regulation of blood pressure. In plants they may function as gravity sensors in gravitropism, whereas in bacteria they constitute a mechanism that prevents excessive water inflow and build-up of excessive turgor pressure by acting as mechano-electrical switches, which open in response to cell membrane deformations caused by osmotic forces under hypotonic conditions. Among the MS channels studied to date the best characterised are bacterial MscL and MscS channels, the MS channels of large (L) and small (S) conductance (Martinac, 2004). Their 3D structure was determined by X-ray crystallography allowing for in-depth studies of the gating mechanism in these channels. In particular, the structure, function and structural dynamics of MscL channel has been well characterized by a number of techniques including the patch-clamp technique, electronparamagnetic resonance (EPR) spectroscopy, molecular dynamics simulations and most recently FRET spectroscopy. MscL and other prokaryotic MS channels are gated by bilayer deformation forces indicating that mechanism of mechanotransduction in these channels is defined by both local and global asymmetries in the transbilayer pressure profile and/or bilayer curvature at the lipid protein interface (Perozo et al., 2002a, 2002b). Moreover, eukaryotic MS ion channels found in non-specialized mechanotransducer cells, such as TREK-1 (Patel et al., 2001) and TRPC1 (Maroto et al., 2005), have also been shown to be gated by membrane tension purely developed in the lipid bilayer. The implication of these findings is that the lipid bilayer is much more than a neutral solvent by actively modulating the specificity and fidelity of signalling by membrane proteins. Maroto, R., Raso, A., Wood, T.G., Kurosky, A., Martinac, B. & Hamill, O.P. (2005) Nature Cell Biology 7(2): 179-185. Patel, A.J., Honoré, E. & Lazdunski, M. (2001) Current Opinions in Cell Biology 13: 422-428. Martinac, B. (2004) Journal of Cell Science 117: 2449-2460. Perozo, E., Kloda, A., Cortes, D.M. & Martinac, B. (2002a) Nature Structural Biology 9: 696-703. Perozo, E., Cortes, D.M., Sompornpisut, P. Kloda, A. & Martinac, B. (2002b) Nature 418: 942-948.

Proceedings of the Australian Physiological Society

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The mechanism of fast gating in ClC chloride channels B. Corry1, D. Bisset2 and S.H. Chung2, 1Chemistry. School of Biomedical, Biomolecular and Chemical Science, The University of Western Australia, Crawley, WA 6009, Australia and 2Research School of Physical Sciences and Engineering, The Australian National University Canberra, ACT 0200, Australia. ClC proteins are a ubiquitous family of chloride channels and transporters that perform diverse functions such as the stabilisation of membrane potentials and the regulation of cell volumes. The most widely studied member of this family is ClC-0 from the Torpedo electric ray that has been shown to contain two independent ion conductive pores and have two distinct voltage gating mechanisms. The ‘slow’ or inactivation gate operates on both pores in the dimer simultaneously, whereas the ‘fast’ gate acts on each pore individually and opens and closes at a much faster rate. We have investigated the hypothesis that the side chain of a single glutamate residue acts as the fast gate in these channels using molecular dynamics simulations. We find that the motion of this side chain can indeed gate the channel, and furthermore demonstrate that this mechanism explains the dependence of channel gating on extracellular Cl- concentration, membrane potential and pH. Using the crystal structure of a bacterial ClC protein as a template we first create a putative open state configuration of the ClC-0 channel. Using this open state model we then conduct molecular dynamics simulations to study the motion of the central glutamate side chain. We find that when the side chain extends towards the extracellular end of the channel it presents an electrostatic barrier to Cl- conduction. However, external Cl- can push the side chain into a more central position where, pressed against the channel wall, it does not impede the motion of Cl- ions. Alternatively, the barrier to ion conduction can be removed by a proton from a low pH external solution binding to the side chain and neutralising its charge. Finally we use Brownian dynamics simulations to demonstrate the influence of membrane potential and external Cl- concentration on the open probability of the channel.

Proceedings of the Australian Physiological Society

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Conformational changes associated with glycine receptor activation J.W. Lynch and R.L. Hawthorne, School of Biomedical Sciences, University of Queensland, Brisbane, QLD 4072, Australia. The glycine receptor Cl− channel (GlyR) mediates inhibitory neurotransmission in the central nervous system. Like other members of the ligand-gated ion channel family, functional GlyRs comprise 5 subunits arranged symmetrically around an ion-conducting pore. Each subunit consists of a large external ligand-binding domain and 4 α-helical transmembrane domains (M1-M4). The external M2-M3 linker domain is crucial for transmitting the agonist-induced conformational change to the channel gate. Consistent with this role, a substituted cystine accessibility study on the M2-M3 linker of the α1 GlyR showed that the surface accessibility of 6 contiguous cysteine-substituted residues (R271C to K276C) was increased in the open state (Lynch et al., 2001). Thus, the conformation of the M2-M3 domain depends on the degree to which the GlyR is activated by agonist. This study investigated whether the closure of the channels by picrotoxin preserves the relationship between domain conformation and fractional peak current magnitude that is seen in its absence. If this relationship is not preserved, it may be concluded that picrotoxin closes the channel by inducing a novel conformational change in this domain. HEK293 cells were transfected with WT and mutant GlyR cDNA using the calcium phosphate precipitation method. Transfection solution was removed after 24 h and glycine-gated currents were recorded using whole-cell patch clamp techniques over the following 24−72 h. The surface accessibility of the introduced cysteines was assessed via their reaction rate with the sulfhydryl modifying agent, methanethiosulfonate ethyltrimethylammonium (MTSET) as previously described (Lynch et al., 2001). Picrotoxin significantly slowed the reaction rate of MTSET with A272C, S273C and L274C, although it had no measurable effect on R271C, P275C or K276C. Before interpreting this result as a picrotoxin-specific conformational change, it was necessary to eliminate the possibility of steric competition between picrotoxin and MTSET. One way of achieving this is to identify the location of the picrotoxin binding site. Accordingly, we showed that picrotoxin and the structurally-unrelated pore blocker, bilobalide, were both trapped in the R271C GlyR in the closed state and that a point mutation to the pore-lining T6’ residue abolished inhibition by both compounds. We also demonstrated that the picrotoxin dissociation rate was linearly related to the channel open probability. These observations constitute a strong case for picrotoxin binding in the pore. By binding in the pore, picrotoxin cannot sterically hinder MTSET from reacting with M2-M3 domain cysteines. We therefore conclude that picrotoxin changes the MTSET reaction rate by changing the intrinsic reactivity rates of the introduced cysteines. Because PTX changes the relationship between equivalent concentration and cysteine reactivity, we conclude that it alters the conformation of the GlyR M2-M3 domain in a way that cannot be achieved by simply varying the glycine concentration alone. This result implies that the M2-M3 domain integrates information from multiple categories of binding sites and sends a net signal to the activation gate. This reveals a hitherto unexpected complexity in the role of the M2-M3 domain. Lynch, J.W., Han, N.-L. R., Haddrill, J.L., Pierce K.D. & P.R. Schofield. (2001) Journal of Neuroscience, 21, 2589-2599.

Proceedings of the Australian Physiological Society

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AuPS/ASB Meeting - Canberra 2005 Symposium 2: Functional Roles of Potassium Channels in the Vasculature Wednesday September 28 2005 Chair: Mike Hill

Endothelium-dependent vasodilatation: Fundamental role of SKCa and IKCa potassium channels

C.J. Garland, Vascular Pharmacology Group, Department of Pharmacy & Pharmacology, University of Bath, Bath BA2 7AY, U.K.

Activation of vascular potassium (K) channels underlies both the radial and axial spread of dilatation within the artery wall. Radial spread, or an endothelium-dependent hyperpolarizing factor (EDHF) response, is initiated by agonist activation of endothelial cells, while axial, or spreading dilatation, can follow local hyperpolarization in either the endothelial or the smooth muscle cells. EDHF describes the endothelium dependent smooth muscle hyperpolarization persisting in the presence of inhibitors of nitric oxide (NO) synthase and cyclooxygenase, and causes smooth muscle relaxation by closing voltage-operated calcium channels. Originally assumed to reflect the action of a diffusible factor or factors, with analogy to EDRF or NO, the term is now also taken to encompass the possibility of passive spread of hyperpolarization from the endothelium (Busse et al., 2002). Key to understanding this pathway is the observation that EDHF-evoked hyperpolarization and associated smooth muscle relaxation can be blocked with a combination of apamin (blocks small conductance calcium-activated K channels, SKCa) and charybdotoxin (blocks intermediate and large calcium-activated K channels, IKCa and BKCa, plus delayed rectifier channels, KV). Alone, these toxins partially blocked EDHF responses, but in combination they totally abolished the response. Although initially taken to indicate that SKCa and BKCa on the smooth muscle were responsible for hyperpolarization (to a diffusible EDHF), iberiotoxin was unable to substitute for charybdotoxin (see Busse et al., 2002 for review). Furthermore, direct membrane potential measurements from endothelial cells in situ revealed that apamin and charybdotoxin are acting on K channels in these cells (Edwards et al., 1998). Pharmacological studies (using 1-EBIO and TRAM-34/39) then showed that the target for charybdotoxin is in fact the IKCa channel. Thus, agonist activation of the endothelium, leading to increases in [Ca2+]i, activates both SKCa and IKCa (which may be spatially separated, Crane et al., 2003) causing hyperpolarization which is transfered by a diffusible factor or passive spread through myoendothelial gap junctions to the adjacent smooth muscle, where relaxation is evoked. In addition to radial spread, axial spread of hyperpolarization is well described in the microcirculation (see Segal 2005). However, it seems to reflect an inherent property of resistance arteries as well (Takano et al., 2004). In small mesenteric arteries, selective activation of endothelial cell hyperpolarization, or of the KATP channels localized in the smooth muscle, results in hyperpolarization which spreads along the endothelium causing distant upstream dilatation. Interestingly, spread of hyperpoarization is not associated with an increase in endotheial cell [Ca2+] (Takano, 2004). It also may in part reflect KIR activity (Goto et al., 2004). Vascular potassium channels therefore play a crucial role in the spread of dilator signals through the artery wall, and disruption of this role may underlie alterations in vascular function in cardiovascular disease. Busse, R., Edwards, G., Feletou, M., Fleming, I., Vanhoutte, P.M. & Weston, A.H. (2002) Trends in Pharmacological Sciences 23, 374-380. Crane, G.J., Gallagher, N.T., Dora, K.A. & Garland C.J. (2003) Journal of Physiology 553, 183-189. Edwards, G., Dora, K.A., Gardener, M.J., Garland, C.J. & Weston, A.H. (1998) Nature 396, 269-272. Goto, G., Rummery, N.M., Grayson, T. & Hill, C.E. (2004) Journal of Physiology 561, 215-231. Segal, S.S. (2005) Microcirculation 12, 33-45. Takano, H., Dora, K.A., Spitaler, M.M. & Garland, C.J. (2004) Journal of Physiology 556, 887-903.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/7P

Functional effects of vascular KIR channels

C.G. Sobey, Department of Pharmacology, The University of Melbourne, Parkville, Victoria 3010, Australia. (Introduced by M. Hill) Potassium ion (K+) channel activity is one of the major determinants of vascular muscle cell membrane potential and thus vascular tone. Four types of K+ channels are functionally important in the vasculature Ca2+-activated K+ channels, voltage-dependent K+ channels, ATP-sensitive K+ channels, and inwardly rectifying K+ (KIR) channels. The latter type will be the subject of this review. Recent advances in vascular KIR channel research indicate that this channel: 1) is present in vascular muscle; 2) modulates basal arterial tone; 3) mediates powerful hyperpolarization and vasodilator responses to small but physiological increases in extracellular K+; 4) may contribute to vasodilatation in response to flowinduced shear stress; 5) may be inhibited by protein kinase C activity; 6) may be involved in vasorelaxation mediated by endothelium-derived hyperpolarizing factor; and 7) may be functionally altered by gender and in cardiovascular diseases. Vascular effects of KIR channels have so far been most extensively studied in the cerebral circulation where KIR function may be important in coupling cerebral metabolism and blood flow. Despite the lack of selective inhibitors of KIR channel subtypes, the use of gene knockout technology is beginning to enable more extensive insight to be gained regarding the functional role of these channels in blood vessels.

Proceedings of the Australian Physiological Society

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Kv as a target for nitroxyl anion (NO−)-mediated vasodilatation

B.K. Kemp-Harper and J.L. Favaloro, Department of Pharmacology, Monash University, VIC 3800, Australia. (Introduced by M. Hill)

Traditionally the vascular effects of nitric oxide (NO) have been attributed to the free radical form of NO (NO•) yet the reduced form of NO (NO−) is also produced endogenously and vasodilates both large conduit and small resistance-like arteries (Irvine et al., 2003). Interestingly, NO• and NO− have been shown to have distinct mechanisms of action in the cardiovascular system, particularly in the heart (Paolocci et al., 2003). This study aimed to determine if the vasorelaxant effects of NO− differed to those of NO• in rat small mesenteric resistance arteries. Male Sprague-Dawley rats were killed via CO2 sedation and cervical dislocation. Mesenteric arteries (∼350m m diameter) were isolated, mounted in small vessel myographs and isometric force and intracellular membrane potential measured simultaneously. Cumulative concentration-response curves to NO• (NO gas), the NO− donor, Angeli’s salt and the NO-independent soluble guanylate cyclase (sGC) activator, YC-1 were examined. Vasorelaxation to Angeli’s salt (pEC50=7.02±0.67 -log M; Rmax=96.0±2.2%, n=4) was accompanied by simultaneous vascular smooth muscle cell hyperpolarisation (pEC50=6.82±0.32, 10m M AS -17.8±4.4 mV, n=4). In contrast, maximal vasorelaxation to NO• (pEC50=6.82±0.39, 92.1±1.3%) was achieved before a small hyperpolarisation response was observed at 1m M NO• (-4.9±2.3 mV, n=5). Both relaxation and hyperpolarisation responses to Angeli’s salt were significantly attenuated (P<0.05, n=5) by the NO− scavenger, L-cysteine (3mM) and virtually abolished by the sGC inhibitor, ODQ (10m M; P<0.05, n=4). In contrast, ODQ only decreased the sensitivity of NO•-mediated vasorelaxation approximately 10-fold (P<0.05, n=4) and failed to affect NO•-mediated hyperpolarisation. The Kv channel inhibitor, 4-aminopyridine (1mM) caused a 4-fold (P<0.05, n=4) decrease in sensitivity to Angeli’s salt and abolished the hyperpolarisation response (P<0.05). Glibenclamide (KATP channel inhibitor) and charybdotoxin (BKCa/IKCa channel inhibitor) were without effect. YC-1 also induced smooth muscle hyperpolarisation (10m M YC-1 -43.0±6.3 mV, n=3) which was attenuated by 4-aminopyridine (10m M YC-1 -23.5±2.3 mV, P<0.05, n=3). In conclusion, in rat small mesenteric arteries, NO− mediates relaxation in part via cGMP-dependent activation of Kv channels. In contrast, NO•-mediated vasorelaxation occurs independently of vascular smooth muscle hyperpolarisation and in part via cGMPindependent pathways. Thus, the redox siblings NO• and NO− have distinct mechanisms of vasorelaxation in resistance-like arteries. Irvine, J.C., Favaloro, J.L. & Kemp-Harper, B.K. (2003) Hypertension 41, 1301-1307. Paolocci, N., Katori, T., Champion, H.C., St John, M.E., Miranda, K.M., Fukuto, J.M., Wink, D.A. & Kass, D. (2003) Proceedings of the National Academy of Science 100, 5537-5542.

Proceedings of the Australian Physiological Society

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The smooth muscle BKCa potassium channel and its interaction with arteriolar myogenic tone

T.V. Murphy1, Y.T. Hwang1, H. Ding2, N. Kotecha2 and M.A. Hill1, 1Physiology and Pharmacology, School of Medical Sciences, University of New South Wales, NSW 2052, Australia and 2Division of Biosciences, School of Medical Sciences, RMIT University, VIC 3083, Australia. Myogenic tone in arterioles, generated by intraluminal pressure, is important in autoregulation of blood flow and in determining the response of arterioles to vasodilator stimuli. The extent of arteriolar myogenic tone at a given intraluminal pressure varies among arterioles in different vascular beds; for example arterioles from skeletal muscle being relatively more constricted than those in the cerebral circulation at similar pressures. This may be due to differing expression or activity of large-conductance Ca 2+-sensitive K+-channels (BKCa) in the smooth muscle cells, which are thought to play a key role in regulating pressure-induced myogenic tone (Wellman & Nelson, 2003). The activity of BKCa channels is also increased by cyclic nucleotides (cGMP, cAMP), suggesting BKCa may be involved in the actions of paracrine dilators such as nitric oxide (NO). The aims of our studies were to compare the roles of BKCa in regulating myogenic tone in cerebral and skeletal muscle arterioles and to examine the importance of BKCa in endothelium-dependent dilation in vessels possessing different levels of myogenic tone. Functional studies in skeletal muscle arterioles from both rats and mice showed pressure-dependent vasoconstriction indicating the presence of myogenic tone. Over the pressure range 0 to 150 mmHg, a steep sigmoidal relationship was observed between the extent of myogenic tone (0 to 53.0 ± 7.8 %) and smooth muscle Em (-55.3 ± 4.1 mV to -29.4 ± 0.7 mV). Compared with data from published studies in cerebral vessels the slope of this relationship was both steeper and shifted towards more depolarised values. The selective BKCa inhibitor iberiotoxin (0.1 m M) caused a slight but significant vasoconstriction and depolarisation. Iberiotoxin treatment did not, however, alter the fundamental relationship between myogenic responsiveness and Em. Immunohistochemistry (IHC) demonstrated the presence of BKCa channels in smooth muscle cells of rat cerebral and cremaster muscle arterioles, without any difference in the expression pattern or levels. Real-time PCR, performed on mouse arterioles, demonstrated the expression of various Ca2+-activated K+-channels in the order sK3 > IK > BKCa. There was no difference, however, in BKCa expression (normalized to actin) between cerebral and skeletal muscle vessels. The data suggest that while BKCa channels are expressed in skeletal muscle arterioles they are not as tightly coupled to myogenic responsiveness as has been suggested for cerebral vessels. This may relate to important differences in vessel function as skeletal muscle vessels under normotensive conditions typically exhibit a high vascular resistance whereas the cerebral circulation tends to maintain a lower vascular resistance to ensure continuity of blood supply. With respect to the possible role of BKCa in endothelium-mediated dilation, responses to the endotheliumdependent dilator acetylcholine (ACh) were measured at differing levels of intraluminal pressure (50 and 120 mmHg) in isolated arterioles from the rat cremaster muscle. Dilation to ACh was significantly inhibited at the higher pressure, yet the magnitude of the ACh-induced hyperpolarization was not altered. In vessels maintained at 50 mmHg, EDHF made a substantial contribution to endothelium-dependent dilation with minor role for NO. At the higher intraluminal pressure (120 mmHg) the relative contribution of EDHF was reduced however, with a corresponding increase in the importance of NO/cGMP-mediated dilation. Further studies showed dilation to cGMP alone was enhanced at the higher pressure, suggesting an increased sensitivity to cGMP. We suggest this is due to a cGMP-induced increase in activity of BKCa channels (Schubert & Nelson, 2001), which is more pronounced with increased pressure-induced myogenic tone and, in part, counteracts the inhibitory effect of increased intraluminal pressure and membrane potential on K+-channel activity. Schubert, R. & Nelson, M.T. (2001) Trends in Pharmacological Sciences, 22, 505-512. Wellman, G.C. & Nelson, M.T. (2003) Cell Calcium, 34, 211-229.

Proceedings of the Australian Physiological Society

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Potassium channels in vascular dysfunction C.R. Triggle1, A. Ellis2, L. Ceroni3, W. Wiehler3 and H. Ding1, 1School of Medical Sciences, RMIT University, Melbourne, VIC, Australia, 2Division of Chinese Medicine, School of Health Sciences, RMIT University, Melbourne, VIC, Australia and 3Smooth Muscle Research Group, University of Calgary, Canada. (Introduced by Michael Hill) Intermediate and small conductance calcium-activated potassium channels, IKCa and SKCa respectively, play a critical role in the regulation of endothelium-derived hyperpolarizing factor (EDHF)-mediated endothelium-dependent vasodilatation (EDV). Connexins (Cx) 37, 40, 43 and 45 are expressed in vascular tissue and also contribute to EDHF-mediated EDV in a tissue dependent manner. In the wild type control (WT) C57BL/6J mouse the contribution of EDHF increases, relative to NO, from 1st to 2nd and greatest in 3rd order vessels. Changes in the contributions of nitric oxide (NO) and EDHF have also been reported in disease states, such as diabetes, and may reflect an important contribution to the pathophysiology (Pannirselvam et al., 2002). In this study we have compared EDV initiated by acetylcholine (ACh) in resistance vessels (small mesenteric arteries – SMA) from male eNOS-null mouse (eNOS-/-), that present with a hypertensive and insulin resistant phenotype, to the hypertensive, insulin resistant and hyperglycaemic type 2 diabetic db/db mouse and the type 1 diabetic apoE-null- streptozotocin (STZ) mouse. In SMA from the eNOS-/- mouse EDV, initiated by ACh, is mediated entirely by EDHF and similarly in the db/db, leptin receptor mutant type two diabetic mouse. Despite the absence of a contribution from NO to EDV in the db/db mouse no difference was found in either mRNA or protein levels of eNOS. In the STZ-induced type 1 diabetic apoE-null mouse the contribution of EDHF to EDV is reduced and the expression of eNOS is increased. The combination of the IKCa channel blockers, charybdotoxin (ChTx) or TRAM-34, and the SKCa blocker apamin inhibits a large portion of the contribution of EDHF to ACh-mediated EVD in eNOS-/-, db/db, and the STZ-apoE-/- mice with a small component remaining that is sensitive to iberiotoxin, IbTx. The data with IbTx indicates a role for the large conductance BKCa channel and this, likely, reflects an action on the vascular smooth muscle cells mediated by a cytochrome P450 metabolite. The presence of the putative myoendothelial gap junction (MEGJs) inhibitor, b -glycyrrhetinic acid (b -GA), produced a significant inhibition of EVD in the eNOS-/- but not in the WT mouse. These data suggest that a component of the EDHF-mediated EDV in the eNOS-/-, but not the WT, is mediated by MEGJs. Real time PCR was also conducted to determine mRNA expression for the KCa channels: the large conductance BKCa, IKCa, and the SKCa SK1, SK2 and SK3 subtypes in 1st, 2nd and 3rd vessels from eNOS-/- and WT mice; however, no difference, relative to the housekeeping gene b -actin was found. Similarly for the expression of mRNA for Cx 37, 40, 43 and 45 – no differences in expression levels were found. In contrast, in SMA from the STZ-apoE mouse, expressions levels of SK2, SK3 and Cx37 were significantly reduced as was the functional contribution of EDHF to EDV, whereas eNOS levels were increased We conclude that type 1 and type 2 diabetic states have different effects on EDV with type 1 decreasing the contribution of EDHF and type 2 decreasing the bioavailability of NO. Western blots to determine protein levels have not been consistently successful for interpretation reflecting the low protein yield from the SMA. Pannirselvam, M., Verma, S., Anderson, T.J. & Triggle, C.R. (2002) British Journal of Pharmacology 136, 255-263.

Proceedings of the Australian Physiological Society

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AuPS/ASB Meeting - Canberra 2005 Free Communications 1: Exercise Physiology Wednesday September 28 2005 Chair: David Allen

Sudomotor responses during isometric exercise appear to be intensity- and muscle massdependent C.J. Gordon, C.D. Haley, J.N. Caldwell and N.A.S. Taylor, Department of Biomedical Science, University of Wollongong, Wollongong, NSW 2522, Australia. Cardiac frequency, mean arterial pressure and skin sympathetic nerve activity during isometric exercise, increase in proportion to exercise intensity (Vissing et al., 1991), while pressor responses also appear to be modulated by the size of the active muscle mass (Ray and Wilson, 2004). Sweating also responds to exercise intensity (Kondo et al., 2000), however, there is no information relating to the affect of muscle mass recruitment on sudomotor function. The hypothesis was tested that non-thermal sudomotor drive in the heat would be influenced by both exercise-intensity and the size of the recruited muscle mass. Seven, resting (upright) males were heated (60 min) using a water-perfusion suit (37.2°C) and a climatecontrolled chamber (36.7°C, 58% relative humidity) to induce steady-state sweating. Body temperature was clamped thereafter. Isometric handgrip and knee extension activations (60 s with 10 min rest) were performed at ˙ sw) was measured (1 Hz: 30% and 50% maximal voluntary contraction (MVC) in a balanced order. Sweat rate (m 2 3.16 cm capsules) at four sites (forehead, chest, and inactive forearm and thigh), and averaged. Cardiac frequency was monitored continuously (0.2 Hz), and mean arterial pressure was measured beat-by-beat. Oesophageal and mean skin temperatures did not change during either rest or isometric exercise, verifying the veracity of the thermal clamp. Cardiac frequency displayed both an intensity- and a mass-dependence, resulting in the following pre- to post-activation changes (1 min): handgrip (5.9±1.4, 22.4±2.0 b.min-1, 30 and 50% MVC; P<0.05); knee extension (14.5±1.4, 26.6±2.5 b.min-1, 30 and 50% MVC; P<0.05). Similar to cardiac frequency, mean arterial pressure increased significantly during handgrip (10.1±1.9, 23.7±4.1 mmHg, 30 and 50% MVC; P<0.05), and knee extension (20.1±1.6, 32.1±2.8 mmHg, 30 and 50% MVC; P<0.05). Whilst ˙ sw from baseline, were intensity-dependent, ˙ sw baselines were similar, normalised increases in m pre-activation m but not mass-dependent: handgrip (0.093±0.027 and 0.212±0.035 mg.cm-2.min-1, 30 and 50% MVC; P<0.05); knee extension (0.140±0.017 and 0.198±0.026 mg.cm-2.min-1; P>0.05). However, the integrated sudomotor responses during isometric exercise appeared to reveal an intensity- and muscle mass-dependency: handgrip (3.15±0.70 mg.cm-2 and 4.61±0.87 mg.cm-2, 30 and 50% MVC); knee extension (4.17±0.48 and 5.53±0.89 mg.cm-2). Whilst differences between handgrip and knee extension were non-significant (30% MVC P=0.09; 50% MVC P=0.08), post hoc analyses reveal our design to be under-powered; further testing is underway. In ˙ sw remained elevated compared to handgrip exercise. The possibility exists addition, following knee extension, m ˙ that the delayed msw recovery, was mediated by intramuscular changes, which may be mass-dependent. This study provides evidence that sudomotor responses to isometric exercise, during heat stress, may be exercise-intensity and muscle mass-dependent. If real, this latter observation is both novel and significant. Nonthermal factors have been suggested to modulate sweating during isometric exercise (Kondo et al., 2000). We now propose that motor unit recruitment may also influence sweating. In addition, the continued elevation of ˙ sw, but not body temperature, after isometric exercise, in particular knee extension exercise, may indicate that m metaboreceptor stimulation, or an unidentified thermal factor, has augmented post-exercise sweating. This appears to also be mass-dedendent. Kondo, N., Tominaga, H., Shibasaki, M., Aoki, K., Okada, S. & Nishiyasu, T. (2000) Journal of Applied Physiology 88: 1590−1596. Ray, C.A. & Wilson, T.E. (2004) Journal of Applied Physiology 97: 160-164. Vissing, S.F., Scherrer, U. & Victor, R.G. (1991) Circulation Research, 69: 228-238.

Proceedings of the Australian Physiological Society

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Hydration indices in exertional heat stress A.T. Garrett1,2, N.G. Goossens1, N.J. Rehrer1, M.J. Patterson3 and J.D. Cotter1, 1School of Physical Education, University of Otago, Dunedin, New Zealand, 2College of Education, University of Canterbury, Christchurch, New Zealand, and 3Defence Science and Technology Organisation, Melbourne, Australia. Introduction. Hydration is a multi-factorial and dynamic phenomenon relating to the volume and composition of bodily fluid compartments. Nonetheless hypohydration (lower than normal body water content) can be associated with reduced cognition and endurance exercise performance especially in the heat, and possibly with increased propensity or severity of heat illnesses. In regard to heat illness and performance, functionally relevant measures of hydration may be most validly measured during or immediately after the dehydrating stress. The reduction in body mass (%) is a traditional index of hydration, but plasma volume (PV) and plasma osmolality (Osmop) might be viewed as having a more functional role in maintaining homeostasis under prolonged exertional heat stress. Purpose. To examine the relationship between indices of hydration during dehydrating exercise in the heat, with variable rehydration. Methods. Eighteen males (mean ±SD age 25 ±6 y, mass 74.9 ±4.4 kg, cycling peak oxygen uptake 4.7 ±0.3 L min-1) undertook two to six 90-min heat exposures involving intermittent exercise in hot humid conditions (39.5°C, 60% r.h.) or continuous exercise in warm, moderately-humid conditions (35°C, 60% r.h.), with rehydration varying from none to full water replacement orally. Hydration-related indices measured before during and after exposures included plasma indices (AVP, Aldosterone, Na+, Osmolality, D PV), thirst, urine (specific gravity (SGU), colour, osmolality), and body mass. Results. Baseline reliabilities (mean difference) were variable between measures; AVPp 2.2%, Aldosteronep 25.8%, Na+p 0.3%, Osmop 1.5%, thirst 15.9% and SGU 0.1%. Linear relations between hydrationrelated indices are shown in the table. Measure 1 (min - max)

Measure 2 (min - max)

r2

P

D body mass (-2.8 - 0.9%)

D PV (-20.7 - 1.0%) Osmop (275 - 319 mosmol kg-1) AVPp (1 - 26 pg mL-1) Aldosteronep (20 - 993 pg mL-1) Na+p (136 - 149 mmol L-1) Proteinp (61 : 113 mg mL-1) SGU (1.000 - 1.030 units) %D body mass PV Osmop Na+p Proteinp Osmop

14% 4% 15% 94% 69% 16% 3% 81% 13% 7% 6% 46% 14%

0.02 0.36 0.03 0.00 0.00 0.00 0.24 0.00 0.22 0.45 0.36 0.00 0.11

Thirst (4 - 9 units)

D PV

Conclusion. Statistically-significant associations were evident between most pairs of hydration-related measures under conditions of dynamic exercise and ambient heat stress with varied rehydration. However, most associations were weak, and plasma osmolality, which is considered the most criterion measure, showed little association with other functional measures or with fieldable measures. The close relation between thirst and change in body mass has functional value but, oddly, was not reflective of a similarly close relation for factors that stimulate thirst. Supported by research grants from DSTO and University of Otago.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/13P

Plasma ammonia responses during heavy-intensity constant-load cycling in young and older individuals S. Sabapathy, D.A. Schneider and N.R. Morris, School of Physiotherapy and Exercise Science, and Heart Foundation Research Centre, Gold Coast campus, Griffith University, Southport, QLD 4215, Australia. A delayed and slowly increasing component of O2 uptake kinetics may be observed when performing high intensity constant-load exercise (Barstow, 1994). Additionally, the increase in plasma ammonia concentration ([NH3]) during high intensity cycling has been associated with the recruitment of type II fibres (Dudley et al., 1983). This study sought to examine the relationship between the slow component of O2 uptake kinetics and plasma [NH3] during constant-load cycling in healthy young and older individuals. Seven young (mean age ± SD: 21.4 ± 2.8 yr) and 8 older healthy male adults (71.7 ± 2.7 yr) performed 7 min of heavy constant-load exercise. The power output for the constant-load tests was quantified as 50% of the difference between the power output attained at the gas exchange threshold and that achieved at peak O2 uptake. The kinetics of O2 uptake measured during constant-load exercise (including the slow component amplitude) were characterised using established non-linear regression modelling techniques (Sabapathy et al., 2004), as illustrated in the Figure. Plasma [NH3] was measured at rest, following 3 min of unloaded cycling, and at 3 and 7 min of constant-load exercise.

The amplitude of the slow component was 406 ± 65 mL/min in the young and 217 ± 59 mL/min in the older subjects. Plasma [NH3] values measured after 3 min of unloaded cycling and at 3 min of constant-load exercise were not significantly different from resting values, but increased significantly (P<0.01) between 3 and 7 min of exercise in both groups and correlated significantly (P<0.05) with the slow component (Young: r = 0.79; Older: r = 0.75). While these findings do not indicate a causal link between the two variables, they could be related to a common physiological mechanism. The increase in [NH3] observed is consistent with a progressive recruitment of type II muscle fibres during the slow component phase of exercise in both young and older individuals. The measurement of plasma [NH3] during high-intensity exercise could provide a relatively non-invasive index of muscle fiber recruitment patterns. Barstow T.J. (1994) Medicine and Science in Sports and Exercise, 26: 1327-1334. Dudley G.A., Staron R.S., Murray T.F., Hagerman F.C. & Luginbuhl, A. (1983) Journal of Applied Physiology, 54: 582-586. Sabapathy S., Schneider D.A., Comadira G., Johnston I. & Morris N.R. (2004) Respiratory Physiology and Neurobiology, 139: 203-213.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/14P

Abnormal muscle Na+,K+-pumps, plasma K+, and exercise limitation in renal failure patients A.C. Petersen1, M.J. Leikis2, K.T. Murphy1, J.A. Leppik1, X. Gong1, A.B. Kent2, L.P. McMahon2 and M.J. McKenna1, 1Muscle, Ions and Exercise Group, Centre for Ageing, Rehabilitation, Exercise and Sport, School of Human Movement, Recreation and Performance, Victoria University, Melbourne, VIC 8001, Australia and 2Department of Nephrology, Royal Melbourne Hospital, Department of Medicine, University of Melbourne, Melbourne, VIC 3052, Australia. Patients with chronic kidney disease demonstrate an abnormally low exercise performance, which has been linked to impaired extrarenal K+ regulation (Sangkabutra et al., 2003). The cause of the impaired skeletal muscle K+ regulation is unknown, but skeletal muscle Na+,K+-ATPase activity is subnormal in uraemic rats (Goecke et al., 1991). In renal transplantation recipients (RTx), exercise performance is improved. Whether this is due to improved extrarenal K+ regulation is unknown. Therefore, this study investigated whether plasma K+ regulation during an incremental cycle test to fatigue was 1) impaired in haemodialysis patients (HD), 2) improved in RTx compared to HD, and 3) correlated to exercise performance. We also investigated whether skeletal muscle Na+,K+-ATPase activity and content were impaired in HD and RTx. Ten HD, nine RTx, and ten age-, body mass-, height- and gender-matched controls (CON) performed incremental cycle exercise to fatigue, to measure peak oxygen consumption (VO2peak) and arterial (HD, RTx) or arterialised-venous (RTx, CON) plasma [K+] during exercise, corrected for plasma volume shifts. Leg-extensor isokinetic muscle strength was measured at 0, 60, 120, 180, 240, 300, and 360°.s-1 and fatiguability determined by the percentage decline in peak torque during 30 maximal contractions at 180°.s-1 and 0.5 Hz. Thigh muscle cross-sectional area (CSA) was measured by CT-scan. A resting biopsy was taken from the vastus lateralis muscle and analysed for Na+,K+-ATPase content (3H-ouabain binding site content) and maximal activity (K+-stimulated 3-O-methylfluorescein phosphatase activity). [Hb] was not different between the groups (HD 13.3±1.4 (mean ± SD), RTx 13.4±0.9, and CON 14.5±1.3 g dl-1). VO2 peak was higher in CON than HD and RTx, by 35% and 32%, respectively (35.7±4.0, 26.4±6.0, 27.0±9.6 ml kg-1 min-1, respectively, P<0.01). Leg-extensor muscle strength relative to CSA did not differ between groups, but was higher in CON when expressed relative to body mass (P<0.05). Leg-extensor fatiguability was lower in CON than in HD and RTx (13.3±5.9, 25.2±4.3, 23.8±10.7 %, respectively, P<0.01). The rise in plasma [K+] with exercise (D [K+]) only differed between groups at fatigue where CON was higher than HD and RTx (P<0.01). The D [K+]-to-work ratio was not different between groups (HD 14.9±8.5, RTx 20.8±15.6, CON 15.6±10.4 nmol L-1 J-1, P=0.53) and was not correlated to VO2 peak or leg-extensor fatiguability. Muscle 3H-ouabain binding site content did not differ between HD, RTx, or CON (285 ± 77, 275 ± 46, 284 ± 56 pmol g wet wt-1, respectively). The Figure shows higher maximal K+-stimulated 3-O-MFPase activity in CON by 44% and 38%, compared to HD and RTx, respectively (P<0.05). For pooled data (n=28) 3-O-MFPase activity was correlated 3 (P<0.05) with H-ouabain binding site content (r = 0.42), VO2peak (r = 0.45), maximum workrate (r = 0.43), total work done (r = 0.39), and D [K+] during incremental exercise (r = 0.41), as well as kidney function measured by creatinine clearance (r = 0.44). Whilst HD and RTx exhibited lower VO2peak and higher leg-extensor fatiguability compared to CON, their + D [K ]-to-work ratio during incremental exercise was not impaired. Muscle Na+,K+-ATPase content was normal in the patients, however muscle maximal Na+,K+-ATPase activity was reduced suggesting an abnormality in skeletal muscle Na+,K+-ATPase in renal failure patients. Furthermore, muscle maximal Na+,K+-ATPase activity was correlated to VO2peak, suggesting a link with impaired incremental exercise performance in uraemia. Goecke, I.A., Bonilla, S., Marusic, E.T. & Alvo, M. (1991) Kidney International 39, 39-43. Sangkabutra, T., Crankshaw, D.P., Schneider, C., Fraser, S.F., Sostaric, S., Mason, K., Burge, C.M., Skinner, S.L., McMahon, L.P. & McKenna, M.J. (2003) Kidney International 63, 283-290.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/15P

The effect of eccentric exercise on plasma K+ regulation and skeletal muscle Na+,K+-ATPase activity and content J.A. Bennie1, C.A. Goodman1, M.J. Leikis2 and M.J. McKenna1, 1Muscle, Ions and Exercise Group, School of Human Movement, Recreation and Performance, Centre for Ageing, Rehabilitation, Exercise and Sport, Victoria University, PO Box 14428, Melbourne, VIC 8001, Australia and 2Department of Nephrology, Royal Melbourne Hospital, Department of Medicine, University of Melbourne, Melbourne, VIC, Australia. Intense exercise results in considerable muscle K+ efflux, with consequently increased muscle interstitial and reduced intracellular [K+]. These changes and the reduction in transcellular [K+] gradient have been linked with impaired skeletal muscle excitability and contractility (Sejersted & Sjøgaard, 2000). Eccentric exercise causes damage to involved muscles, with a commonly observed consequence being a reduction in the structural integrity of the sarcolemma and T-tubular system and release of intracellular proteins (Allen et al., 2005). Hence, it is possible that the Na+,K+-ATPase inserted in these membranes may also be impaired, which may therefore also affect plasma [K+] and Na+ and K+ regulation in skeletal muscle. There have been no published investigations into the effects of unaccustomed eccentric exercise on plasma [K+] or on Na+,K+-ATPase activity and content and these were therefore investigated here. It was hypothesized that eccentric exercise would progressively increase plasma [K+] and depress both the Na+,K+-ATPase activity and content immediately post-exercise. Six healthy subjects (3 males, 3 females) performed a single bout of one-legged, eccentric, knee extensor exercise, comprising 300 repetitions of maximal eccentric contractions, at 30o/s. The eccentric exercise bout was conducted on an isokinetic dynamometer, and consisted of 10 sets of 30 repetitions, with a 1 min recovery period separating each set. Maximal isometric knee extensor torque was assessed pre-and immediately postexercise. Plasma [K+] was measured in arterialised blood sampled from a dorsal hand vein immediately prior to exercise and at the end of sets 1, 2, 4, 6, 8, and 10. Muscle biopsies were taken from the vastus lateralis muscle at rest and immediately post-exercise and analysed for maximal Na+,K+-ATPase (3-O-MFPase) activity and Na+,K+-ATPase ([3H]-ouabain binding) content. Maximal isometric torque of the knee extensors was depressed (P <0.05) immediately post-exercise by 26 ± 11% (Mean ± SD). Total work performed by the knee extensors during each set remained constant from sets 1 to 5 after which it was reduced for all subsequent sets (P <0.05). Plasma [K+] was elevated above rest by the end of the first set (P <0.05). However, despite the declining work output, plasma [K+] remained elevated throughout the remainder of the exercise bout. The rise in [K+] above rest (D [K+]) expressed relative to the amount of work performed (D [K+]/work ratio), increased from set 2 to set 4 (P <0.05) and then remained elevated through to set 10. Although tendencies for declines were noted, no significant change was found after eccentric exercise in maximal 3-O-MFPase activity (P = 0.095) or [3H]-ouabain binding site content (P = 0.074). In conclusion, the observation that plasma [K+] remained elevated despite a decrease in work performed by the knee extensor muscles suggests an impairment in K+ regulation during prolonged maximal eccentric exercise. This may reflect a reduction in muscle Na+,K+-ATPase and/or damage to the muscle membranes.

[K+]

Allen, D.G., Whitehead, N.P. & Yeung, E.W. (2005) Journal of Physiology In press - published July 7, 2005, 10.1113/jphysiol.2005.091694 Sejersted, O. M. & Sjøgaard, G. (2000) Physiological Reviews 80, 1411-1481.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/16P

N-acetylcysteine infusion enhances skeletal muscle Na+,K+-ATPase activity and plasma K+ regulation, and delays fatigue, during prolonged submaximal exercise in well-trained individuals C.A. Goodman1, I. Medved1, M.J. Brown2, A.R. Bjorksten3, K.T Murphy1, A.C Petersen1, S. Sostaric1, X. Gong1 and M.J. McKenna1, 1Muscle, Ions & Exercise Group, Centre for Ageing, Rehabilitation, Exercise and Sport, School of Human Movement, Recreation and Performance, Victoria University, PO Box 14428, Melbourne, VIC 8001, Australia, 2Department of Anaesthesia, Austin Health, Heidelberg, VIC, Australia and 3Department of Anaesthesia and Pain Management, Royal Melbourne Hospital, Melbourne, VIC, Australia. The production of reactive oxygen species (ROS) in skeletal muscle has been linked with muscle fatigue (for review see Reid, 2001). Recently, we showed that intravenous infusion of the antioxidant N-acetylcysteine (NAC) increased each of muscle NAC (total and reduced), cysteine and glutathione (reduced) and improved prolonged submaximal exercise performance in well-trained individuals (Medved et al., 2004). We have found depressed Na+,K+-ATPase activity in skeletal muscle during exercise, which may contribute to disturbed muscle ionic homeostasis and fatigue (Leppik et al., 2004). This study investigated whether ROS may be involved in this process, by examining the effect of NAC infusion on skeletal muscle Na+,K+-ATPase activity and potassium (K+) regulation during prolonged submaximal endurance exercise, in well trained individuals. Eight well-trained subjects participated in a double blind, randomised, crossover design study, receiving either an NAC or saline (CON) infusion into a superficial forearm vein (Medved et al., 2003). NAC was intravenously infused at 125 mg.kg-1.hr-1 for 15 min, then 25 mg.kg-1.hr-1 for 20 min prior to and throughout exercise, which was continued until fatigue. Subjects completed cycling exercise comprising 45 min at 70% VO2peak, then to fatigue at 90% VO2peak. Muscle biopsies were taken from the vastus lateralis before exercise, at 45 min and at fatigue and analysed for maximal in vitro Na+,K+-ATPase activity (maximal K+-stimulated 3-Omethyfluorescein phosphatase, 3-O-MFPase). Blood was sampled at pre-infusion, immediately prior to exercise, during exercise at 15, 30, 45 min and at fatigue. Blood was analysed for plasma [K+] as well as blood haemoglobin concentration ([Hb]) and hematocrit (Hct). Time to fatigue at 90% VO2peak was reproducible in preliminary trials (CV 5.6±0.6%) and with NAC was enhanced by 20.8±9.1% (NAC 6.4±0.6 vs CON 5.3±0.7 min, P<0.05) (Medved et al., 2004). Maximal 3-OMFPase activity decreased by 21.6±2.8% at 45 min and by 23.9±2.3% at fatigue when compared to rest (P<0.05). NAC attenuated the percentage change in maximal 3-O-MFPase activity at 45 min (P<0.05) compared to control but not at fatigue. However, the change in 3-O-MFPase activity to work ratio was attenuated by NAC both at 45 min and at fatigue (P<0.005). The rise in plasma [K+] and plasma D [K+]-to-work ratio during exercise were both attenuated by NAC (P<0.05). There was no significant correlation between time to fatigue and each of maximal 3-O-MFPase, rise in plasma [K+] and plasma D [K+]-to-work ratio. In conclusion, our data show that NAC infusion in well-trained individuals attenuated the depression in muscle Na+,K+-ATPase and enhanced K+ regulation, which may be important in delaying fatigue during prolonged submaximal exercise. This suggests that ROS play a role in skeletal muscle fatigue and specifically in Na+,K+-ATPase regulation and K+ regulation during submaximal exercise. Leppik, J.A., Aughey, R.J., Medved, I., Fairwhether, I., Carey, M.F. & McKenna, M.J. (2004) Journal of Applied Physiology, 97, 1414-1423. Medved, I., Brown, M.J., Bjorksten, A.R., Leppik, J.A., Sostaric, S. & McKenna, M.J. (2003) Journal of Applied Physiology, 94, 1572-1582. Medved, I., Brown, M.J., Bjorksten, A.R., Murphy, K.T., Petersen, A.C., Sostaric, S., Gong, X. & McKenna, M.J. (2004) Journal of Applied Physiology, 97, 1477-1485. Reid, M.B. (2001) Journal of Applied Physiology, 90, 724-731.

Proceedings of the Australian Physiological Society

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AuPS/ASB Meeting - Canberra 2005 Free Communications 2: Ion channels Wednesday September 28 2005 Chair: Peter Barry

Investigating the mechanism of proton transfer through the bacterial ClC transporter M. O’Mara, J. Yin, M. Hoyles and S.H. Chung, Research School of Physical Sciences and Engineering, Australian National University, Canberra, ACT 0200, Australia. The ClC chloride channel family is a ubiquitous, yet highly unique family of ion channels, involved in a diverse range of physiological functions. Accardi & Miller (2004) showed that the bacterial ClC channel, ClCec1, is not a simple chloride channel, but a chloride / proton exchange transporter, exchanging two chloride ions for every one proton. More recent experimental studies have shown that two eukaryotic members of the family, ClC-4 and ClC-5, are also chloride / proton exchange transporters (Picollo & Pusch, 2005; Scheel et al., 2005). Computational investigations have provided a detailed description of the mechanism of chloride permeation through several ClC isoforms (Cohen & Schulten, 2004; Corry et al., 2004). However, there is very little information describing the transport of protons through ClC-ec1, or the location of the proton translocation pathway. It is known, however, that Glu148, Ser107 and Tyr455 are involved in the translocation pathways of both chloride and protons (Accardi & Miller, 2004). Here we use computational techniques to probe ClC-ec1 to determine the most energetically favourable translocation pathway for protons. First we ran multiple searches using the HOLE program (Smart et al., 1993) to identify every continuous pathway through the protein with a radius greater than 0.6Å. Our results converged on four possible pathways through each protein dimer. We then used a Poisson-Boltzmann calculation to determine which of these pathways was energetically favourable for protons. Our investigations reveal a narrow fissure through each dimer, 0.75 Å in radius, close to the dimer interface. The protein surrounding these fissures is relatively rich in polar and ionizable amino acids, creating an environment favourable for protons. In support of the experimental evidence, we find that Glu148, Ser107 and Tyr455 are pore-lining residues of our proposed proton translocation pathway, as well as the chloride translocation pathway. Electrostatic calculations of the unoccupied ClC-ec1 transporter show that our proposed proton translocation pathway contains an electrostatic potential barrier to proton permeation, in the intracellular region of the pathway, effectively barring proton permeation. However, when two chloride ions occupy the chloride pathway, the potential energy barrier in the proton translocation pathway is converted to an electrostatic potential energy well of approximately 18 kT, deep enough to hold one proton in a stable configuration. This occupancy pattern, confirmed by Brownian dynamics simulations, supports the experimentally predicted exchange rate of one proton for every two chloride ions (Accardi & Miller, 2004). Accardi, A. & Miller, C. (2004) Nature 427, 803-807. Cohen, J. & Schulten, K. (2004) Biophysical Journal 86, 836-845. Corry, B., O’Mara, M. & Chung, S.H. (2004) Biophysical Journal 86, 846-860. Picollo, A. & Pusch, M. (2005) Nature 436, 420-423. Scheel, O., Zdebik, A.A., Lourdel, S. & Jentsch, T.J. (2005) Nature 436, 424-427. Smart, O.S., Goodfellow, J.M. & Wallace. B.A. (1993) Biophysical Journal 65, 2455-2460.

Proceedings of the Australian Physiological Society

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An electrostatic basis for valence selectivity in cationic channels T. Vora1, B. Corry2, and S.H. Chung1, 1The Department of Theoretical Physics, RSPhysSE, The Australian National University, Canberra, ACT 0200, Australia and 2Chemistry, School of Biomedical and Chemical Science, The University of Western Australia, Crawley, WA 6009, Australia. It is well known that the charge distribution surrounding the pore is responsible for ion selectivity in cation channels. Negative charges lining the pore region present an energy well for cations and a barrier of a similar magnitude for anions, thereby selecting one species and excluding the other. Less well known is how these channels select between cations with differing magnitudes of charge. Here we have constructed models of the KcsA, sodium and calcium channels and compared the permeation of monovalent and divalent ions to understand the basis of this valence selectivity. For both the KcsA and sodium channels, monovalent ions readily pass through the channel. However, as soon as a divalent ion enters the selectivity filter it binds strongly, rarely leaving and thereby almost completely preventing the permeation of monovalent ions. This effect is equally important for inward and outward currents in the KcsA channel, but more pronounced for the inward current in the sodium channel. In contrast, calcium channels conduct monovalent ions in the absence of any divalent ions, but when a mixture of monovalent and divalent ions is present, they allow only the divalent ions to pass. This phenomenon of selectivity between monovalent and divalent ions can be attributed to electrostatics. We have used simulations of Brownian dynamics and electrostatic calculations to show that in all three channel types, the distribution of charges in the protein creates an energy well that attracts many ions into the channel, making conduction a three ion process for sodium and calcium channels and a four ion process for the KcsA channel. But for the case of divalent ions, the energy well in the KcsA and sodium channels is very deep. Once a divalent ion has entered it finds it difficult to leave, even with the aid of a repulsive kick from other ions in the channel. Thus divalent ion block of the channel causes the currents to plummet. On the other hand, for the calcium channel a second divalent ion entering the channel presents enough repulsion to push a resident divalent ion out of the channel, but a monovalent ion does not. Thus, the calcium channel is able to select divalent ions in a divalent-monovalent ion mixture.

Proceedings of the Australian Physiological Society

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A current source and a cation conductance are components of an electrical circuit connected across the plasma membrane of the malaria parasite Plasmodium falciparum R.J. W. Allen, K.J. Saliba and K. Kirk, School of Biochemistry and Molecular Biology, Linnaeus Way, Australian National University, Canberra, ACT 0200, Australia. Like most cells, the intraerythrocytic malaria parasite Plasmodium falciparum requires a high intracellular concentration of K+ (∼135 mM) for normal development. Using 86Rb+ and the potential-sensitive compound 3HTPP+, we have shown that the parasite’s mechanism of K+ uptake is electrophoretic, mediated by a pathway with characteristics of a K+ channel. The driving force, the parasite’s membrane potential, ∆ψ, originates from the extrusion of H+ by a (V-type) H+-ATPase on the plasma membrane. However, we have also shown that ∆ψ is modulated (partially offset) by extracellular K+, indicating an interdependence between K+ influx and ∆ψ. Investigations into the kinetics of K+ uptake have shown that between 5 mM – 130 mM K+, the influx of + K remains constant, despite there being a reduction in ∆ψ with increasing concentrations of extracellular K+. These phenomena may be reconciled by considering the H+-ATPase as an ‘ideal’ current source, and the + K channel as a ‘variable’ conductance, the latter a function of the extracellular concentration of K+ (see figure). In this electrical model, the inward current carried by K+ influx through the K+ channel, ‘Iin’, is equal to the outward current carried by the (net) export of H+ via the H+-ATPase, ‘Iout’ (i.e. Iin = Iout). As the K+ conductance of the membrane is varied by altering the extracellular concentration of K+, the offset to ∆ψ caused by the influx of K+ also varies, so that the equality Iin = Iout remains satisfied.

During its growth phase, the accumulation of K+ by the parasite is achieved in the context of a >10-fold decrease in the concentration of K+ (from ∼140 mM) within the host red cell (itself a result of the parasite manipulating the permeability of the host cell membrane). The mechanism we describe is able to explain the parasite’s ability to generate a stable influx of K+, neither overwhelmed by, nor starved of, K+, as the concentration of K+ within the red cell undergoes a dramatic reduction. Largely on the basis of sequence homology to the canonical selectivity filter of homotetrameric K+ channels, two putative K+ channel genes have been identified in the Plasmodium falciparum genome database. Hydropathy profiles suggest that both channels have additional transmembrane domains over and above the 6 characteristic of voltage-gated K+ channels, a feature shared by several members of the ‘slo’ K+ channel family. The function of these domains is unknown. Both channels are unusual for their great size (the larger has ∼2000 residues per subunit), and have large hydrophilic domains which are predicted to reside cytosolically, the functions of which are also unknown. The larger protein has an ‘S4’ segment containing 3 regularly spaced arginines, in a pattern consistent with a (perhaps degenerate?) voltage sensor of a voltage-gated K+ channel. Immunofluorescence studies demonstrate localisation of this protein to be predominantly at the host cell membrane, suggesting that it is not the K+ uptake pathway in the parasite membrane discussed above, but perhaps plays a role in the alteration of the ionic makeup of the host cell cytosol by the parasite. No data currently exists for the location of the smaller protein. These putative K+ channels are the subject of continuing investigations.

Proceedings of the Australian Physiological Society

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Role of protein flexibility in gramicidin A channel permeability Turgut Bastug, School of Physics, University of Sydney, NSW 2006, Australia. Proteins have a flexible structure, and their atoms exhibit considerable fluctuations under normal operating conditions. However, apart from some enzyme reactions involving ligand binding, our understanding of the role of flexibility in protein function remains mostly incomplete. Here we investigate this question in the realm of membrane proteins that form ion channels. Specifically, we consider ion permeation in the gramicidin A channel (GA), and study how the energetics of ion conduction changes as the channel structure is progressively changed from fixed NMR structure to a completely flexible one as obtained from molecular dynamics (MD) simulations. For each channel structure, the potential of mean force for a permeating potassium ion is determined from MD simulations. Using the same MD data for completely flexible gramicidin A, we also calculate the average densities and fluctuations of the GA atoms and investigate the correlations between these fluctuations and the motion of a permeating ion. Our results show conclusively that peptide flexibility plays an important role in ion permeation in the gramicidin A channel, and hence it cannot be modeled using continuum electrostatics with an average, fixed structure.

Proceedings of the Australian Physiological Society

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Ca2+ influx through store-operated Ca2+ channel in mouse sinoatrial node Y.K. Ju1, H. Chaulet2, R.M. Graham2 and D.G. Allen1, 1School of Medical Sciences, University of Sydney, NSW 2006, Auatralia and 2Victor Chang Cardiac Research Institute, NSW 2010, Australia. In both excitable and non-excitable cells, the depletion of intracellular Ca2+ stores causes a flux of Ca2+ into the cells and refills the Ca2+ store to its original level. The inward Ca2+ flux resulting from depletion of Ca2+ store is through the store-operated cation channels (SOCCs). There is growing evidence that SOCCs play an important role in muscle cell signalling (for review see Gailly, 2002). In previous studies, we found that intracellular Ca2+ stores are involved in cardiac pacemaking (for review see Ju & Allen, 2001). To examine if store–operated Ca2+ entry is present in cardiac pacemaker tissue and its possible role in regulating heart rate, sinoatrial node (SAN) tissue was dissected from mouse right atria of the heart and loaded with the Ca2+ indicator indo-1 AM. In the presence of extracellular Ca2+ ([Ca2+]o ), cyclopiazonic acid (CPA), a selective sarcoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor, significantly increased resting [Ca2+]i and gradually reduced the amplitude of [Ca2+]i transients. Incubating SAN in Ca2+ free solution caused a substantial decline in resting [Ca2+]i and stopped pacemaker activity. Reintroduction of Ca2+ (1.8 mM) to the perfusate in the presence of CPA evoked a striking increase in resting [Ca2+]i, a characteristic of SOCC activity. The Ca2+ influx in response to reintroduction of [Ca2+]o was 7.1 ± 3.2 fold greater in the presence of CPA than in its absence (p < 0.03, n = 11), which suggested that the Ca2+ influx was dependent on the SR store depletion. It is known that the Na+-Ca2+ exchanger exists in cardiac pacemaker tissue. After a period of incubation in zero Ca2+ solution, the reintroduction of Ca2+ could also activate the reverse mode of Na+-Ca2+ exchanger and increase Ca2+ influx. To test this possibility, we applied Na+-Ca2+ exchanger inhibitor KBR -7943. We found that in the presence of KBR -7943, there was still a significant rise of [Ca2+]i in response to the depletion of SR the Ca2+ store. Moreover, gadolinium (100 µM), a known SOCC inhibitor, significantly inhibited 72 ± 8% of Ca2+ influx in the present of CPA (P< 0.01, n = 4). Recent studies have suggested that SOCCs might be related to the transient receptor potential canonical (TRPC) gene family. We examined SAN mRNA expression of the seven known mammalian TRPC isoforms by RT-PCR. mRNA for TRPC1, 2, 3, 4, 6 and 7 was detected in SAN, whereas that for the TRPC5 was not. These results suggest that cardiac pacemaker tissue exhibits store-operated Ca2+ activity which may be due to expression of TRPCs in these cells. Gailly P. (2002) Biochimica et Biophysica Acta 1600, 38-44. Ju, Y.K. & Allen, D.G. (2001) Clinical and Experimental Pharmacology and Physiology 28, 703-8. Supported by NH&MRC

Proceedings of the Australian Physiological Society

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A hydrogen peroxide insult causes sustained alteration in the sensitivity of the L-type Ca2+ channel to β-adrenergic receptor stimulation in ventricular myocytes L.C. Hool, H.M. Viola and P.G. Arthur, School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, Crawley, WA 6009, Australia. We have shown previously that mitochondrial-derived hydrogen peroxide (H2O2) regulates the function of the L-type Ca2+ channel. A decrease in mitochondrial-derived H2O2 is associated with an increase in the sensitivity of the channel to the beta-adrenergic receptor agonist isoproterenol (Iso) and exposing myocytes to H2O2 attenuates the response. Here we examine the effect of a hydrogen peroxide insult on the function of the L-type Ca2+ channel. Ventricular myocytes were isolated from hearts excised from anaesthetised guinea-pigs as approved by the Animal Ethics Committee of The University of Western Australia and in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (NHMRC). The cells were exposed to 30 µM H2O2 for 5 min followed by 10U/ml catalase for 5 min to degrade the H2O2, and then the response of the channel to Iso was examined. In the absence of a peroxide insult, 10 nM Iso elicited a current that was 72.1±8.0% of the current elicited by 1 µM Iso (a maximally stimulating concentration of the agonist) within the same cell (n=6). However, after exposure of cells to peroxide 10 nM Iso elicited a current that was just 18.6 ± 10.0 % of the response elicited by 1 µM Iso within the same cell (n=6; P<0.05) suggesting that the peroxide insult significantly decreased the sensitivity of the channel to Iso. More importantly this effect persisted for several hours after the peroxide insult. We examined whether the effect was a result of enhanced production of reactive oxygen species by the cell. Cellular production of superoxide was measured using the fluorescent indicator dihydroethidium. Exposing cells to 30 µM H2O2 for 5 min followed by 10U/ml catalase for 5 min caused a 61.1±14.0% increase in superoxide production (n=13; P<0.05) compared to controls exposed to catalase only (n=8). The increase in superoxide was completely attenuated when cells were exposed to the mitochondrial inhibitor myxothiazol (6-10 nM; n=14; P<0.05). The NAD(P)H oxidase inhibitor apocynin (300 µM, n=5) did not alter the increase in superoxide associated with a peroxide insult nor did 100 µM of the xanthine oxidase inhibitor, allopurinol (n=5). We propose that a hydrogen peroxide insult causes a further increase in hydrogen peroxide production from the mitochondria and the increase in peroxide results in a sustained decrease in sensitivity of the channel to β-adrenergic receptor stimulation.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/23P

Structure and gating mechanism of the nicotinic acetylcholine receptor N. Unwin, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. The nicotinic acetylcholine (ACh) receptor is the neurotransmitter-gated ion channel at the nerve-muscle synapse. It serves as a model for other members of the Cys-loop superfamily, including neuronal ACh, GABAA, glycine and 5-HT3 receptors. Structures of the ACh receptor in the closed-channel form (Unwin, 2005; Miyazawa, Fujiyoshi & Unwin, 2003) and in the open-channel form (Unwin, 1995) are now known to resolutions of 4Å and 9Å respectively, from electron crystallographic studies of Torpedo postsynaptic membranes. The receptor is a large (290kD) glyco-protein, assembled from a ring of homologous subunits (α, g , α, b , d ) and divided into three domains: a large N-terminal extracellular ligand-binding domain, a membranespanning pore, and a smaller intracellular domain, giving it a total length of about 160Å normal to the membrane plane. The ligand-binding domain shapes a long, ∼20Å diameter central vestibule and has two binding sites for ACh, which are in the α subunits at interfaces with the g and d subunits, on opposite sides of the pore. The pore makes a narrow water-filled path across the membrane and contains a hydrophobic gate, which breaks open when ACh occupies both binding sites. The intracellular domain shapes another, smaller vestibule, having narrow lateral openings for the ions. The inner surfaces of both vestibules are negatively charged, creating a cation-stabilising environment at either entrance to the membrane pore. We show that the ligand-binding portions of the two α subunits have a ‘distorted’ conformation relative to the b , g and d subunits when the binding sites are empty and the channel is closed. Binding of ACh causes a local rearrangement in which loops B and C of the α subunits are drawn in around the bound ligand to enable coordination of relevant side-chains. The local rearrangement overcomes the distortions in the α subunits, stabilising an alternative conformation which is like that of the non-α subunits. This transition is accompanied by rotations of the inner sheet of the b sandwich composing the ligand-binding portions of the α subunits. The rotations are communicated to the pore-lining α-helices in the membrane, triggering cooperative movements which disrupt the gate of the channel, allowing ions to flow through. Thus gating in this channel appears to occur by fast cooperative movements of helices lining the membrane pore, whereas the ligand-binding domain appears to function as a controlling device that either disenables or facilitates these movements. Unwin, N. (2005) Journal of Molecular Biology 346, 967-989. Miyazawa, A., Fujiyoshi, Y. & Unwin, N. (2003) Nature 423, 949-955. Unwin, N. (1995) Nature 373, 37-43.

Proceedings of the Australian Physiological Society

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AuPS/ASB Meeting - Canberra 2005 Free Communications 3: Ligand-Gated Ion Channels Wednesday September 28 2005 Chair: Louise Tierney

Analysis of a GABAAg 2 (R43Q) knock-in mouse model of familial epilepsy

S. Petrou, H. Tan, P. Davies and S. Murphy, Howard Florey Institute, The University of Melbourne, VIC 3010, Australia. (Introduced by L. Tierney) Epilepsy is a common neurological disorder that causes paroxysmal electrical discharges in the brain. It can be difficult to treat, with up to 30% of all patients unable to achieve satisfactory pharmacological control of their seizures, and many others living with a host of adverse side effects such as sedation and cognitive impairment. Genetic analysis of familial forms of epilepsy led to the discovery of ion channel gene mutations linked to a range of epilepsy syndromes, thus pointing to changes in ion channel function as the primary causative agent of many common forms of epilepsy. Lessons learned from the analysis of these mutations and how they alter neurobiology and neurophysiology are vital to our gaining knowledge of the fundamental mechanisms of seizure genesis, and provide the key to developing better strategies to control epilepsy. We have begun this process with a detailed examination of an autosomal dominant mutation of a GABAAg 2 receptor subunit [GABRg 2(R43Q)] found in a large family with GEFS+ (Generalised Epilepsy with Febrile Seizures plus). The study of mouse models harbouring human epilepsy causing mutations is one way in which a more direct link between genes and phenotypes can be made that not only permits the molecular study of mutated genes in vivo, but which may also provide a direct link to phenotype. We therefore generated a knock-in mouse model with the GABRg 2(R43Q) mutation, to investigate the in situ functional consequences of this genetic lesion on inhibitory synapses, and to assess the potential involvement of this mutation in the development of epilepsy. Wild type mice (g 2R43/R43) and mice heterozygous for the GABRg 2(R43Q) mutation (g 2R43/Q43) were anaesthetised i.p. with ketamine (100 mg/kg) and xylazine (15 mg/kg), and instrumented for EEG recording. Video and EEG analysis of recordings revealed absence seizures, with correlated 3-7 Hz spike and wave discharges (SWD) in the mutant animals. Human patients carrying the GABRg 2(R43Q) mutation display absence seizures with 3 Hz SWD; recapitulation of the human phenotype in our mouse model is a vital validation step, and suggests that underlying pathological mechanisms may be shared between mouse and human. The heterozygous mutant mice also showed an elevated sensitivity to challenge with the pro-convulsant drug, pentylenetetrazol (40-120 mg/kg, s.c.). Miniature Inhibitory Postsynaptic Currents (mIPSCs) were analysed in Layer 2/3 cortical neurons using the whole cell patch clamp technique, in acute brain slices obtained from P14-16 mice decapitated after anaesthesia by inhalation of isoflurane. Analysis of wild type, heterozygous and homozygous mutant (g 2Q43/Q43) mice demonstrated a reduction in amplitude (g 2R43/R43 = 67.5 ± 2.91 pA, 2R43/Q43 = 58.4 ± 2.22 pA, g 2Q43/Q43 = 40.0 ± 2.62 pA), and a slower rate of decay (g 2R43/R43 = 4.7 ± 0.15 ms, g 2R43/Q43 = 5.9 ± 0.32 ms, g 2Q43/Q43 = 31.4 ± 2.62 ms). The heterozygous phenotype was much more subtle than would be expected from a simple gene dosing effect. Interestingly, the frequency of detectable synaptic events was also drastically reduced in the homozygous mutant animals (g 2R43/R43 = 7.17 ± 0.83 Hz, g 2Q43/Q43 = 0.85 ± 0.15 Hz). We are currently characteristing mIPSCs in the thalamocortical relay circuit, which has been implicated in the generation of the SWD of absence epilepsy. To determine whether the altered characteristics of the inhibitory currents were due to a pre- or postsynaptic pertubation, immunohistochemical analysis was performed in brain slices obtained from P12-P16 mice killed after pentobarbitone anaesthesia (100 mg/kg, i.p). In homozygous mutant mice, punctate staining of glutamic acid decarboxylase (GAD), the marker of GABAergic synaptic inputs, was unaltered compared to wild type controls. However, in contrast, immunoreactivity for GABAA α1 and g 2 subunits was virtually absent from most brain regions, including the cortex, thalamus and cerebellum. The distribution of g 2 subunits was investigated further in cortical neurons, maintained in primary cultures for 14-16 days. Pregnant female mice were killed by cervical dislocation, and cortices dissected from decapitated E15-E16 embryos. To determine the location of GABAA receptor subunits in cultured neurons, extracts of total cellular proteins and cell surface proteins were prepared: the cell surface proteins were biotinylated in living cultures, and subsequently extracted on an immobilised avidin column. In heterozygous and homozygous mutant animals, the g 2 subunit was detected in extracts of total cellular protein, but its expression was reduced in cell surface protein extracts. Our studies suggest that a reduction in cell surface expression of the g 2 subunit, along with a reduction in amplitude and altered deactivation kinetics of mIPSCs, are the key molecular deficits responsible for absence epilepsy in this model.

Proceedings of the Australian Physiological Society

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The receptor-associated protein, rapsyn, and regulation of postsynaptic acetylcholine receptor packing density and turnover at the neuromuscular synapse W.D. Phillips and O.L. Gervásio, Department of Physiology, Institute for Biomedical Research, University of Sydney, NSW 2006, Australia. Rapsyn is a membrane-associated protein that binds the cytosolic M3-M4 loop domain of the nicotinic acetylcholine receptor (AChR). During embryogenesis, neural agrin (a proteoglycan secreted by the nerve terminal) is thought to coordinate the spatially-appropriate activation of Muscle Specific Kinase (MuSK). This initiates divergent intracellular pathways that result in 1) formation of an AChR cluster (a process that depends upon rapsyn) and 2) possibly also the transcriptional activation of synaptic genes. The precise signaling pathways for these effects remain ambiguous (Bezakova & Ruegg, 2003). We have been studying the role of rapsyn in the homeostasis of the established synapse. Rapsyn was tagged by fusing enhanced green fluorescent protein (EGFP) to its C-terminus (Gervásio & Phillips, 2005). Rapsyn-EGFP functioned like unmodified rapsyn since it assembled into AChR clusters when cultured myotubes were treated with neural agrin. To study its role in vivo we anaesthetized 4-week old female FVB mice with 5m l/g I.P. of a mixture of ketamine (10mg/ml) and xylazine (10mg/ml). The tibialis anterior muscle was exposed and electroporated with expression plasmid for rapsyn-EGFP, followed by subcutaneous injection of 30 m l of buprenorphine (300m g/ml). Rapsyn-EGFP targeted to the dispersed Golgi elements in the muscle fibre where it may normally assemble with the newly synthesized AChR. Rapsyn-EGFP also targeted directly to the postsynaptic AChR cluster where it increased the stoichiometry of rapsyn to AChR. This was associated with a slowing in the metabolic turnover of synaptic AChR (Gervásio & Phillips, 2005). Rapsyn-AChR stoichiometry can also be increased by neural agrin treatment, suggesting a possible physiological mechanism that might regulate retention of AChRs within the postsynaptic AChR cluster. What do we mean when we speak of a postsynaptic receptor cluster? These are often defined in papers merely as bright spots or puncta of anti-receptor antibody staining. Intracellular receptors are often located immediately beneath the postsynaptic membrane. These may be confused with functional, surface-exposed receptors in routine immunostaining. In the case of the neuromuscular synapse, small <1m m AChR clusters are found at the lips of the post-junctional membrane infoldings. Here AChRs are packed tightly together (10,000/m m2; Salpeter & Harris, 1983) and mediate the postsynaptic current. Clusters interdigitate with postjunctional membrane in-foldings in which a reserve of AChRs are present, but at 10-fold lower density. While exposed to the extracellular fluid, these ‘unclustered’ AChRs are unlikely to contribute greatly to the normal postsynaptic current. Unclustered AChRs seem to be in continual interchange with the neighboring, clustered AChRs. Alterations in the efficiency of rapsyn-mediated AChR clustering might change the fraction of ‘synaptic’ AChRs that are engaged in the postsynaptic AChR cluster, and thereby the amplitude of the postsynaptic current. To gauge the efficiency of postsynaptic AChR clustering we have developed a Fluorescence Resonance Energy Transfer (FRET) technique. FRET is a sensitive method for detecting situations where two fluorescently labeled proteins come within 10nm of each other. AChRs are labeled with a mixture of TRITC-α-bungarotoxin (FRET donor) and Alexafluor647-α-bungarotoxin (acceptor). These are allowed to bind randomly to the AChR. The two binding sites on each AChR channel are separated by about 8nm and yield only weak intramolecular FRET. FRET efficiency increases 5-fold due to intermolecular FRET when AChRs come together in a cluster. FRET efficiency was low at synapses in newborn mice but increased approximately twofold during postnatal development, coincident with a similar increase in the rapsyn-AChR stoichiometry at the synapse. This suggests that the efficiency with which AChRs are partitioned within postsynaptic AChR clusters may be regulated by rapsyn-AChR stoichiometry. Bezakova, G. & Ruegg, M.A. (2003) Nature reviews of Cell and Molecular Biology 4, 295-308. Gervásio, O.L. & Phillips, W.D. (2005) Journal of Physiology 562.3, 673-685. Salpeter, M.M. & Harris, R. (1983) Journal of Cell Biology 96, 1781-1785.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/26P

Functional consequences of clustering GABAA receptors

M.L. Tierney, T. Luu, A.E. Everitt, P.W. Gage, Membrane Physiology & Biophysics Group, The John Curtin School of Medical Research, The Australian National University, Canberra 0200, Australia.

Inhibitory signals in mammalian brains are mediated primarily by g -aminobutyric acid type A receptors (GABAAR). Different subtypes of these receptors, distinguished by their subunit composition, are either concentrated at postsynaptic sites where they mediate phasic inhibition or found at non-synaptic (extrasynaptic) sites where they mediate tonic inhibition. Neurons, therefore, require discrete trafficking mechanisms to regulate the subcellular distribution of GABAAR subtypes. Although its precise role in vivo is yet to be clearly defined, the GABAA receptor-associated protein GABARAP has been shown to participate in trafficking and membrane fusion events that underlie organisational processes at GABAergic synapses (Kittler et al., 2001; Kneussel, 2002). Co-expression of GABARAP has been shown to cluster recombinant GABAA receptors (Chen et al., 2000; Everitt et al., 2004) and, as a consequence of this ordered packing arrangement, the recombinant GABAA receptors function differently. At the single-channel level we have shown that GABAA receptors coexpressed with recombinant GABARAP in L929 cells may display high conductances (>40 pS) (Everitt et al., 2004) which is in stark contrast to the conductance of αbg receptors expressed without GABARAP (<40 pS) (Luu et al., 2005). Single-channel amplitude distribution histograms and open probabilities were analysed to examine the effects of drugs such as GABA and diazepam. The presence of the soluble intracellular protein GABARAP influences recombinant GABAA channels such that they may open to multiple discrete states and the maximum single-channel conductance is dependent on the effective GABA concentration. It remains a fundamental issue as to what these multiple conductance states represent. Is it a single channel or multiple channels opening in concert? An important conclusion is that recombinantly expressed αbg receptors behave more like native receptors when cotransfected with GABARAP. Chen, L., Wang, H.B., Vicini, S. & Olsen, R.W. (2000) Proceedings of the National Academy of Sciences of the United States of America, 97, 11557-11562. Everitt, A.B., Luu, T., Cromer, B., Tierney, M.L., Birnir, B., Olsen, R.W. & Gage, P.W. (2004) Journal of Biological Chemistry, 279(21), 21701-21706. Kittler, J.T., Rostaing, P., Schiavo, G., Fritschy, J.M., Olsen, R., Triller, A. & Moss, S.J. (2001) Molecular and Cellular Neurosciences, 18, 13-25. Luu, T., Cromer, B.C., Gage, P.W. & Tierney, M.L. (2005) Journal of Membrane Biology, 205, in press. Kneussel, M. (2002) Brain Research - Brain Research Reviews, 39, 74-83.

Proceedings of the Australian Physiological Society

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The charge of the P-loop glutamate controls cation-anion selectivity in CNG channels W. Qu1, A.J. Moorhouse1, M. Chandra1, K.D. Pierce2, T.M. Lewis1 and P.H. Barry 1, 1Dept of Physiology and Pharmacology, School of Medical Sciences, The University of New South Wales, NSW 2052, Australia and 2Neurobiology Research Program, The Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, NSW 2010, Australia. Cyclic-nucleotide-gated (CNG) channels play a critical role in olfactory and visual transduction. We have used site-directed mutagenesis and inside-out patch-clamp recordings to investigate ion permeation and selectivity in two mutant homomeric rat olfactory CNGA2 channels expressed in HEK 293 cells. We showed that a single point mutation of the negatively-charged pore-loop (P-loop) glutamate to either a positivelycharged lysine or arginine did produce functional channels, which consistently responded to either cGMP or cAMP, although the currents were extremely small. We found that the concentration-response curve of the lysine mutant channel was very similar to that of wild-type (WT) channels, suggesting no major structural alteration to the mutant channels. Reversal potential measurements, during cytoplasmic NaCl dilutions, showed that both the lysine and the arginine mutations switched the selectivity of the channel from cations (PCl/PNa = 0.07 [WT]) to anions (PNa/PNa = 15 [Lys] or 10 [Arg]). In addition, we showed that these mutant channels seem to have an extremely small single-channel conductance, measured using noise analysis to be about 1 pS, compared to a WT value of about 25 pS. Our results indicated that it is predominantly the charge of the E342 residue in the P-loop, rather than the pore helix dipoles, which controls the cation-anion selectivity of this channel. However, the outward rectification displayed by both mutant channels in symmetrical NaCl solutions suggests that the negative ends of the pore helix dipoles probably play a role in reducing the outward movement of Cl- ions through these anion-selective channels. Such a postulated mechanism is also supportive of the mutations only causing local effects within the selectivity filter region of the channel. These results may have general implications for the determinants of anion-cation selectivity in the large family of P-loop containing channels. We also showed from measurements of reversal potentials, with different halide ion substitutions, that the relative permeability of the halide ions increases with ionic radius in these E342K and E342R mutant CNG channels. Since ionic radius is inversely related to hydrated ion size, this result indicates that it is dehydration of these ions, as they pass through the selectivity region of the mutant channel, that is the major factor determining their relative permeability, as is also observed in anion-selective GABAA and glycine receptor channels (FatimaShad & Barry, 1993). Fatima-Shad, K. & Barry, P.H. (1993) Proceedings of the Royal Society (London) B, 253, 69-75.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/28P

Structure and dynamics of the periplasmic loop of the MscL mechanosensitive channel studied by electron paramagnetic resonance spectroscopy G. Meyer1,2,3, E. Perozo2 and B. Martinac1,3, 1School of Medicine and Pharmacology, University of Western Australia, Crawley, WA 6009, Australia and 2Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22906, USA. 3(Present address: School of Biomedical Sciences, University of Queensland, Brisbane, QLD 4072, Australia). The periplasmic loop of the bacterial mechanosensitive channel of large conductance (MscL) is one of the five structural domains of the channel, which has been suggested to play a significant role in gating of the channel by mechanical force (Ajouz et al., 2000; Maurer & Dougherty, 2003). The structure of the loop has however, not been fully characterised. After the structural details of the MscL transmembrane helices, TM1 and TM2, were determined by crystallography and electron paramagnetic resonance (EPR) spectroscopy (Chang et al., 1998; Perozo et al., 2001), a model of the complete structure of MscL of E. coli was proposed (Sukharev et al., 2001). The model provided basis for characterisation of the MscL gating by molecular dynamics (MD) simulations (Gullingsrud et al., 2001). Recent MD simulations (Meyer et al., 2004) suggested further an important role for the periplasmic loop in the MscL channel gating. Using the methods of cysteine scanning mutagenesis, spin labelling and EPR spectroscopy on MscL reconstituted into liposomes, we carried out an initial study towards characterisation of the structural dynamics of the loop. The EPR spectra recorded from the channel in its closed configuration and in the open state induced by lysophosphatidylcholine (LPC) (Perozo et al., 2002), indicated that significant structural rearrangements in the loop region occurred during channel opening. Our results thus appear consistent with the findings of the MD simulation studies of the structural dynamics of the channel. Future experiments are aiming to provide a complete structure of the periplasmic loop in the open and closed states of the MscL channel, which should allow obtaining a more accurate model of the gating mechanism of this channel. Ajouz, B., Berrier, C., Besnard, M., Martinac, B., & Ghazi, A. (2000) Journal of Biological Chemistry 275, 1015-1022..in 0 Chang, G., Spencer, R., Lee, A., Barclay, M. & Rees, D. (1998) Science 282(5397):2220-2226..in 0 Gullingsrud, J., Kosztin, D. & Schulten K. (2001) Biophysical Journal 80, 2074-2081. Maurer, J.A. & Dougherty, D.A. (2003) Journal of Biological Chemistry 278, 13336-13342..in 0 Meyer, G.R., Gullingsrud, J., Martinac, B. & Schulten, K. (2004) Biophysical Journal 84, A2832..in 0 Perozo, E., Kloda, A., Cortes, D. & Martinac, B. (2001) Journal of General Physiology 118(2):193-206..in 0 Perozo, E., Kloda, A., Cortes, D.M. & Martinac, B. (2002) Nature Structural Biology 9, 696-703..in 0 Sukharev, S., Durell, S. & Guy, H. (2001) Biophysical Journal 81(2), 917-936.

Proceedings of the Australian Physiological Society

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AuPS/ASB Meeting - Canberra 2005 Free Communications 4: Skeletal Muscle Regulation: From Molecular Mechanism to Physiology Wednesday September 28 2005 Chair: Brett Cromer, David Allen

Calcium-phosphate precipitation in the sarcoplasmic reticulum reduces action potentialmediated Ca2+ release in mammalian skeletal muscle T.L. Dutka, L. Cole and G.D. Lamb, Department of Zoology, La Trobe University, Victoria 3086, Australia. Rapid ATP buffering during vigorous activity is predominantly achieved by the enzyme creatine kinase and the substrate creatine phosphate (CrP), which is present at ∼40 mM (Allen et al., 1995). As ATP is hydrolysed to ADP and inorganic phosphate (Pi), CrP donates its phosphate to the ADP to resynthesize ATP, and the [Pi] within the cytoplasm of fast-twitch muscle fibres may reach ³ 30 mM. Evidently Pi can enter the sarcoplasmic reticulum (SR) passively (Posterino & Fryer, 1998), via small conductance chloride channels that conduct Pi (Laver et al., 2001). It has been proposed (Fryer et al., 1995) that once inside the SR, Pi could bind to Ca2+ forming a calcium-phosphate (Ca-P) precipitate. We examined whether Ca-P precipitate formed in the SR and whether it reduced normal action potential (AP)-mediated Ca2+ release, and hence could contribute to the later stages of metabolic muscle fatigue that result from a failure of Ca2+ release (Allen et al., 1995). Long-Evans hooded rats were killed under deep anaesthesia (2% v:v halothane) and the extensor digitorum longus (EDL) muscles were excised. Single fibres were mechanically-skinned, connected to a force transducer and immersed in a standard K-HDTA ‘control’ solution (1mM free Mg2+; 8 mM total ATP; 10 mM creatine phosphate (CP) at pH 7.10, containing 75 m M EGTA, pCa 6.9). Individual fibres were then stimulated: 1) electrically (75 V cm-1, 20 pulses of 2 ms duration) to produce tetanic (50 Hz) force responses, or 2) by exposure to a 30 mM caffeine-0.05 mM Mg2+ solution with 0.5 mM EGTA present, which produced a submaximal longer-lasting force response (e.g. ∼10 sec). 30 mM Pi solutions (replacing 23 mM HDTA with 30 mM Pi, and adjusting the total [Mg2+]) were made similar to the standard K-HDTA solution (with or without 10 mM CrP present). The fibre was exposed to either no Pi (control), 10 or 30 mM Pi for 10 s, then immersed in paraffin oil (1 min), placed back into the same solution (10 s) as before and then transferred back into the oil (1 min). This procedure created a ‘closed’ system around the fibre and prevented any appreciable net Ca2+ uptake or loss by the SR from the weakly Ca2+-buffered solution trapped inside the fibre. The fibre was then washed (30 s) in standard solution to remove any Pi in the cytoplasm before stimulating the fibre. Total SR Ca2+ content was ascertained by pre-equilibrating the fibre for 20 s in standard solution with a known [BAPTA] present and then lysing all membranous compartments within the fibre by exposure to an emulsion of Triton-X100 (10% v:v) in paraffin oil (Owen et al., 1998). All experiments were performed at 24 ±1 °C. After a 2 min exposure to 30 mM Pi (with, n=4, or without, n=6, 10 mM CrP present) the total amount of 2+ Ca released from the SR by caffeine-low [Mg2+] stimulus was significantly (P<0.05) reduced by ∼20%, and the initial rate of force development slowed (∼55%). Peak tetanic (50 Hz) force was also significantly reduced by ∼25% and ∼45% after 10 and 30 mM Pi exposures respectively, n=4 for 10 mM Pi and n=14 for 30 mM Pi). Tetanic force responses produced after 30 mM Pi exposure were nearly identical to those seen in the same fibre following depletion of total SR Ca2+ by ∼35% (using a tetanic stimulus in the presence of 2 mM BAPTA, the total Ca2+ remaining in the SR was 0.75 ± 0.03 mM, n=5). Ca2+ content assays revealed that the total amount of Ca2+ remaining in the SR was not detectably changed after 30 mM Pi exposure (initially 1.16 ± 0.04 mM, n=9 and 1.16 ± 0.07 mM, n=3 after 30 mM Pi exposure) thus indicating that Ca2+ had not leaked out of the SR but instead formed a precipitate with the Pi, thereby reducing the amount of available Ca2+ for rapid release. These results suggest that Ca-P precipitation occurring within the SR may contribute to the failure of Ca2+ release observed in the later stages of metabolic muscle fatigue. They also demonstrate that a drop in the amount of total SR Ca2+ to a level substantially below the normal endogenous level will appreciably reduce tetanic force. Allen, D.G., Lannergren, J. & Westerblad, H. (1995) Experimental Physiology 80(4): 497-527. Fryer, M.W., Owen, V.J., Lamb, G.D. & Stephenson, D.G. (1995) Journal of Physiology 482(1): 123-40. Laver, D.R., Lenz, G.K. & Dulhunty, A.F. (2001) Journal of Physiology 535(3): 715-28. Owen, V.J., Lamb, G.D., Stephenson, D.G. & Fryer, M.W. (1997) Journal of Physiology 498(3): 571-86 Posterino, G.S. & Fryer M.W. (1998) Journal of Physiology 512(1): 97-108.

Proceedings of the Australian Physiological Society

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Digoxin effects on muscle strength, fatigue and K+ fluxes during exercise in healthy young adults M.J. McKenna1, S. Sostaric1, M.J. Brown2, C.A. Goodman1, X. Gong1, A.C. Petersen1, J. Aw3, J. Leppik1, C.H. Steward1, S.F. Fraser4, R.J. Snow5 and H. Krum3, 1Muscle, Ions and Exercise Group, School of Human Movement, Recreation and Performance, Centre for Ageing, Rehabilitation, Exercise and Sport, Victoria University, PO Box 14428, Melbourne, VIC 8001, Australia, 2Department of Anaesthesia, Austin Health, Heidelberg, VIC, Australia, 3Department of Epidemiology and Preventive Medicine, Monash University, Alfred Hospital, Melbourne, VIC, Australia, 4School of Medical Sciences, RMIT University, Bundoora, VIC, Australia and 5School of Exercise Science and Nutrition, Deakin University, Burwood, VIC, Australia. The Na+,K+ATPase enzyme constrains muscle K+ loss and Na+ gain and is vital for skeletal muscle contractility, but our recent studies have found that maximal Na+,K+ATPase activity is depressed with fatigue. We investigated the effects of the specific Na+,K+ATPase inhibitor digoxin on muscle strength, fatiguability and performance; and on K+ fluxes across active and inactive muscles during exercise. Ten active, but not well-trained healthy volunteers (9 M, 1 F), with normal ECG, plasma electrolytes, renal function, and no history of adverse cardiovascular events gave written informed consent. A series of exercise tests were performed after taking digoxin (DIG, 0.25 mg.d-1) or a placebo (CON) for 14 d, in a randomised, counterbalanced, cross-over, double blind design study, with trials separated by 4 weeks. Quadriceps muscle strength (peak torque at 0-360°/s) and fatiguability during 50 maximal contractions (fractional decline in peak torque at 180°/s) were measured on day 13 on a Cybex isokinetic dynamometer. All subjects performed incremental cycle ergometer exercise to measure VO2peak and to determine 33, 67 and 90% VO2peak work rates. Subjects also performed an incremental test using concentric, dynamic finger flexor contractions to determine their peak work rate (WRpeak). On day 14 subjects completed two invasive trials separated by ∼2 h. A finger flexion exercise trial comprised three 1-min bouts, then a final bout to fatigue, at 100% WRpeak. Two-legged cycling comprised 10 min each at 33% and 67% VO2peak, then to fatigue at 90% VO2peak. Radial arterial (a) and deep antecubital venous (v) blood was sampled simultaneously at rest, before and during each exercise bout and in recovery, for both exercise trials. Serum digoxin was 0.7±0.2 nM at day 13 and 0.8±0.2 nM at day 14 (Mean±SD) in the DIG trial, and < 0.4 nM for CON. Muscle peak torque and the fatigue index (CON 0.57±0.10 vs DIG 0.54±0.09) were unchanged by digoxin. Time to fatigue during finger flexion exercise was not significantly affected by digoxin (CON 236±211 vs DIG 157±118 s, n=9). During finger flexion exercise, each of [K+]a, [K+]v and [K+]a-v were greater with exercise in CON (by 0.37±0.21, 1.29±0.84 and -0.89±0.69 mM), and similarly with DIG (by 0.34±0.36, 1.12±0.87 and -0.69±0.69 mM). The unchanged [K+]a-v suggests unaltered K+ release from contracting muscles with DIG. Time to fatigue during leg cycling exercise was not significantly affected by digoxin (CON 254±125 vs DIG 262±156 s). During leg exercise, each of [K+]a, [K+]v and [K+]a-v were greater with exercise than at rest in CON (by 2.51±0.83, 1.22±0.52 and 1.29±0.68 mM), but none were modified by DIG (by 2.62±0.57, 1.18±0.73 and 1.43±0.78 mM). The unchanged [K+]a-v suggests unaltered K+ uptake by inactive muscles with DIG. In summary, DIG at therapeutic levels did not adversely affect muscle performance, [K+] or K+ fluxes during exercise in healthy young adults. Whether this reflects inadequate digitalization, a safety tolerance to small reductions in functional Na+,K+ATPase, or limited adverse effects of digitalization when muscle Na+,K+ATPase is normal (i.e. high) is unclear. Funded by NH&MRC.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/31P

The peak tetanic force-[K+]o relationship in mouse fast- and slow-twitch muscle: modulation with [Na+]o or [Ca2+]o

S.P. Cairns, Division of Sport & Recreation, Auckland University of Technology, Auckland 1020, New Zealand. Potassium (K+) is frequently postulated to cause skeletal muscle fatigue. Indeed, the trans-sarcolemmal gradient falls during high-intensity exercise (Sejersted & Sjøgaard, 2000) and experimentally raising extracellular [K+], ([K+]o) causes depolarisation and reduces force in non-fatigued muscle (Cairns et al., 1997, 1998). However, large elevations of [K+]o are necessary to cause a severe reduction of force (Cairns et al., 1997). The aim of the present study was to extend our understanding of the role of K+ in fatigue, by testing for interactive effects between raised [K+]o and other ionic changes that occur during intense exercise (Cairns et al., 1998; Sejersted & Sjøgaard, 2000), i.e., diminished trans-sarcolemmal sodium (Na+) and calcium (Ca2+) gradients. Isometric contractions were evoked by supramaximal electric field stimulation (parallel plate electrodes) in isolated slow-twitch soleus (SOL) or fast-twitch extensor digitorum longus (EDL) muscles of mice. Muscles were bathed in control Krebs solution (4 mM K+, 147 mM Na+, 1.3 mM Ca2+, 128 mM Cl-) at 25°C. With raised K+ solutions NaCl was replaced with KCl, with lowered Na+ solutions NaCl was replaced with N-methylD-glucamine, and with altered Ca2+ solutions Ca2+ was replaced with Mg2+, or CaCl2 was added. Maximum tetanic force was achieved at 125 Hz in SOL and 200 Hz in EDL. Fatigue was induced with repeated tetanic stimulation at 125 Hz for 500 ms, every 1 s, for 100 s. When [K+]o was raised from 4 to 7 mM fatigue was exacerbated, and when [K+]o was lowered to 2 mM the fatigue was slowed in SOL. The relative force at 100 s of stimulation (mean value) was 50% initial at 2 mM K+, 40% at 4 mM K+, and 23% at 7 mM K+. The relationship between peak tetanic force and [K+]o (8-12 mM) was established in non-fatigued muscles. At raised [K+]o (i) increasing the stimulation pulse strength (20 to 26 V) increased force in SOL but not EDL, (ii) increasing the stimulation pulse duration (0.1 to 0.15 to 0.25 ms) progressively restored force, but to a greater extent in SOL than EDL, and (iii) stimulating with transverse wire rather than parallel plate electrodes resulted in a greater force loss, especially in SOL. When [K+]o was raised to 8 mM and [Na+]o lowered to 100 mM, synergistic depressive effects occurred on peak tetanic force in both SOL and EDL, i.e., the peak tetanic force-[K+]o relationship shifted leftwards towards lower [K+]o. Force could then be partially restored by lowering the stimulation frequency but only in SOL. Raising the [Ca2+]o (1.3 to 2.5 to 10 mM) shifted the peak tetanic force-[K+]o relationship in SOL rightwards towards higher [K+]o. Conversely, lowering [Ca2+]o shifted the relationship leftwards, e.g., at 8 mM K+ the peak force was 77% initial at 1.3 mM Ca2+ and 52% initial at 0.5 mM Ca2+. In summary, moderate changes of [K+]o clearly influence the rate of fatigue in SOL which implicates K+ in the fatigue process. The peak tetanic force-[K+]o relationship in non-fatigued muscle depended on the stimulation pulse parameters and stimulation electrode type, and more so in SOL than EDL. Muscles are more susceptible to K+-induced force depression at slightly lowered [Na+]o and slightly lowered [Ca2+]o which is a likely physiological scenario. Thus, when such ionic shifts occur simultaneously during exercise, they are likely to act together to impair muscle force production, i.e., cause fatigue.

K+

Cairns, S.P., Hing, W.A., Slack, J.A., Mills, R.G. & Loiselle, D.S. (1997) American Journal of Physiology, 273, C598-C611. Cairns, S.P., Hing, W.A., Slack, J.A., Mills, R.G. & Loiselle, D.S. (1998) Journal of Applied Physiology, 84, 1395-1406. Sejersted, O.M. & Sjøgaard, G. (2000) Physiological Reviews, 80, 1412-1465. This work was supported by a grant from the New Zealand Lottery Grants Board.

Proceedings of the Australian Physiological Society

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The effect of dithiothreitol (DTT) application on isolated mouse muscle fatigued at 37°C T.R. Moopanar and D.G. Allen, School of Medical Sciences, University of Sydney F13, NSW 2006, Australia. We have previously shown that muscle fatigue at 37°C in vitro is associated with a reduction in calcium sensitivity and that this process could be prevented by the antioxidant Tiron (Moopanar & Allen, 2005). Previous studies have also found that application of the reducing agent dithiothreitol (DTT) to the rat diaphragm improves recovery post-fatigue (Diaz et al., 1998). The aim of the current study was to determine whether DTT could reverse the effect of temperature- and fatigue-induced myofibrillar desensitization. Single muscle fibres were isolated from the foot of balb-C mice and were attached to a force transducer. The temperature in the muscle chamber was raised to 37°C prior to each experiment. Fibres were microinjected with indo-1 to measured intracellular calcium ([Ca2+]i), and were stimulated at a range of frequencies (20, 30, 50, 70 and 100 Hz and 100 Hz in the presence of 10 mM caffeine) to establish myofibrillar sensitivity to calcium. The preparation was then fatigued and sensitivity was immediately reassessed. Finally, the muscle preparation was treated with DTT (0.5 mM) for two minutes and myofibrillar sensitivity was again tested. The Ca50, which is the level of [Ca2+]i that produces half maximum force and a measure of 2+ Ca -sensitivity, was initially found to be 649 ± 40 nM (n=18). This value was increased post fatigue to 872 ± 40 nM (n=9). There was no change to the Ca50 in the absence of fatiguing stimuli. Application of DTT to the fatigued muscle caused the Ca2+- sensitivity to return to prefatigue values (683 ± 40 nM (n=6)). In order to determine whether the decline in muscle function was due to a change in maximum calcium activated force (Fmax), fibres were stimulated at 100 Hz in the presence of caffeine (10 mM). There was no significant change in Fmax. These results indicate that the process of myofibrillar desensitization at 37°C requires repeated stimulation to occur. In addition, we show that the desensitization can be reversed by DTT. This suggests that a protein involved in calcium sensitivity has critical S-H groups which can be oxidized to form disulphide bonds (S-S) with loss of Ca2+-sensitivity. This reaction can be reversed by the reducing agent DTT. Diaz, P.T., Costanza, M.J., Wright, V.P., Julian, M.W., Diaz, J.A. & Clanton, T.L. (1998) Medicine and Science in Sports and Exercise 30, 421-426. Moopanar, T.R. & Allen, D.G. (2005) Journal of Physiology 564, 189-199.

Proceedings of the Australian Physiological Society

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Cytoplasmic ATP-sensing CBS domains regulate gating of skeletal muscle ClC-1 chloride channels B. Bennetts1, G.Y. Rychkov2, H-L. Ng1, C.J. Morton1, D. Stapleton3, M.W. Paarker1 and B.A. Cromer 1, 1St. Vincent’s Institute, Fitzroy, VIC 3065, Australia,2 The University of Adelaide, Adelaide, SA 5005, Australia and 3Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC 3010, Australia. ClC proteins are a family of chloride channels and transporters that are found in a wide variety of prokaryotic and eukaryotic cell-types. The mammalian voltage-gated chloride channel ClC-1 is important for controlling the electrical excitability of skeletal muscle. Reduced excitability of muscle cells during metabolic stress can protect cells from metabolic exhaustion and is thought to be a major factor in fatigue. Here we identify a novel mechanism linking excitability to metabolic state by showing that ClC-1 channels are modulated by ATP. The high concentration of ATP in resting muscle effectively inhibits ClC-1 activity by shifting the voltage-gating to more positive potentials. ADP and AMP had similar effects to ATP but IMP had no effect, indicating that the inhibition of ClC-1 would only be relieved under anaerobic conditions such as intense muscle activity or ischaemia, when depleted ATP accumulates as IMP. The resulting increase in ClC-1 activity under these conditions would reduce muscle excitability, thus contributing to fatigue. We show further that the modulation by ATP is mediated by cystathionine-b -synthase-related (CBS) domains in the cytoplasmic C-terminus of ClC-1. This defines a function for these domains as gating-modulatory domains sensitive to intracellular ligands, such as nucleotides, a function that is likely to be conserved in other ClC proteins.

Proceedings of the Australian Physiological Society

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Modelling diffusive O2 supply to isolated muscle preparations

C.J. Barclay, Muscle Energetics Laboratory, School of Physiotherapy & Exercise Science, Griffith University Gold Coast, PMB50 Gold Coast Mail Centre, Gold Coast, Queensland 9726, Australia. The sole source of oxygen (O2) to an isolated muscle preparation is by diffusion from the muscle surface. A.V. Hill (1928) derived equations that described the spatial and temporal dependencies of intramuscular O2 partial pressure (PO2) for muscles of various shapes and showed solutions for frog muscles. The purpose of the current study was to use Hill’s diffusion equation for cylindrical muscles to assess the adequacy of diffusive O2 supply into commonly-used mammalian cardiac and skeletal muscles during typical experimental contraction protocols. The diffusion equation was solved numerically to give (1) the maximum O2 diffusion distances during steady-state activity at various contraction duty cycles and temperatures and (2) the time, in more severe contraction protocols, before central anoxia would develop during the rest-to-work transition. The effects of incorporating myoglobin-facilitated O2 diffusion were also assessed. The analysis was performed for soleus, extensor digitorum longus (EDL) and cardiac papillary muscles from the rat and mouse and for frog sartorius muscle using published metabolic data. The results indicated that for all the preparations considered, it would be difficult to ensure adequate O2 supply using whole muscles; adequate O2 supply can only be ensured over a reasonable range of duty cycles by using preparations with radii substantially smaller than those of whole muscles. Reducing experimental temperature is an effective strategy for enhancing O2 supply to skeletal muscle. However, diffusive O2 supply to isolated papillary muscles is not greatly affected by temperature because increasing temperature has opposite effects on active and resting metabolic rates of cardiac muscle. Taking account of O2 supply from myoglobin had only minimal effects on oxygenation under typical isolated muscle conditions. Hill, A.V. (1928) Proceedings of the Royal Society of London Series B: Biological Sciences 104, 39-96.

Proceedings of the Australian Physiological Society

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AuPS/ASB Meeting - Canberra 2005 1615 - Poster Session, Afternoon Tea and Drinks Wednesday September 28 2005

Effects of gadolinium and static magnetic fields on MscL channel activity E. Petrov, Z.-W. Liu and B. Martinac, School of Biomedical Sciences, University of Queensland, St Lucia, Queensland 4072 Australia. All biological tissues are highly penetrable for static magnetic fields (SMF). There are a number of hypotheses concerning the cellular and/or subcellular target of these fields. One possibility is that they target the cell membrane. It was shown that applying a SMF of 80 mT affected the open probability (Po) and gating of the bacterial Mechanosensitive channel of Large conductance (MscL) reconstituted into liposomes (Hughes et al., 2005). Since phospholipid molecules possess diamagnetic anisotropy (Rosen, 2003), the SMF effect on MscL could originate from the reorientation of the lipid molecules perpendicularly to the direction of the magnetic field. Taking into account that thousands of phospholipid molecules form well ordered arrays in the bilayer the effect of SMF thus becomes amplified affecting the embedded MscL protein. Another possible effect of SMF could be via membrane-bound ions, such as Ca2+ (Del Moral & Azanza, 1994). To test this hypothesis we examined if SMF could modulate the ability of Gd3+ ions (non-specific blocker of mechanosensitive channels (Hamill & McBride, 1996)) to inhibit MscL gating, since Gd3+ ions interact with phospholipid molecules in a similar way as Ca2+ ions (Ermakov et al., 2001). Single channel patch-clamp experiments were carried out using the MscL channels reconstituted into liposomes and effect of Gd3+ on MscL activity was recorded. The results showed that Gd3+, in a dosedependent manner, caused an increase in the negative pressure required to open the MscL channels. 50 m M Gd3+ in the bath partially blocked the MscL channel, whereas 400 m M Gd3+ blocked the channels completely. Gd3+ also prolonged the duration of the single channel openings by decreasing the frequency of the channel opening and reducing channel flickering. Next we studied the effect of SMF on the MscL activity and MscL block by Gd3+. Negative pressures of 40-50 mmHg were required to stretch liposome patches and activate the MscL channels. Only patches were examined which exhibited stable channel activity during the initial 5-7 minutes of an experiment. A rare-earth NdFeB magnet was positioned at a distance of 2 mm from the tip of the pipette. The estimated strength of SMF was 400 mT. Application of the SMF had a two-fold effect on the channel activity: (1) a decrease of the open probability NPo (N, unknown number of channels in a patch) during application of the SMF to 70.6±8.3% (mean±S.E., n=10) of the initial steady-state level before the application of SMF; and (2) an increase of NPo upon removal of the SMF to 119.0±10.8% (n=10). The effects of the SMF were slowly developing over approximately 10 minutes upon application /release of the SMF. The time-dependence of the SMF effect may be explained by formation and destruction of ordered phospholipid clusters in the bilayer. Variability in the extent of the observed effects in our experiments might be due to the fact that the patch membrane is not flat when suction is applied to the pipette (Sukharev et al., 1999), so that the peripheral and central parts of a patch are at different angles to the SMF vector. In most of the examined patches a partial blockade of the MscL activity by 50 m M of Gd3+ increased in the presence of SMF. After removal of SMF the channel activity recovered to the previous level and often increased further regardless of the presence of Gd3+ ions. In some patches the channel activity did not increase after the removal of SMF, but had already done so in its presence. Our results suggest that ordering of phospholipid molecules in the bilayer by SMF could cause a displacement of Gd3+ ions bound to phospholipid molecules due to the electrostatic repulsion between the ions, which resulted in reduction of the MscL channel block by Gd3+. Del Moral, A. & Azanza, M.J. (1994) Progress in Neurobiology 44, 517-601. Ermakov, Y.A., Averbakh, A.Z., Yusipovich, A.I. & Sukharev, S. I. (2001) Biophysical Journal 80, 1851-1862. Hamill, O.P. & McBride, D.W. Jr. (1996) Pharmacological Reviews 48, 231-252. Hughes, S., El Haj, A.J., Dobson, J. & Martinac, B. (2005) European Biophysics Journal (in press). Rosen, A.D. (2003) Cell Biochemistry and Biophysics 39, 163-173. Sukharev, S.I., Sigurdson, W.J., Kung, C. & Sachs F. (1999) Journal of General Physiology. 113, 525-540.

Proceedings of the Australian Physiological Society

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Conformational changes involved in MscL channel gating measured using FRET spectroscopy B. Corry1, P. Rigby2 and B. Martinac3, 1Chemistry, School of Biomedical, Biomolecular and Chemical Science, 2Biomedical Imaging and Analysis Facility and 3School of Medicine and Pharmacology, The University of Western Australia, Crawley, WA 6009, Australia. Transmembrane channels facilitate the movement of small molecules and ions across cell membranes. Obtaining detailed structural information about such proteins has been difficult, due not only to the complications of crystallisation, but also because they adopt multiple conformational states that are not easy to probe with static x-ray images. We demonstrate that fluorescence resonance energy transfer spectroscopy (FRET) is a powerful tool for in situ structural analysis of multimeric membrane proteins by measuring the conformational changes involved in gating the mechanosensitive ion channel MscL. MscL channels act as safety valves in bacterial cells, opening wide pores to prevent cell death during hypo-osmotic stress. The MscL protein monomers are fluorescently labelled by randomly attaching AlexaFluor 488 (AF488) and AlexaFluor 568 (AF568) to a single cysteine residue introduced via site directed mutagenesis. As the channel protein is a pentamer, this mutation introduces five identical cysteine sites each equally likely to be occupied by AF488 or AF568. The protein is reconstituted into artificial phosphatidylcholine liposomes and imaged in a laser scanning confocal microscope. The channels are normally closed in their resting state, but may be forced into the open conformational state with the addition of lysophosphatidylcholine (LPC) which inserts in the outer layer of the liposome membrane bilayer promoting membrane curvature and/or a change in the transbilayer pressure profile, thus leading to channel opening. The liposomes are imaged in both AF488 (donor) and AF568 (acceptor) emission bands before and after bleaching of the acceptor AF568. The intensity of the AF488 donor emission increases after bleaching indicating FRET is taking place. The proportion of energy being transferred from the donor to the acceptor is then related to the pentamer radius in both the closed and open channels using a Monte-Carlo ensemble analysis program. This accounts for each channel protein containing a random mix of five donors and acceptors and the fact that energy transfer could arise between fluorophores attached to different proteins. As illustrated schematically in the figure, we find that the diameter of the fluorescently labelled MscL channel increases by 16Å upon activation, creating a large pore and representing one of the largest known conformational changes in membrane proteins.

Proceedings of the Australian Physiological Society

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C-terminal charged cluster of the mechanosensitive channel MscL, RKKEE, functions as a pH sensor Anna Kloda and Boris Martinac, School of Biomedical Sciences, University of Queensland, Brisbane, Queensland 4072 Australia. A highly conserved cluster of charged residues, RKKEE, located within the C-terminus of the bacterial mechanosensitive channel MscL is essential for the channel gating. The mutated protein lacking these amino acid residues is not functional (Häse et al., 1997). This structural motif is a part of a cytoplasmic helix and is proposed to serve as a stabilizing element of the closed configuration of the MscL channel (Perozo et al., 2001). The crystal structure of MscL was obtained at low pH showing the channel in its closed state (Chang et al., 1998). In the crystal structure the charged residues are facing each other inside the C-terminal helical bundle. However an independent study of the channel closed structure (Perozo et al., 2001) shows that at neutral pH these residues are outwardly oriented facing the aqueous medium. This suggests that the orientation of the Cterminal helices relative to the aqueous medium is pH dependent. Thus, it is possible that the RKKEE cluster functions as a pH sensor. In the present study we examined the effects of pH as well as of charge reversal and substitution within the RKKEE cluster on mechanosensitivity of E. coli MscL reconstituted into liposomes using the patch-clamp technique. Charge reversal mutations did not affect the free energy of activation or activation pressure of the channel (D G0 = 15.8 kT, p1/2 = 76.3 mmHg and D G0 = 16.4 kT, p1/2 = 84.2 mmHg) for the RKKEE wild type and the EEKKR mutant respectively. Protonation of E107 and E108 residues, achieved by decreasing the experimental pH or replacement of negative charges by glutamine, significantly increased free energy of activation for the MscL channel due to an increase in activation pressure p1/2 (D G0 = 26.9 kT, p1/2 = 120.2 mmHg for wild type MscL at pH 5.5 and D G0 = 26.1 kT, p1/2 = 130.3 mmHg for the RKKQQ mutant channel at pH 7.0). A similar increase in D G0 was observed when positive charges were substituted by glutamine (D G0 = 23.8 kT, p1/2 = 118.6 mmHg at pH 7.0) or the overall charge of the cluster was neutralized by increasing experimental pH of the RKKQQ mutant (D G0 = 25.7 kT, p1/2 = 124.0 pH 9.5). Interestingly, protonation of the positively charged residues of the RKKQQ mutant by lowering the experimental pH to 5.5 resulted in p1/2 (89.0 mmHg) and D G0 (13.0 kT) comparable to the wild-type MscL at physiological pH of 7.0 suggesting the importance of the preservation of the total charge of the cluster. Our data indicate that the RKKEE charged cluster acts as a pH sensor that regulates the stability of the cytoplasmic helix. Our data further suggest that, in contrast to the gating model proposed by Anishkin et al. (2003) the cytoplasmic helix is not only functioning as a size-exclusion filter but also substantially influences channel gating. Anishkin, A., Gendel, V., Sharifi, N.A., Chiang, C.S., Shirinian, L., Guy, H.R. & Sukharev, S. (2003) Journal of General Physiology, 121, 227-244. Chang, G., Spencer, R.H., Lee, A.T., Barclay, M.T. & Rees, D.C. (1998) Science, 282, 2220-2226. Häse, C.C., Ledain, A.C. & Martinac, B. (1997) Journal of Membrane Biology, 157, 17-25. Perozo, E., Kloda, A., Cortes, D.M. & Martinac, B. (2001) Journal of General Physiology, 118, 193-205.

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The effects of eriochrome cyanine R on the mechanosensitive channels of E. coli T. Nguyen1,2, B. Clare2, L. Hool2 and B. Martinac3, 1School of Medicine and Pharmacology, University of Western Australia, WA 6009, Australia2, School of Biomedical, Biomolecular and Chemical Sciences, University of Western Australia, WA 6009, Australia and 3School of Biomedical Sciences, University of Queensland, QLD 4072, Australia. The existence and characteristics of the mechanosensitive channel of large conductance (MscL) and small conductance (MscS) of E.coli have been well documented and extensively studied (Sukharev et al., 1997). MscL and MscS have been found to play an important role in osmoregulation and the two channels are capable of compensating the absence of each other (Berrier et al., 1992; Levina et al., 1999). Though similar in physiological function, the channels differ somewhat in structure and characteristic. The MscL gene comprises of 136 amino acid residues amounting to a 15-kDa protein (Sukharev et al., 1994) to form a homopentamer (Chang et al., 1998). Whereas, MscS, is a 286-residue membrane protein with its 3D structure revealing a homoheptamer (Bass et al., 2002). MscS is gated at pressures approximately half of which MscL is activated and has a conductance of ∼1nS (Martinac et al., 1987; Sukharev et al., 1993) compared to MscL conductance of ∼3nS (Sukharev et al., 1994). We have previously shown that parabens, which are food and cosmetic preservatives, were able to spontaneously activate MscL reconstituted in liposomes and MscS in giant spheroplasts, and also increased the mechanosensitivity of MscL (Nguyen et al., 2005). Our studies therefore, broaden to other compounds which would bind to the MscL gate with greater affinity than parabens. Based on our in-silico data, eriochrome cyanine R bound to the MscL channel gate with a Gibbs free energy of -47.03kJ mol-1 which is a much lower value than that of parabens, indicating a greater affinity to the channel. The patch-clamp studies with eriochrome cyanine R were shown to complement in-silico results with spontaneous MscL activity observed in 78% of the patches. The Boltzmann distribution curve of MscL in the presence of eriochrome cyanine R was markedly shifted to the left of the control curve and the Boltzmann parameters namely, α, p½ and D Go were also significantly lowered (p < 0.05) in the presence of eriochrome cyanine R compared to control values. That is, the mechanosensitivity of MscL was greatly increased in the presence of eriochrome. Based on our study, it is possible that eriochrome cyanine R could be used as a lead compound for the development of a novel type of antibiotic. An antibiotic which would act by gating the mechanosensitive (MS) channels resulting in the leakage of essential ions and cell osmoticants out of the bacterium and thus, prevent its growth and survival. Bass, R. B., Strop, P., Barclay, M. & Rees, D. C. (2002) Science 298, 1582-1587. Berrier, C., Coulombe, A., Szabo, I., Zoratti, M. & Ghazi, A. (1992) European Journal of Biochemistry 206, 559-565. Chang, G., Spencer, R.H., Lee, A.T., Barclay, M.T. & Rees, D.C. (1998) Science 282, 2220-2226. Levina, N., Totemeyer, S., Stokes, N.R., Louis, P., Jones, M.A. & Booth, I.R. (1999) EMBO Journal 18, 1730-1737. Martinac, B., Buechner, M., Delcour, A.H., Adler, J. & Kung, C. (1987) Proceedings of the National Academy Science USA 84, 2297-2301. Nguyen, T., Clare, B., Guo, W. & Martinac, B. (2005) European Biophysics Journal in press. Sukharev, S.I., Blount, P., Martinac, B., Blattner, F. R. & Kung, C. (1994) Nature 368, 265-268. Sukharev, S. I., Blount, P., Martinac, B. & Kung, C. (1997) Annual Review of Physiology 59, 633-657. Sukharev, S.I., Martinac, B., Arshavsky, V.Y. & Kung, C. (1993) Biophysical Journal 65, 177-183. Supported by NH&MRC and a UWA Postgraduate Award to T.N.M. Nguyen.

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Mutations within the selectivity filter of the NMDA receptor channel influence voltage-dependent block by extracellular 5-hydroxytryptamine Anna Kloda and David Adams, School of Biomedical Sciences, University of Queensland, Brisbane QLD 4072, Australia. The NMDA receptor is a tetrameric cation channel which mediates important physiological processes such as long-term potentiation, synaptic plasticity and neurodegeneration via conditional Ca2+ signalling. The ionic influx through the open channel pore coincides with the presynaptic release of glutamate and postsynaptic membrane depolarization, which relieves voltage-dependent Mg2+ block. The asparagine residue on the NR2 subunit corresponding to position 596 (N+1 site) contributes to the Mg2+ binding site and affects channel rectification due to block by extracellular Mg2+. In contrast, asparagine at position 598 (N0 site) on the adjacent NR1 subunits barely affects such events (Wollmuth et al., 1998). Both residues are located near the tip of the M2 re-entrant loop and line the selectivity filter of the channel pore. Recently we reported voltage-dependent inhibition of NMDA receptor currents by 5-hydroxytryptamine (5-HT) (Kloda & Adams, 2005). The voltage sensitivity of the block indicated that 5-HT, similar to Mg2+, binds within the membrane electric field. In the present study, we assessed the effects of NR1(N0S) and NR2A(N+1Q) mutations of NMDA receptors expressed in Xenopus oocytes on the block by extracellular 5-HT using the two-electrode voltage clamp recording technique The mutation within the NR1 subunit of the NR1(N0S)-NR2A receptor combination, strongly reduced the magnitude of the block by 0.3 mM 5-HT and abolished the voltage dependence of block. The corresponding mutation within the NR2 subunit of the NR1-NR2A(N+1Q) receptor channels reduced the block by 5-HT to a lesser extent although the rectification of the I-V curve was similar to that observed for the wild type. This is opposite to the block produced by external Mg2+ where a substitution of the NR2A(N+1) site asparagine but not the NR1 N-site significantly reduces the block (Wollmuth et al., 1998). Furthermore, the NR1 and NR2 mutant channels differed in their sensitivities to the 5-HT block compared to wild type. The IC50 values for 5-HT block at -120 mV were 59 µM for wild type but increased to 230 µM and 1.5 mM for the NR1 and NR2A mutants, respectively. These data indicate that the block by 5-HT is attenuated by corresponding asparagine mutations in the NR1 and NR2 subunits. The effect of the asparagine substitution in the NR1 and NR2 subunits on 5-HT block suggests that, in contrast to the Mg2+ block, 5-HT block critically depends on the NR1 asparagine residue and to a lesser extends on the NR2 residue. Thus, the binding of 5-HT to key residues in a narrow constriction of the channel pore may provide a significant barrier to ionic fluxes through the channel. Kloda, A. & Adams, D.J. (2005) British Journal of Pharmacology 144, 323-330. Wollmuth, L.P., Kuner, T., & Sakmann, B. (1998) Journal of Physiology 506, 13-52.

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Ion selectivity of glycine receptors with mutations of charged residues in the intracellular portals T.M. Lewis1, Sugiharto1, J.A. Peters2, J.J. Lambert2, P.H. Barry1 and A.J. Moorhouse1, 1School of Medical Sciences, The University of New South Wales, NSW 2052, Australia and 2Department of Pharmacology and Neuroscience, The University of Dundee, Dundee DD1 9SY, United Kingdom. The glycine receptor (GlyR) is a member of the nicotinic-like family of ligand gated ion channels that are comprised of five subunits, each with a similar topology of a large N-terminal extracellular domain and four transmembrane domains (TM1 to TM4). The prototypical member of this family is the muscle endplate nicotinic acetylcholine receptor (nAChR). Cryo-electron microscopy studies of nAChRs from the Torpedo electric ray provide the best structural information of this receptor (Unwin, 2005) and by homology, a good template for other members of the family, including the GlyR. The nAChR structure shows that the intracellular loop between TM3 and TM4 is, in part, an α helix (designated ‘MA’) and the MA helix from each subunit comes together to form an inverted ‘tee-pee’ underneath the intracellular mouth of the ion channel pore. Charged residues in the MA helix are hypothesized to influence the ion size and charge selectivity of the channel as a consequence of lining the ‘portals’ or windows that form between adjacent MA helices. In the serotonin receptor (5-HT3R), these residues influence the channel conductance (Kelley et al., 2003). We investigated homologous residues in the MA helix of the α1 GlyR to see if they influenced ion selectivity or conductance. Homology alignment of the amino acid sequence for the α1 GlyR subunit and the α subunits of the Torpedo nAChR and 5-HT3R was used to identify the likely charged residues in the GlyR that line the MA helix and face the portals. Four positively charged residues were identified: Arg377, Lys378, Lys 385 and Lys386. Two mutant α1 GlyR subunits were created: one where all four of these residues were substituted for neutral alanine (‘4A’) and another where they were substituted for the negatively charged glutamate (‘4E’). The cDNA for the wild-type, 4A and 4E GlyR mutants were separately transfected into 293 cells using a polyethylenimine reagent (jetPEI™) and glycine activated currents were recorded using standard whole-cell patch-clamp techniques. Current-voltage (I-V) curves were determined from a voltage-step protocol (100 ms steps) performed during the continued application of non-desensitizing concentration of glycine. I-V curves were initially determined in an extracellular solution containing (in mM): NaCl, 145; glucose, 10; HEPES, 10 (adjusted to pH 7.4). I-V curves were subsequently determined in solutions with 50% NaCl (75 mM) and 25% NaCl (37.5 mM) that were osmotically balanced with sucrose. The internal (pipette) solution was (in mM): NaCl, 145; CaCl2, 2; EGTA, 5; HEPES 10 (adjusted to pH 7.4). The reversal potentials (Vrev) were determined from the I-V curves for each NaCl dilution and corrected for liquid junction potentials. The shifts in Vrev for each dilution were fitted with the Goldman-Hodgkin-Katz equation to estimate the relative permeability for chloride ions with respect to sodium ions (PCl/PNa). The mean values for the Vrev obtained from the wild-type, 4A and 4E (n=7 in each case) are shown in the following table:

100% NaCl 50% NaCl 25% NaCl

wild-type -0.3±0.2 11.6±0.7 22.6±1.5

Vrev (mV) 4A -0.3±0.6 10.7±0.5 22.4±1.3

4E -0.8±0.3 11.5±0.7 22.8±1.4

The corresponding PCl/PNa values determined were similar in all three cases: 7.5±0.2 for the wild-type, 7.0±0.5 for 4A, and 7.6±0.3 for 4E. These results indicate that the substitution of the four charged residues in the MA helix had no significant effect upon the ion selectivity of the α1 GlyR. Preliminary data suggest that both the 4A and 4E mutations reduce the single channel conductance. Further studies are required to address the possibility that other charged residues lining the portals may be involved in ion selectivity. Unwin, N. (2005) Journal of Molecular Biology 346, 967-989. Kelley S.P., Dunlop J.I, Kirkness E.F., Lambert J.J. & Peters J.A. (2003) Nature 424, 321-324.

Proceedings of the Australian Physiological Society

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A molecular determinant of tropisetron inhibition of the glycine receptor Cl− channel Z. Yang, A.D. Ney and J.W. Lynch, School of Biomedical Sciences, University of QLD, Brisbane QLD 4072, Australia. Tropisetron, an antagonist of the 5-HT3 receptor cation channel, is used clinically as an anti-emetic drug. It also has potent effects on the structurally related glycine receptor Cl− channel (GlyR). At low (submicromolar) concentrations, tropisetron potentiates the GlyR and at higher concentrations it produces inhibition (Supplisson & Chesnoy-Marchais, 2000). Since prostaglandins increase the transmission of pain impulses to the brain via downregulation of spinal glycinergic neurotransmission, the GlyR has emerged as a novel target for therapies directed at neuropathic pain (Harvey et al., 2004). As a potentiating agent, tropisetron is a lead compound for the development of novel analgesic therapeutics directed at the GlyR. However, the locations of the inhibitory and potentiating tropisetron binding sites on the GlyR are unknown. This study sought to identify the tropisetron inhibitory binding site on homomeric α1 and heteromeric α1b GlyRs. HEK293 cells were transfected with WT and mutant GlyR cDNA using the calcium phosphate precipitation protocol. When co-transfecting α1 and b subunits, their respective cDNAs were combined in a ratio of 1:10. The transfection solution was removed after 24h and glycine-gated currents were recorded using whole-cell patch clamp techniques over the following 24-72 h. Heteromeric GlyRs were identified by GFP fluorescence coupled to b subunit expression and by their reduced sensitivity of heteromeric GlyRs to picrotoxin. We first confirmed that sub-micromolar concentrations of tropisetron elicited potentiation and that concentrations above 100 µM inhibited the WT α1 GlyR (Figure, left panel). We then used 500 µM tropisetron to screen a large number of mutant GlyRs in which various known ligand binding sites were abolished. We investigated the principal ligand-binding domain A (via mutations I93A, A101H/C, N102A/C/D/Q), domain B (F159A, Y161C) and domain C (K200A, H201A, Y202F, N203A). We also serially eliminated the zinc binding sites (H107N, H109N) and the alcohol binding site (S267C). The four N102 mutations were the only tested mutations that abolished inhibition and in each case this was achieved without affecting tropisetron potentiation (Figure, right panel). When the N102Q mutant α1 subunit was co-expressed with the WT b subunit, tropisetron inhibition returned to near normal potency. N125 in the b subunit residue corresponds to N102 in the α1 subunit. When the N102Q mutant α1 subunit was co-expressed with the N125D mutant b subunit, tropisetron inhibition was also normal.

We conclude that N102 in the α1 subunit is a specific determinant of tropisetron inhibition. Its location in the agonist binding pocket implies that it may be a tropisetron binding site. Our results indicate that b subunits also contain tropisetron inhibitory sites. However, the location of the b subunit site does not correspond to its location in the α1 subunit. Supplisson, S. & Chesnoy-Marchais, D. (2000) Molecular Pharmacology, 58, 763-777. Harvey, R.J., Depner, U.B., Wassle, H., Ahmadi, S., Heindl, C., Reinold, H., Smart, T.G., Harvey, K., Schutz, B., Abo-Salem, O.M., Zimmer, A., Poisbeau, P., Welzl, H., Wolfer, D.P., Betz, H., Zeilhofer, H.U. & Muller, U. (2004) Science, 304, 884-887.

Proceedings of the Australian Physiological Society

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Subunit-specific inhibition of recombinantly expressed glycine receptors by ginkgolides and bilobalide R.L. Hawthorne and J.W. Lynch, School of Biomedical Sciences, University of Queensland, Brisbane QLD 4072, Australia. Extracts from the ginkgo biloba tree have been used in traditional Chinese medicine for centuries. Major active components of these extracts include the ginkgolides A, B and C (GA, GB, GC) and bilobalide (BB). GA, GB and GC are terpene trilactones which differ only in the number and placement of their hydroxyl groups. They share some structural similarity with BB and with the known glycine receptor Cl− channel (GlyR) inhibitor, picrotoxin. The ginkgo compounds have recently been shown to have potent inhibitory effects on GlyRs endogenously expressed in cultured central neurons (Kondratskaya et al., 2001; Ivic et al., 2003). Their use- and voltage-dependence suggest they may be pore-blockers (Kondratskaya et al., 2001; Ivic et al., 2003). The aim of the present study was to investigate the specificity of these compounds for recombinantly expressed α1, α2, α1b and α2b GlyRs. Their use-dependence, voltage-dependence and agonist concentration dependence of inhibition were also examined. HEK293 cells were transfected with GlyR cDNAs by the calcium phosphate precipitation protocol. The α and b subunits were co-expressed in a 1:10 ratio. The transfection solution was removed after 24 h and glycinegated currents were recorded by whole-cell recording over the following 24−72 h. Heteromeric GlyRs were identified by GFP fluorescence and by their reduced sensitivity to picrotoxin inhibition. In homomeric α2 GlyRs, inhibition by GA, GB and GC was more pronounced at positive voltages whilst BB showed no significant voltage-dependence. Lower concentrations of glycine markedly increased the inhibitory potency of BB, whereas glycine concentration changes had no significant effect on the degree of inhibition by GA, GB and GC. All four extracts showed use- dependence with no inhibition observed in the absence of glycine. As with picrotoxin, the potency of BB inhibition was drastically reduced upon co-expression of the b subunit with either the α1 or α2 subunits. On the contrary, co-expression of the b subunit with either the α1 or α2 subunits caused a significantly increased sensitivity to GB and GC. The sensitivity to GA was significantly increased in the α2b relative to the α2 GlyR, but was not significantly changed in the α1b relative to the α1 GlyR. The α1 subunit mutation T6¢ F abolished inhibition by all compounds. The use-dependence, voltage-dependence and the sensitivity to the pore-lining T6¢ F mutation, all suggest that the 4 tested ginkgo biloba extracts bind at a site in the GlyR pore. The results however indicate a different mechanism of inhibition by BB compared to that of GA, GB and GC. BB inhibition of the GlyR appears to mimic the effects of picrotoxin, despite these compounds sharing little structural similarity. The subunitspecificity of these compounds may be of use in defining the subunit composition of native neuronal GlyRs. Further investigations are required to examine the molecular basis of the observed differences in their mechanisms and subunit-specificity of action. Kondratskaya, E.L., Lishko, P.V., Chatterjee S.S. & Krishtal, O.A. (2002) Neurochemistry International 40, 647-653. Ivic L., Sands, T.T., Fishkin, N., Nakanishi, K., Kriegstein, A.R. & Stromgaard, K. (2003) Journal of Biological Chemistry 278, 49279-49285.

Proceedings of the Australian Physiological Society

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Crosslinking of α1b 1 GABAA receptor subunits via cysteines introduced into the transmembrane domain T.I. Webb, Z. Yang and J.W. Lynch, School of Biomedical Sciences, University of QLD, Brisbane QLD 4027, Australia. GABAA receptors belong to the ligand gated ion channel superfamily. Activation of these channels is thought to involve movement of the pore lining M2 domain. A model for α1b 1 GABAA receptor activation has been proposed on the basis of disulphide bond trapping experiments at an M2 residue (T6¢ ) located near the activation gate (Horenstein et al., 2001). This residue was mutated in both subunits to produce αT6¢ C and b T6¢ C subunits. Western analysis of GABAA receptors expressed in HEK293 cells plus electrophysiology of the same receptors in Xenopus oocytes suggested that disulphide bonds between adjacent b subunits lock the channels in the open state. The authors conclude that activation is mediated by an asymmetric rotation of adjacent b subunits. Data from our laboratory presented previously (Shan et al., 2002) and here question this model. To facilitate Western analysis, all αT6¢ C subunits were tagged with a FLAG epitope and all b T6¢ C subunits were tagged with a myc epitope. HEK293 cells were transfected with different combinations of wild type α, wild type b , αT6¢ C and b T6¢ C subunits and investigated by whole-cell electrophysiological recording. The oxidising agent, copper phenanthroline (Cu:phen), was used to promote formation of disulphide bonds and the reducing agent, dithiothreitol (DTT), was used to break them. Crude membrane preparations of the same transfected subunit combinations were used for Western analyses. Surface expression of functional receptors was confirmed by immunocytochemistry. Disulphide bond formation between b T6¢ C subunits was observed in the closed state. In cells expressing channels containing b T6¢ C subunits, GABA-gated currents decreased irreversibly upon Cu:phen treatment. As this effect was reversed only upon DTT application, we conclude that the disulphide bonds lock the channel closed. These bonds also formed spontaneously at a much slower rate. Western analysis provided direct evidence that formation of disulphide bonds in the closed state occurs between b subunits. The inclusion of DTT at several stages of channel protein preparation was sufficient to reduce the inter-subunit disulphides. Similarly, when Cu:phen was applied in combination with GABA, cells expressing channels containing b T6¢ C subunits again drastically reduced GABA current magnitude. Since the channels could only be reopened upon a subsequent application of DTT, we conclude that disulphide bonds lock the channel in a closed conformation. Since channels containing b T6¢ C subunits desensitize rapidly relative to wild type channels, the conclusion that the b T6¢ C subunits are crosslinked in the open state (simply because GABA is present) should be treated with caution. Due to the very short lifetime of the open state, we suggest that disulphide bond formation occurs predominantly in the desensitized state. Western analysis again provided direct evidence that formation of disulphide bonds in the desensitized state occurs between b subunits. No inter-subunit disulphides were observed with channels containing only αT6¢ C subunits and b wild type subunits. Indeed, αT6¢ C subunits were observed to form DTT-sensitive intramolecular disulphides. However, a weak band corresponding to a small population of mixed disulphides was observed with channels containing both αT6¢ C subunits and b T6¢ C subunits. Because we find no evidence for a state-dependent disulphide trapping of T6¢ C residues, our results are inconsistent with the model for LGIC activation proposed by Horenstein et al. (2001). Horenstein, J., Wagner, D.A., Czajkowski, C. & Akabas, M.H. (2001) Nature Neuroscience, 4, 477-485. Shan, Q., Haddrill, J.L. & Lynch, J.W. (2002) Journal of Biological Chemistry, 277, 44845-44853.

Proceedings of the Australian Physiological Society

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Etomidate alters the single-channel properties of GABAA receptors in newborn rat hippocampal neurons V.A.L. Seymour, P.W. Gage and M.L. Tierney, The John Curtin School of Medical Research, The Australian National University, Canberra, ACT 2601, Australia. The GABAA receptor is a GABA activated chloride channel that belongs to the superfamily of cysteineloop ligand-gated ion channels. The receptor is largely responsible for fast inhibitory neural transmission in the mammalian brain and is the target of many drugs including barbiturates, benzodiazepines and general anaesthetics such as etomidate. Etomidate is a carboxylated imidazole general anaesthetic. It is used as an induction agent in rapid sequence intubation in the emergency department because of its fast activation and hemodynamic stability (Bergen & Smith, 1997; Smith et al., 2000). Whole cell data has shown that etomidate modulates the GABAA receptor in a number of ways; at clinical concentrations (1-10m M) etomidate potentiates the GABAA receptor’s response to GABA; at higher concentrations (10-1000m M) etomidate can directly activate and desensitize the receptor; and at even higher concentrations (>1000m M) it produces an inhibitory affect (Zhang et al., 2002). To investigate how etomidate affects the properties of single GABAA receptors single-channel currents activated by GABA and etomidate are being recorded from hippocampal pyramidal neurons. Neurons are cultured from newborn Wistar rats (<24hours old) and experiments performed from seven days after culture. Preliminary results indicate that at clinical concentrations (1-10m M) etomidate potentiates the GABA induced current by increasing channel open time, open probability and channel conductance. The ability of the general anaesthetic etomidate to increase the maximum channel conductance to >40pS adds to our growing list of drugs that are capable of affecting the conductance of GABAA receptors. Together with diazepam, pentobarbitone, propofol and now etomidate, which may all increase the maximum conductance of GABAA channels, our data suggest that such drugs are acting through a common molecular mechanism inherent in GABAA receptors. Bergen, J.M. & Smith, D.C. (1997) Journal of Emergency Medicine 15, 221-230. Smith, D.C., Bergen, J.M., Smithline, H. & Kirschner, R. (2000) Journal of Emergency Medicine 18, 13-16. Zhang, Z.X., Lu, H., Dong, X.P., Liu, J. & Xu, T.L. (2002) Brain Research 953, 93-100.

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GABAA αb receptors open spontaneously when the conserved M2 leucine 9¢ residue is mutated to a threonine T.L. Luu, M.L. Tierney and P.W. Gage, Division of Molecular Bioscience, The John Curtin School of Medical Research, The Australian National University, ACT 2601, Australia. Inhibitory neurotransmission in the central nervous system of the brain is largely mediated by the g -aminobutyric acid type A (GABAA) receptor. This pentameric receptor is selectively permeable to chloride ions when activated by agonist. The simplest functional GABAA receptor is composed of α and b subunits. Each subunit has 4 transmembrane α-helices, of which the second transmembrane helix (M2) from all 5 subunits forms the pore. At the 9¢ position of the M2 is a leucine residue that is conserved in all ligand-gated ion channels. A single-channel study was performed to examine the effect of substituting the conserved leucine 9¢ residue (L9¢ ) to a threonine in the M2 domain in GABAA αb receptors. Site-directed mutagenesis was performed on the human α1 and b 1 subunit cDNAs. L929 mouse fibroblasts were transfected with either wild type or mutant GABAA α and b subunits and green fluorescent protein (GFP) plasmids. Successfully transfected cells showed bright green fluorescence and were targeted for single-channel outside-out patch-clamp recordings. Wild type GABAA αb receptors showed single-channel activity when activated by agonist. By contrast, αb receptors that contained the L9¢ T substitution in either the α, b or both subunits had spontaneous channel activity. The single-channel activity recorded from wild type αb receptors in the presence of 1 m M GABA consisted predominantly of brief open time events with a main single-channel conductance of 15 pS. Singlechannel activity was recorded from mutant α(L9¢ T)b and αb (L9¢ T) receptors in the absence of agonist. The spontaneous single-channel activity from these two mutant combinations had significantly longer open time events compared to wild type channels, and there was no change in the single-channel conductance. Application of GABA to the mutant α(L9¢ T)b and αb (L9¢ T) receptors led to an increase in single-channel activity. In contrast to the behaviour of α(L9¢ T)b and αb (L9¢ T) receptors, outside-out recordings from the mutant α(L9¢ T)b (L9¢ T) receptor showed only a spontaneous leak current with no single-channel closures. The application of penicillin to this spontaneous leak induced very brief closures. The data suggest that in wild type αb receptors the functional role of the L9¢ residue is to stabilise the closed state of the channel. When mutated to a threonine the equilibrium of the receptor is pushed towards the open state as revealed by the ability of mutant receptors to now open spontaneously.

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GABARAP influences the conductance of recombinant GABAA channels

A.B. Everitt, M.L. Tierney and P.W. Gage, The John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia. ‘Native’ GABAA receptors display distinct electrophysiological properties not always seen in recombinant receptors irrespective of subunit composition. Native channels can have conductances over 40pS (Gray & Johnson, 1985; Smith et al., 1989: Curmi et al., 1993). Moreover, the conductance of some channels can be increased by modulating drugs such as diazepam, pentobarbitone and propofol (Eghbali et al., 1997; Guyon et al., 1999; Eghbali et al., 2003). By contrast, conductances of recombinant channels have never exceeded 35pS and, although their open probability can be increased by modulating drugs, channel conductance was not enhanced by drugs. A GABAA receptor-associated protein, GABARAP, is an intracellular protein that can interact with the GABAA g 2 subunit. When co-expressed with GABAA subunits, GABARAP caused clustering of the receptors and changes in whole-cell current kinetics in QT6 fibroblasts (Wang et al., 1999). Recent observations have shown that over-expression of GABARAP in Xenopus oocytes and in neurons increases the level of GABAA receptors detected at the plasma membrane, indicating that GABARAP is involved in the trafficking of GABAA receptors (Chen et al., 2005; Leil et al., 2004). It has been suggested that high channel conductances may represent co-operative openings of clustered channels resulting in an apparent high single channel conductance (Laver & Gage, 1997). We tested this hypothesis in an expression system by co-expressing GABARAP, known to cluster GABAA receptors, with GABAA receptor subunits in L929 mouse fibroblasts. Immunofluorescent studies revealed that co-expression of GABARAP with GABAA subunits, showed a punctate pattern of staining of surface receptors compared to a diffuse pattern in control cells. We recorded single channel currents in the cell-attached (c/a) configuration 24-72 hours post transfection. Control patches expressing GABAA α1, b 1 and g 2s subunits alone had a mean conductance of 22.3 ± 1.2 pS (n=15). In 16 out of 25 patches recorded from cells co-transfected with GABAA α1, b 1 and g 2s subunits and GABARAP, single channel conductances were above 40pS (g = 60.7 ± 4.3pS, n=16). These ‘high’ conductance channels were never seen in control patches. In the remaining 9 patches, the mean conductance was 29.1 ± 1.9 pS. Both high and low conductance channel activities were blocked by 100m M bicuculline. The current-voltage relationship of high conductance channels showed outward rectification of the current, similar to that seen in native receptors. Diazepam and pentobarbital have been shown to increase both open probability and conductance of GABAA channels. In patches from cells co-expressing GABAA α1, b 1 and g 2s subunits and GABARAP, both of these effects were seen irrespective of initial channel conductance. In control patches where GABARAP was not expressed, application of diazepam or pentobarbital increased the channel open probability with no effect on single channel conductance. Our results show that co-expression with GABARAP has changed the properties of recombinant GABAA channels. It is possible that clustered receptors may be able to couple and open cooperatively by virtue of their close physical proximity. Curmi, J.P., Premkumar, L.S., Birnir, B. & Gage, P.W. (1993), Journal of Membrane Biology, 136, 273-280. Eghbali, M., Curmi, J.P., Birnir, B. & Gage, P.W. (1997), Nature, 388, 71-75. Eghbali, M., Gage, P.W. & Birnir, B. (2003), European Journal of Pharmacology, 468 (2): 75-82. Gray, R., Johnston, D. (1985) Journal of Neurophysiology, 54: 134-142. Guyon, A., Laurent, S., Paupardin-Tritsch, D., Rossier, J. & Eugen, D. (1999), Journal of Physiology, 516, 719-737. Smith, S.M., Zorec, R. & McBurney, R.N. (1989) Journal of Membrane Biology, 108, 45-52. Wang, H., Bedford, F.K., Brandon, N.J., Moss, S.J. & Olsen, R.W. (1999), Nature, 397, 69-72. Chen, Z., Chang-Sheung, S., Leil, T.A., Olcese, R. & Olsen, R.W. (2005), Molecular Pharmacology, 68(1), 152-159. Leil, T.A., Chen, Z., Chang-Sheung, S. & Olsen, R.W. (2004), Journal of Neuroscience, 24(50): 11429-11438. Laver, D.R.. & Gage, P.W. (1997), Prog. Biophsy. Mol. Biol, 67: 99-140.

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C-Terminal peptide of M protein from dengue virus (DVM-C) forms ion channels A. Premkumar, C.R. Horan and P.W. Gage, Division of Molecular Biosciences, John Curtin School of Medical Research, Australian National University, PO Box 334, Canberra City, ACT 2601, Australia. The dengue virus belongs to the family of Flaviviridae and causes an infectious disease carried by mosquitoes - dengue fever. There is no specific treatment for dengue fever, and most people recover completely within 2 weeks. Like the alphaviruses and influenza viruses, the dengue virus enters cells in an endocytotic vesicle. The M, or membrane protein, of the dengue virus may have a similar function to that of the M2 protein of Influenza A and assist in the entry of the virus by functioning as an ion channel. The M protein is one of the structural proteins of the virus and is 75 amino acids in length. The C-terminal end of the protein from amino acids 48 to 70 contains a predicted transmembrane region, thought to anchor the protein in the lipid bilayer (Kuhn et al., 2002). The function of the M protein is still unknown. To test the hypothesis that the M protein of dengue functions in a similar manner to the M2 protein of influenza A we tested the C-terminal end of the M protein containing the putative transmembrane region for channel activity. The C-terminus of the M protein of the Dengue virus type 1 strain Singapore S275/90 (DVM-C) was chemically synthesised and tested for channel forming properties in artificial lipid bilayers. We have found that the DVM-C peptide from Dengue virus forms ion channels in lipid bilayer membranes. The channels were selective for monovalent cations over monovalent anions but were relatively impermeable to calcium ions. Amantadine (10 m M) and 100 m M Hexamethylene amiloride (HMA) block channels formed by DVM-C. The dengue virus M protein can be added to an increasing list of virus proteins that have been shown to form ion channels in artificial lipid bilayers. Kuhn, R.J., Zhang, W., Rossmann, M.G., Pletnev, S.V., Corver, J., Lenches, E., Jones, C.T., Mukhopadhyay, S., Chipman, P.R., Strauss, E.G., Baker, T.S., Strauss & J.H. 2002. Cell 108:717-25.

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The role of the M1-P1 loop in acid sensitive two-pore domain potassium (TASK) channel regulation Catherine E. Clarke1,2, Alistair Mathie3 and Jamie I. Vandenberg1,2, 1St Vincent’s Hospital Clinical School, University of New South Wales, Victoria Road, Darlinghurst NSW 2010, Australia, 2Electrophysiology and Biophysics Unit, Victor Chang Cardiac Research Institute, 384 Victoria Street, Darlinghurst, NSW 2010, Australia and 3Department of Biological Sciences, Imperial College London, Exhibition Road, London SW7 2AZ, UK. Background potassium channels control the resting membrane potential of many cells and regulate their excitability. Two-pore-domain potassium (2PK) channels have been shown to underlie a number of such background currents. Although often classed as “leak” channels, currents through 2PK channels are tightly regulated. For example, the acid sensitive 2PK (TASK) channels are inhibited by extracellular acidification with pKa’s for TASK-1, TASK-2 and TASK-3 ranging from 6.5 to 8.5. TASK channels show relatively high homology in their transmembrane domains, but very little homology in extracellular domains, most notably in the ∼50 residue linker between the first transmembrane domain and the first pore domain (the M1P1 loop). The M1P1 loop has previously been shown to be involved in the differential sensitivity of TASK channels to block by zinc (Clarke et al., 2004). The aim of the present study was to test the hypothesis that the M1P1 loop contributes to the differential pH sensitivity of TASK channels. Chimaeric TASK channels were constructed by swapping M1P1 loops between family members and expressed in Chinese Hamster Ovary cells. Channels were characterised using standard whole cell patch clamp techniques. The chimaera formed from TASK-3 with the TASK-1 M1-P1 loop (T3/T1-M1P1) had a pKa for inhibition that was much more similar to that of TASK-1 than TASK-3 (T1/T3 M1P1: pKa = 7.0 ± 0.05, TASK-1: pKa = 7.1 ± 0.04; TASK-3, pKa = 6.7 ± 0.07). This indicates that the M1P1 loop contributes to the differential pH sensitivity of TASK-1 and TASK-3 channels. All other chimaeras, however, were non-functional. This further suggests that the M1P1 loops are important for function and/or structure. However, further work needs to be done to assess whether these non-functional chimaeras are reaching the membrane and whether function can be restored through re-addition of further regions of the channel, such as the P-loop. Clarke, C.E., Veale, E.L., Green, P.J., Meadows, H.J. & Mathie, A. (2004) Journal of Physiology 560, 51-62.

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Structural studies of chloride intracellular ion channel proteins A.V. Mynott1, D.R. Littler1, P.M.G. Curmi1, L.J. Brown2 and S.N. Breit3, 1School of Physics, University of New South Wales, NSW 2052, Australia, 2Department of Chemistry and Biomolecular Sciences, Macquarie University, NSW 2109, Australia and 3St Vincent’s Hospital, Sydney, NSW 2010, Australia. The chloride intracellular channel (CLIC) family of proteins belong to a new class of chloride ion channels. They are generally localised on the nuclear membrane, but are also found in the cytoplasm and on the cell membrane. They have the unique feature of existing in both soluble and membrane associated forms. The structural conformation of these proteins may be affected by various biological mechanisms including pH, redox and interactions with binding partners. For example, suggestions of CLIC translocation to the nucleus under stress has led to speculation of a direct interaction of CLIC with components of the nuclear import machinery. Furthermore, the location of the conserved cysteine residues in the 3-dimensional structure of the CLIC proteins may also prove crucial to the understanding of the structure-function relationship. In this study, members of the CLIC family have been examined using various biophysical techniques, including circular dichroism spectroscopy, to reveal possible structural conformational changes that may occur under variations in environmental conditions. The conditions examined include binding partner interactions, oxidation and mutagenesis of the conserved cysteine residues. Results suggest that changes in these conditions, particularly mutation of Cys178 in CLIC1, lead to conformational instability and structural differences.

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Photochemical behaviour and Na+,K+-ATPase sensitivity of voltage-sensitive styrylpyridinium fluorescent membrane probes S. Amoroso1, V.V. Agon1, T. Starke-Peterkovic1, M.D. McLeod1, H.-J. Apell2, P. Sebban3 and R.J. Clarke1, 1School of Chemistry, University of Sydney, NSW 2006, Australia, 2Faculty of Biology, University of Constance, D-78434 Constance, Germany and 3Laboratory of Physical Chemistry, UMR 8000, University of Paris XI, Orsay 91405, France. Styrylpyridinium dyes are widely used probes of electric field strength changes in biological membrane systems. They can be used as optical probes in the imaging of electrical activity in nerve and muscle cells, and for the investigation of the mechanisms of electrogenic ion pumps. A limit to their application is, however, their photochemical stability. Probes with improved stability and voltage sensitivity are required in order to extend their areas of application. Exposure of the voltage-sensitive membrane probe RH421 to continuous illumination with 577 nm light from an Hg lamp leads to an increase in its steady-state fluorescence level when bound to lipid membranes. The increase occurs on the second time-scale at typical light intensities and was found to be due to a single-photon excited-state isomerisation. In order to suppress this undesirable reaction, modifications to the dye structure are, therefore, necessary. The related probe ANNINE 5, which has a rigid polycyclic structure, shows no observable photochemical reaction when bound to DMPC vesicles on irradiation with 436 nm light. The voltage sensitivity of ANNINE 5 was tested using Na+,K+-ATPase membrane fragments. As long as ANNINE 5 is excited on the far red edge of its visible absorption band, it shows a similar sensitivity to RH421 in detecting chargetranslocating reactions triggered by ATP phosphorylation. Unfortunately, the wavelengths necessary for ANNINE 5 excitation are in a region where the Hg lamps routinely used in stopped-flow apparatus have no significant lines available for excitation.

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Applications of styrylpyridinium dyes in elucidating ion-transport mechanisms in plant cells S. Amoroso1, R.J. Clarke2, A. Larkum1 and R. Quinnell1, 1School of Biological Sciences, University of Sydney, NSW 2006, Australia and 2School of Chemistry, University of Sydney, NSW 2006, Australia. Styrylpyridinium dyes have been used in a range of biological applications, including quantitative electrophysiology and qualitative microscopy for the detection of the membrane electric field strength changes. Examples of these dyes include RH421 and di-4-ASPBS. The aims of our project are to extend the applications of styrylpyridinium dyes and to elucidate ion-transporting mechanisms that take place within algal systems. The dyes RH421, di-4-ASPBS, Annine V, RH414, RH795 and RH237 have been tested on a freshwater photosynthetic system, Chara corallina and preliminary measurements have shown that all of these dyes are successfully taken up by the membranes of Chara cells. The potential of these molecules to detect ATPase activity is under examination. In addition, we are examining the ability of these molecules to detect the effects of membrane transport inhibitors and protonophores. So far, we have concluded that the styrylpyridinium dye, di-4-ASPBS is the most useful dye for resolving these processes in a freshwater alga cell. These preliminary results are encouraging, as these particular dyes have not previously been applied in photosynthetic systems.

Proceedings of the Australian Physiological Society

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The crystal structure of Pichia pastoris lysyl oxidase at 1.23Å reveals a lysine-lysine covalent cross-link, dehydrolysinonorleucine A.P. Duff1, A.E. Cohen2, P.J. Ellis2, D.B. Langley1, D.M. Dooley3, H.C. Freeman1 and J.M. Guss1, 1School of MMB, University of Sydney, NSW 2006, Australia, 2Stanford Synchrotron Radiation Laboratory, CA, USA and 3Chemistry and Biochemistry, Montana State University, Bozeman MT, USA. We have refined the structure of Pichia pastoris lysyl oxidase (PPLO) in a new crystal form at 1.23Å resolution with R = 0.112 and Rfree = 0.146. PPLO is a copper amine oxidase (CuAO) containing a trihydroxyphenylalanine quinone (TPQ) cofactor. PPLO is unusual in being able to oxidise the side chain of lysine residues in a polypeptide. In this respect, it is functionally related to another class of CuAOs of unrelated sequence, which contain the related quinone cofactor, lysine tyrosylquinone (LTQ). The asymmetric unit comprises residues 43 to 779 of the polypeptide, 7 carbohydrate residues, the active-site Cu atom, an imidazole molecule bound in the substrate-binding site, 2 buried Ca2+ ions, 5 surface Mg2+ ions, 5 surface Cl- ions, and 1045 water molecules. The cofactor, TPQ, and some other active site residues are poorly ordered, in contrast to the high degree of order of their other neighbours. A covalent cross-link between two lysine residues, Lys 778 and Lys 66, is observed. The cross-link, dehydrolysinonorleucine, is formed by the oxidation of Lys 778, followed by spontaneous reaction with Lys 66.

Proceedings of the Australian Physiological Society

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Molecular dynamics study of conformational changes in human serum albumin by binding of fatty acids S. Fujiwara and T. Amisaki, Department of Biological Regulation, Faculty of Medicine, Tottori University, 86 Nishi-machi, Yonago, 683-8503, Japan. Human serum albumin (HSA) is a major protein component of blood plasma. Binding of drugs to HSA is one of the most important factors determining pharmacokinetics of drugs (Kragh-Hansen et al., 2002). When measuring binding affinity of a drug to HSA in vitro, defatted HSA is usually used. On the other hand, under normal physiological conditions, HSA binds with fatty acid. So far, there is little information on conformational changes of HSA upon binding of fatty acids. The present study was aimed to elucidate the conformational changes as well as the structure and dynamics of HSA, based on the molecular dynamics (MD) simulations with explicit water molecules. Materials and methods. The initial coordinates of unliganded HSA and HSA-myristate (HSA-MYR) complex were obtained from Protein Data Bank (unliganded HSA: PDB entry 1AO6, HSA-MYR: PDB entry 1BJ5). A series of MD calculations were carried out using AMBER7 package (Case et al. 2002). A rectangularshaped box of water was constructed. 5 ns MD calculations were carried out for both the unliganded HSA and HSA-MYR complex models under periodic boundary condition. The long-range electrostatic interactions were handled by the particle mesh Ewald algorithm (Darden et al., 1993). The resultant model systems contained 87223 (unliganded HSA) and 99126 (HSA-MYR complex) atoms, respectively. Results and discussion. The root mean square deviation (RMSD) from the X-ray structure over the course of a MD simulation reached plateau at about 2 ns. The RMSD values were as small as about 3.0 Å, which were roughly comparable to the X-ray resolution. Hence, we concluded that significant structural drift from the X-ray structure did not occur during the MD simulations. Binding of MYR to HSA increased the radius of gyration (Rg) of HSA in the MD simulations. Through the structural comparison of the average structures, the dramatic extent of the relative motions of domains I and III, especially those of subdomains IA and IIIB, were observed. Thus, increase in Rg by binding of MYR molecules should be observed for HSA, as a result of the motions of domains I and III. Local protein mobility was analyzed by calculating the time-averaged root mean square fluctuation (RMSF) for each residue, using the trajectory in an equilibrium state. RMSF values at drug binding sites I (subdomain IIA) and II (subdomain IIIA) were increased by binding of MYR. This result implies that binding affinity of drugs at these primary binding sites can be changed by MYR binding. To analyze internal motions of the whole HSA molecule and each domain, principal component analysis for collective coordinates from MD simulations was carried out. The primary internal motions, characterized by the first and the second principal components, PC1 and PC2, were observed mainly at domains I and III. The directional motion projected on PC1 of unliganded HSA was similar with that projected on PC2 of HSA-MYR complex, indicating that the first principal directional motion in unliganded HSA is conserved as the second principal directional motion in HSA-MYR complex. On the other hand, the second principal directional motion in unliganded HSA partially turned into the first principal directional motion in HSA-MYR complex. Conclusion. The present study unraveled possible conformational changes in aqueous solution caused by binding of MYR molecules to HSA, based on the results of the MD simulations. Case, D.A., Pearlman, D.A., Caldwell, J.W., Cheatham, T.E. 3rd, Wang, J., Ross, W.S., Simmerling, C.L., Darden, T.A., Merz, K.M., Stanton, R.V., Cheng, A.L., Vincent, J.J., Crowley, M., Tsui, V., Gohlke, H., Radmer, R.J., Duan, Y., Petera, J., Massova, I., Seibel, G.L., Singh, U.C., Weiner, P.K., Kollman, P.A. (2002) AMBER7. San Francisco: University of California. Darden, T., York, D., Pedersen, L. (1993) Journal of Chemical Physics 98, 10089-10092. Kragh-Hansen, U., Chuang, V.T.G., Otagiri, M. (2002) Biological and Pharmaceutical Bulletin 25, 695-704.

Proceedings of the Australian Physiological Society

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NMR probes of red cell deformation P.W. Kuchel, B.E. Chapman, D.J. Philp and W.A. Bubb, School of Molecular and Microbial Biosciences, University of Sydney, NSW 2006, Australia. The mean circulation time of a red blood cell (RBC) in an adult human is ∼1 minute. Thus each RBC in passing through the peripheral and the pulmonary capillary beds undergoes deformation, from its biconcave-disc shape to an elongated bullet shape, and the reverse, every 30 seconds. 23Na and 133Cs nuclei have spin >1/2 and thus a nuclear electric quadrupole. This renders their NMR resonance frequency sensitive to the presence of electric field gradients at binding surfaces. Such gradients exist in anisotropic media. Gelatine, which sets at temperatures below ∼30°C, was cast inside a silicone rubber tube that in turn was placed inside a glass tube. Thus the gelatine could be stretched by a factor of up to ∼2; in the process the gelatine developed structural anisotropy. This anisotropy was evident as a splitting into three of the 23Na+ NMR resonance, whereas the 133Cs+ NMR resonance was split into a septet. In both cases the residual quadrupolar coupling constant was a linear function of the extent of stretching. RBCs set in the gelatine revealed separate resonances for 133Cs+ inside and outside the cells. And, stretching the gelatine also stretched the RBCs as was apparent from the emergence of quadrupolar splitting of the intracellular 133Cs+ resonance. Finally, the metabolic activity of the RBCs was measured using 13C NMR, with D-[U-13C]glucose as substrate, when the cells were in the stretched or unstretched states. These findings allow comment on the energy cost of the return of an RBC to its equilibrium shape, after distortion.

Proceedings of the Australian Physiological Society

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Current-voltage analysis of response to salt stress by salt-tolerant and salt-sensitive charophyte cells Mary J. Beilby and Virginia A. Shepherd, Biophysics, School of Physics, University of NSW, NSW 2052, Australia. The electrophysiological responses of salt-tolerant charophyte Laprothamnium succinctum and saltsensitive charophyte Chara corallina to NaCl increase in the medium are compared. The modelling of the current-voltage (I/V) curves allow us to resolve the response of various transporter populations from the total clamp current. The proton ATPase I/V profile is fitted by the HGSS (Hansen, Gradmann, Slayman and Sanders) model of the proton pump (Beilby, 1984). The background current is fitted by an empirical model. The inward K+ rectifier in Lamprothamnium is modelled by the GHK (Goldman, Hodgkin, Katz) model supplemented by the Boltzmann distribution (Beilby & Walker, 1996). In both charophytes there is a greater background conductance as Na+ concentration in the medium rises. Lamprothamnium is able to maintain the negative resting PD (potential difference) by increasing the rate of proton pumping. The HGSS pump model rate constants k0io, k0oi and k oi all increase over about 120 min of the salt stress (+ 72 mM NaCl). The inward rectifier activates at more positive PDs. Chara cells, faced with hypertonic medium of 50 mM NaCl and 0.5 mM Ca++ added to the APW (artificial pond water), exhibit more varied responses: (i) pump rate was unchanged, (ii) k0io, k0oi increased but not k oi, (iii) k0io, k0oi and k oi all increased over 80 min. However, the pumping increase in Chara is not enough to keep the membrane from depolarizing. Further, the proton pump becomes very sensitive to both depolarization and hyperpolarization imposed on the membrane. Pump inhibition follows and the recovery is very slow. This instability of the pump impedes recovery of the negative resting PD after voltage clamp to a bipolar staircase command or a spontaneous action potential. It is also impossible to voltage clamp the membrane to sufficiently negative PD levels to investigate the effect of high salt on the inward rectifier. Charophytes are evolutionarily most closely allied to the green algal line that gave rise to higher plants (Graham & Gray 2001; Karol et al., 2001) and therefore make good models for them. The significance of the differences in one type of transporter rendering the plant cell salt-tolerant can be considered in the light of evolutionary adaptations. Beilby M.J. (1984) Journal of Membrane Biology 81, 113-125. Beilby M.J. & Walker N.A. (1996) Journal of Membrane Biology 149, 89-101. Graham, L.E. & Gray J. (2001) In: Gensel, P.G. & D. Edwards (eds), Plants Invade the Land. Evolutionary and Environmental Perspectives. Columbia University Press, New York: 140-159. Karol, K.G., McCourt R.M., Cimino M.T. & Delwiche C.F. (2001) Science 294, 2351-2353.

Proceedings of the Australian Physiological Society

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Oxygen evolution in chimeric spinach photosystem II with cyanobacteria manganese stabilising protein Adele Williamson, Warwick Hillier, Reza Razeghifard and Tom Wydrzynski, Photobioenergetics Group, Research School of Biological Sciences, The Australian National University, Canberra, ACT 0200, Australia. Photosystem II (PSII) is the first multiprotein complex in the photosynthetic electron transfer chain and catalyses the light-driven oxidation of water and reduction of plastoquinone. The PSII protein contains a sub domain termed oxygen evolving complex (OEC), where water is split during photosynthesis, and oxygen released as a by product. The OEC is made up of loop regions of the core membrane spanning subunits, a cluster of Mn4Ca ions which are the site of water oxidation, and three extrinsic proteins which are bound electrostatically to the core proteins. Of these extrinsic proteins, only the 33kDa manganese stabilising protein (MSP) is common to higher plants, algae and cyanobacteria. The MSP is essential for stabilising Mn binding and provides optimal O2 evolution. However the MSP does not appear to provide any ligands to any of these ions and its function was proposed to facilitate O2 production by forming a channel which controls entry of the substrate water to the active site or release of the O2 and proton products (De Las Rivas & Barber, 2004; Ferreira et al., 2004; Wydrzynski et al., 1996). In this presentation we have created chimeric PSII complexes by removing all three extrinsic proteins from the PSII complex of spinach, and replacing the native MSP with recombinant MSP from the thermophilic cyanobacterium Thermosynechococcus elongatus. The recombinant protein is either fused with thioredoxin or truncated by 39 amino acids at the C-terminus resulting in a deletion of the conserved water channel residues. Here we present the O2 evolution and tyrosine radical decay kinetics of these chimeric proteins. The results show the importance of these conserved residues to catalytic function and suggest that substrate accessibility is important for this enzyme. De Las Rivas, J. & Barber, J. (2004) Photosynthesis Research 81, 329-343. Ferreira, K.N., Iverson, T.M., Maghlaoui, K., Barber, J., & Iwata, S. (2004) Science 303, 1831-1838. Wydrzynski, T., Hillier, W., & Messinger, J. (1996) Physiologia Plantarum 96, 342-350.

Proceedings of the Australian Physiological Society

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The role of an oil droplet lens in vision enhancement L. Fischer1, M. Vorobyev2, A. Zvyagin1 and T. Plakhotnik1, 1Department of Physics, University of Queensland, QLD 4072, Australia and 2Vision Touch and Hearing Research Centre, University of Queensland, QLD 4072, Australia. The cone photoreceptors of all birds and some reptiles, amphibians, mammals and fish contain both coloured and transparent oil droplets (Walls, 1942). The light incident on the photosensitive region of such receptors is preconditioned by this oil droplet lens. Coloured oil droplets (which form the majority of the oil droplet population) act as long-pass filters and are thus responsible for spectral tuning. The prevalence of transparent oil droplets throughout the vertebrate classes, such as the T-type oil droplets found in the Ultraviolet or Violet-Sensitive (UVS/VS) photoreceptors of birds, suggests an auxiliary dioptric function operating outside of colour filtering (Young & Martin, 1984). It is hypothesized that an oil droplet lens enhances light collection efficiency and - perhaps more importantly - detection directionality. The outstanding features of an idealised photoreceptor can be modelled in the framework of the electromagnetic theory. The table indicates the set of characteristic parameters that are used in the construction of this model. Model Parameter Value (m m) Oil Droplet 2.5 Outer Segment (OS) Length 10.0 OS Base Diameter 1.5 OS Tip Diameter 1.0 Region Refractive Index Cone Outer Segment 1.387 - 0.0011i Extracellular Matrix 1.340 Transparent Oil Droplet 1.480 The geometric optics approximation cannot be applied to this problem since the wavelength of light and the dimensions of the system are of a comparable order of magnitude. The dioptric function of oil droplets has previously been considered in the context of the Mie scattering theory, which provides an analytic solution to Maxwell’s equations of electromagnetics for spherical particles (Ives, Normann & Barber, 1983). Due to the complexity of any realistic photoreceptor structure, a complete analytic solution is not possible. Rather, numerical methods within electromagnetic theory must be employed. The Finite-Difference Time-Domain technique (FDTD) appears to be an attractive alternative to investigate the light coupling efficiency of the photosensitive region in the presence of an oil droplet. FDTD provides a full field solution of Maxwell’s equations for some specific dielectric structure. Numerical data sets have been obtained for the vertebrate photoreceptor structures of rods, cones and cones containing transparent oil droplets under broad-band plane wave excitation. Preliminary results show that in the presence of an oil droplet, cones have an increased light coupling efficiency whilst in the retinal mosaic. The normalised plane wave coupling efficiency of a cone photoreceptor containing an oil droplet is shown in the figure. It is a function of both the wavelength and the angle of incidence of the plane wave. Maximal coupling at normal incidence is significantly blue-shifted when preconditioned by an oil droplet lens. Since the photopigment found in UVS/VS photoreceptors characteristically have absorption maxima between 360 and 430 nm, such peak shifting is indicative of spectral tuning. Both the geometry and dielectric properties of photoreceptors are expected to change during in vitro analysis. Thus, numerical investigation of the model parameters that determine the degree of peak shifting is currently being conducted.

Ives, J.T., Normann, R.A. & Barber, P.W. (1983) Journal of the Optical Society of America 73(12), 1725-1731. Walls, G.L. ed. (1942) The vertebrate eye and its adaptive radiation. Bloomfield Hills, Cranbrook Institute of Science. Young, S.R. & Martin, G.R. (1984) Vision Research 24(2), 129-137. Proceedings of the Australian Physiological Society

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Circular dichroic spectra of the N-terminal region of cardiac myosin binding protein – C C.E. Oakley1, L.J. Brown2 and B.D. Hambly1, 1Department of Pathology, University of Sydney, NSW 2006, Australia and 2Department of Chemistry and Biomolecular Sciences, Macquarie University, NSW 2109, Australia. Myosin binding protein – C (MyBPC) is a multi-domain protein whose role in the sarcomere is complex and not yet fully understood. Three isoforms of human MyBPC have been identified; fast skeletal, slow skeletal and cardiac. The cardiac isoform has been of particular interest because of its link to the heart disease familial hypertrophic cardiomyopathy (FHC). FHC is caused by the expression of abnormal contractile proteins in the heart muscle including numerous mutations in MyBPC. The core structure of cardiac MyBPC consists of eight immunoglobulin (IgI) and three fibronectin (FnIII) domains, numbered 0 – 10 from the N-terminus. MyBPC is phosphorylated up to 3 times near the N-terminus and this is associated with increased contractile force. The binding of MyBPC to myosin S2 in a phosphorylatable-dependent manner is well established, although the role of the extent of phosphorylation is unknown. The location of phosphorylation is a linker of about 100 amino acids between IgI domains 1 and 2. The focus of this work is the effect of phosphorylation and FHC mutations on the structure of the linker or the IgI domains that flank it. The N-terminal IgI motifs, C1C2, have been cloned, expressed, purified and circular dichroic (CD) spectra for (i) the wild type, (ii) the “permanently” phosphorylated mutants (Ser to Asp) and (iii) 9 FHC mutants collected. The spectra indicate that a proportion of the protein is alpha-helix. Modelling of the C1C2 construct also suggested an alpha-helical content and indicated that it is likely to be part of the phosphorylation linker region. Changes in the CD spectra of the FHC mutants indicate a change in secondary structure and may explain the pathogenesis of these mutations. Similarly, a change in spectra due to pseudo-phosphorylation may provide an insight into the functional role of MyBPC in the sarcomere.

Proceedings of the Australian Physiological Society

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Changes in mechanical properties of live cell wall during turgor regulation monitored by atomic force microscopy E.M. Mahomudally1, M.J. Beilby2, V. Shepherd2 and A.R. Moon1, 1Department of Applied Physics, University of Technology, Sydney, NSW 2007, Australia and 2Biophysics Department, School of Physics, University of New South Wales, Kensington, NSW 2052, Australia. (Introduced by M.J. Beilby) Plant cells display mechanosensitivity (Shepherd et al., 2001). Monitoring these mechanical signals via changes in the cell wall is important in the study of control pump activation during hypotonic turgor regulation (Bisson & Beilby, 2002). The atomic force microscope (AFM) has become a useful tool in the study of surface mechanical properties (Burnham & Colton, 1989). In the study of biological samples its great advantage is allowing realtime study of samples in their native state (Radmacher, 1997). We have used the AFM to monitor changes in mechanical properties of the cell wall as the cell is subject to osmotic stress. We report on experiments conducted on small live cells (2-3mm) of Ventricaria ventricosa (Valonia), a well characterised, large single-celled alga. Measurements were taken on the resting cell in artificial seawater (ASW 990 mOsmol•kg-1) prior to initiating turgor regulation. Hypotonic stress of 200 mOsmol•kg-1 was then imposed on the cell. After stabilisation, measurements were collected on the cell surface for approximately 2 hours. The procedure was repeated for an additional hypotonic shock of 590 mOsmol•kg-1. 450

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The figure shows the time response of the cell stiffness. The cell appears to respond to hypotonic shock by rapidly altering its wall. Soon after the onset of hypotonic shock, wall strengthening is observed, then a period of large oscillations between wall strengthening and weakening is seen followed by a period of smaller oscillations. A weak wall compared to the resting cell’s was observed after the oscillation period. The cells examined survived the two levels of hypotonic shock. Bisson, M.A. & Beilby, M.J. (2002) The Journal of Membrane Biology 190, 43-56. Burnham, N.A. & Colton, R.J. (1989) Journal of Vacuum Science and Technology A 7, 2906-2913. Radmacher, M. (1997) IEEE Engineering in Medicine and Biology 16, 47-57. Shepherd, V.A., Shimmen, T. & Beilby, M.J. (2001) Australian Journal of Plant Physiology 28, 551-566.

Proceedings of the Australian Physiological Society

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Effect of temperature on stretch-induced cardiac action potential shortening in the rat heart: involvement of TREK-1 D.R. Kelly, L. Mackenzie and D.A. Saint, Discipline of Physiology, School of Molecular and Biomedical Science, University of Adelaide, SA 5005, Australia. The importance of stretch activated ion channels in the modulation of cardiac electrophysiology is becoming increasingly apparent. Of the stretch activated channels known, both temperature sensitive TREK-1 and non-selective cation channels exist in the rat heart. The present study aimed to evaluate the contribution of these stretch-activated channels to changes in the rat cardiac action potential duration during stretch by taking advantage of the temperature dependent nature of the TREK-1 channel. 6 male Sprague Dawley rats 450-490g were killed by cervical dislocation, their hearts excised and retrogradely perfused on a Langendorf system. Following stabilization, hearts were subjected to three consecutive diastolic pressures (control: 0-5mmHg, moderate: 20-25mmHg and extreme: 50-55mmHg) at two randomised perfusion temperatures (32°C or 37°C). During manipulation of left ventricular diastolic pressure, endocardial and epicardial monophasic action potentials (MAP) were recorded, and the action potential duration at 80% repolarisation (APD80) calculated. Under control conditions of 0-5mmHg pre-load, the endocardial APD80 was 40.3 ±4.2ms, while the epicardial APD80 was 45.4 ±4.4ms at 37°C. Decreasing cardiac temperature to 32°C increased both endocardial and epicardial APD80 to 46.9 ±3.9ms and 61.1 ±2.7ms respectively. The addition of moderate cardiac pre-load (left ventricular diastolic pressure of 20-25mmHg), did not significantly alter epicardial APD80 at either temperature. By contrast, endocardial APD80 reduced by 12.2 ±3.2% and 11.6 ±3.7% from control values at 37°C and 32°C respectively. Extreme left ventricular stretch (50-55mmHg) significantly reduced the APD80 in both epicardial and endocardial recordings at 37°C and 32°C. Epicardial APD80 reduced by 5.9 ±2.5% and 11.5 ±3.9% relative to control conditions at 37°C and 32°C respectively. Similarly, endocardial APD80 decreased by 18.4 ±3.5% and 19.3 ±3.2% at 37°C and 32°C respectively. The reductions in action potential duration observed following extreme stretch at 32°C were not significantly different than those observed at 37°C. It was concluded that a change in cardiac temperature did not affect the magnitude of reduction in action potential duration (as measured by APD80) following moderate (20-25mmHg) or extreme stretch (50-55mmHg) for either endocardial or epicardial recordings when compared to their control. Since TREK-1 channels are temperature sensitive (inactivating at lower temperatures), these results suggest that non-selective stretchsensitive cation channels may be more important in modifying action potential duration during stretch than TREK-1 in rat heart.

Proceedings of the Australian Physiological Society

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Immunohistochemical identification of stretch-sensitive two-pore-potassium (TREK) channels in the human heart S.Y. Yuan, H.P. Zhu and D. Saint, Department of Physiology, School of Molecular & Biomedical Science, University of Adelaide, Adelaide, SA 5005, Australia. Rhythmic contraction and relaxation of cardiac muscle are determined by a regular cycle of depolarisation and repolarization of cardiac myocytes. Outward movement of potassium ions across the membrane is important for the repolarization and maintenance of resting membrane potential. Potassium channels activated by membrane stretch may contribute to maintenance of normal electrical activity of cardiac myocytes. However, so far there is no evidence for the existence of these channels in human cardiac myocytes. In this study, we examined the existence and location of stretch sensitive potassium (TREK-1 and TREK-2) channels in different regions of human heart using immunohistochemical method. Eight pieces (5 ´ 10 ´ 2 mm3 for each piece) of human tissue, four fresh atrial appendages and four frozen ventricles taken from patients with coronary bypass surgery and heart transplantation, were processed for immunohistochemistry after fixation and paraffin section. We found that both TREK-1 and TREK-2 immunohistochemical reactivities were observed as bright punctate granules in most atrial and ventricle cardiac myocytes. These are distributed mainly in the cytoplasm and rarely in the center of the nuclear region. Both TREK-1 and TREK-2 immunoreactive granules were found along the surface of cardiac myocyte membranes and the membrane of T-tubules in the cytoplasm (n=4). No connective tissue was labelled. No immunoreactivity was found in negative control preparations after omission of primary antibody. This study demonstrated that TREK-1 and TREK-2 channels are present in human cardiac myocytes. They may play an important role in regulation of human heart electrical behaviour under physiological and diseased conditions.

Proceedings of the Australian Physiological Society

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More than one type of stretch activated channel contributes to the action potential duration in guinea pig L. Mackenzie, D.R. Kelly and D.A. Saint, Department of Physiology, School of Molecular and Biomedical Science, The University of Adelaide, SA 5000, Australia. Previously, we provided evidence of the differential involvement of stretch activated channels in the shaping of the action potential through the left ventricular wall of the rat heart. We postulated a role for these channels in the control of dispersion of repolarisation. In this study we aimed to establish if this phenomenon holds for a different species, the guinea pig and also to dissect the contributions of non-selective cation channels (SACs) and the family of stretch activated, 2 pore 4 transmembrane domain, potassium channels (TREK). We used Langendorff perfused guinea pig heart preparations (N=6) and recorded monophasic action potentials (MAP) simultaneously from the epicardium and the endocardium of the left ventricular free wall under left ventricular end diastolic pressures of 0-5 mmHg (no stretch), 20-25 mmHg (moderate stretch) and 50-55 mmHg (elevated stretch). Hearts were perfused with Hepes buffered Tyrode’s solution (pH 7.35-7.37) at 37°C bubbled with oxygen and paced at 4 Hz. This was repeated in the presence of the reported SAC channel blocker streptomycin (80µM). Action potential durations (APDs) at 20, 50 and 80% repolarisation were measured and analysed using general linear model ANOVA followed post hoc by Tukey’s pairwise comparisons. APDs were unchanged compared to control following both moderate and elevated stretch in both the epiand endocardial layers when measured at 20 and 50% repolarisation. At APD80 there was a slight increase in duration although this was not statistically significant. Following blockade of SACs by streptomycin there was a decrease in APDs for 20, 50 and 80% of repolarisation which was significant for both levels of stretch at APD80. These results suggest that both SACs and the family of stretch activated, 2 pore 4 transmembrane domain, potassium channels which we interpret to be TREK contribute to the shaping of the action potential in the guinea pig. We further suggest that the guinea pig may be more susceptible to stretch than the rat as the decrease attributed to TREK was already maximal at 20-25mmHg (moderate stretch).

Proceedings of the Australian Physiological Society

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Fatty acid composition of red blood cell membranes as a marker of human heart membrane phospholipid fatty acids Mandy L Theiss1, Salvatore Pepe2 and Peter L. McLennan1, 1Smart Foods Centre, Department of Biomedical Science, University of Wollongong, Wollongong, NSW 2522, Australia and 2Cardiac Surgical Research Lab, Alfred Hospital & Baker Medical Research Institute, Prahran, VIC 3181, Australia. Background. Regular intakes of fish or fish oil are associated with low cardiovascular disease morbidity and mortality. A major effect is in reducing sudden cardiac death (Marchioli et al., 2002). Studies using animals, suggest that the long-chain omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have antiarrhythmic and other cardiac effects which are dependent on their incorporation into myocardial membranes (Pepe & McLennan, 1996). The red blood cell (RBC) EPA+DHA content correlates inversely with adverse cardiovascular outcomes and is proposed as a new cardiovascular disease risk factor (Omega-3 Index) (Harris & von Schacky, 2004) on the premise that it reflects the composition of cardiac cells. Animal studies indicate large differences in membrane fatty acid compositions of different tissues. Objective. To characterise the membrane phospholipid fatty acid composition of human RBC in relation to heart. Design. Membrane phospholipid fatty acids were extracted from atrial biopsy samples and red blood cells, obtained from cardiac surgery patients (n=10). Mixed venous blood samples were obtained preoperatively. Biopsy samples were taken from an atrial appendage during open chest surgery. Phospholipid fatty acids were determined by gas chromatography against known standards. Outcomes. Polyunsaturated fatty acid (PUFA) content of atrial cell membranes was higher (51.13 + 0.75%, values are means + SE) than RBC (34.88 + 0.39%), with PUFA replacing saturated fatty acids. The levels of omega-6 PUFA linoleic acid (LA, 18:2 n-6) 18.89±1.01% and arachidonic acid (AA, 20:4 n-6) 21.32±0.61% were higher in atria than RBC (LA, 6.79±0.34% and AA, 13.96±0.64%). In both atria and RBC, DHA was the major omega-3 fatty acid. Both total omega-3 PUFA and DHA in the atria was highly correlated with RBC EPA+DHA (9.60 + 1.14% (range 4.71-11.45%)). Some patients were supplemented with fish oil prior to surgery and had correspondingly higher omega-3 content in both RBC and atria. Conclusion. The long-chain omega-3 fatty acids EPA and DHA, found in high amounts in fish oil, represent a marker of human atrial omega-3 fatty acid composition and the Omega-3 Index in red blood cells may be a valid marker for human heart composition. Marchioli, R., Barzi, F., Bomba, E., Chieffo, C., Di Gregorio, D., Di Mascio, R., Franzosi, M.G., Geraci, E., Levantesi, G., Maggioni, A.P., Mantini, L., Marfisi, R.M., Mastrogiuseppe, G., Mininni, N., Nicolosi, G.L., Santini, M., Schweiger, C., Tavazzi, L., Tognoni, G., Tucci, C. & Valagussa, F. (2002) Circulation, 105, 1897-1903. Pepe, S. & McLennan, P.L. (1996) Journal of Nutrition, 126, 34-42. Harris, W.S. & von Schacky, C. (2004) Preventative Medicine, 39, 212-220.

Proceedings of the Australian Physiological Society

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Confocal Ca2+ imaging of mouse sinoatrial node Y.K. Ju1, D.G. Allen1 and M.B. Cannell2, 1School of Medical Sciences, University of Sydney, NSW 2006, Australia and 2The Faculty of medical and Health Sciences, University of Auckland, Auckland, New Zealand. Recent studies have demonstrated that intracellular Ca2+ plays an important role in cardiac pacemaking (Ju & Allen, 2001). However, the mammalian sinoatrial node (SAN) is a heterogeneous structure and studies on the centre leading pacemaker region suggest that central pacemaker cells do not require the sarcoplasmic reticulum (SR) calcium release for spontaneous activity (Lancaster et al., 2004). In order to study the Ca2+ dependent pacemaker mechanisms in different regions of mammalian preparation, we developed a new technique to image Ca2+ from intact mouse sino-atrial preparations. Mice (7 -10 weeks) were deeply anaesthetized. The right atrium was opened under a dissecting microscope to expose the crista terminalis, the intercaval area and the interatrial septum as described by Verheijck et al. (2001). The preparation was pinned into a sylgard block with a 3 ´ 5mm open window that allowed microscopic imaging. The SAN preparation was loaded with the fluorescent Ca2+ indicator Fluo-4 AM (10 µM) by incubation in Tyrode solution at 4°C for 5 h. The SAN area was recognized by its anatomic landmarks. 10 mM 2,3–butanedione monoxime (BDM) was used to reduce the motion artifact. The loading of dye into the central region normally was weaker than for peripheral regions for reasons that are unclear. It is known that Na+ channels are required for peripheral but not for the central pacemaker activity. Therefore 100 µM lidocaine was added to the perfusate to block any pacemaker type activity from the periphery of the SAN. Spontaneous Ca2+ signals from the central SAN were imaged using a confocal microscope (LSM410) in XY and XT modes. We found that the SAN Ca2+ signal was modulated by the SR Ca2+ release channel modulator caffeine and by ATP. These results show that it is possible to record Ca2+ signals from the centre of leading pacemaker region in mouse heart. Ju, Y.K. & Allen D.G. (2001) Clinical and Experimental Pharmacology and Physiology 28, 703-8. Lancaster, M.K., Jones, S.A., Harrison, S.M. & Boyett, M.R. (2004) Journal of Physiology 556, 481-494. Verheijck, E.E., van Kempen, M.J.A., Veereschild, M., Lurvink, J., Jongsma, H.J. & Bouman, L.N. (2001) Cardiovascular Research 52, 40-50. This work is supported by NH&MRC Australia & HRC New Zealand

Proceedings of the Australian Physiological Society

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Reactive oxygen species generated from the mitochondria and not NAD(P)H-oxidase regulate Ltype Ca2+ channel function during acute hypoxia in ventricular myocytes L.C. Hool, H.M. Viola, C.A. Di Maria and P.G. Arthur, School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, WA 6009, Australia. Hypoxia and the thiol-reducing agent dithiothreitol increase the sensitivity of the L-type Ca2+ channel (ICa-L) to b -adrenergic receptor stimulation. We examined whether NAD(P)H-oxidase regulates cellular production of reactive oxygen species (ROS) and the function of ICa-L during hypoxia. Ventricular myocytes were isolated from hearts excised from anaesthetised guinea-pigs as approved by the Animal Ethics Committee of The University of Western Australia and in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (NHMRC). The cells were patch-clamped and current was recorded during exposure to the classic NAD(P)H-oxidase inhibitors apocynin or diphenyleneiodonium (DPI) and increasing concentrations of isoproterenol (Iso). DPI caused an increase in the sensitivity of the channel to Iso similar to that of hypoxia, but apocynin did not. In contrast, the K0.5 for activation of the channel by Iso in the presence of AngII, a potent agonist of NAD(P)H-oxidase during hypoxia was 1.7±0.4 nM which was similar to the K0.5 determined during hypoxia alone (1.6±1.1 nM; NS). We measured cellular production of superoxide anion (O2−) using the fluorescent indicator dihydroethidium. Hypoxia was associated with a 41.2±5.2 % decrease in O2− (n=21; P<0.05). In addition, DPI caused a 21.3±4.7 % decrease in O2− (n=16; P<0.05). However, O2− did not increase when cells were exposed to AngII during hypoxia (n=24) or in room oxygen (n=6). When mitochondria were partially uncoupled with FCCP, there was a 31.3±4.5% decrease in O2− (n=23; P<0.05) and a significant increase in the sensitivity of ICa-L to Iso similar to that of hypoxia (n=7). Accounting for the effect of DPI on ICa-L, 10m M DPI caused a 67.2±7.3% decrease in oxygen consumption (n=6; P<0.01) and 28.8±2.1% decrease in O2− in isolated mitochondria (n=4; P<0.05) indicating that DPI is not a specific inhibitor of NAD(P)H-oxidase function. Hypoxia decreased O2− by 69.3±0.8% in isolated mitochondria (n=4; P<0.01). We conclude that a decrease in ROS generated from the mitochondria and not NAD(P)H-oxidase regulates channel function during hypoxia.

Proceedings of the Australian Physiological Society

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Eccentric damage is accentuated in aged dystrophin-deficient EDL muscles from dystrophic mice (MDX) S. Chan and S.I. Head , School of Medical Sciences, UNSW, NSW 2052, Australia. Duchenne Muscular Dystrophy (DMD) is second most common fatal inherited condition of humans affecting 1 in 3500 live male births. Due to the large size and high mutation rate of the dystrophin gene, 1/3 of all cases of DMD are the result of a new spontaneous mutation. DMD is characterized by a severe and progressive loss of skeletal muscle and marked CNS and cognitive defects. Death usually occurs in the late teens or early twenties due to the failure of respiratory muscles or cardiac complications. The most commonly used animal model of DMD is the mdx mouse in which the long M-isoform of dystrophin is absent from the skeletal musculature. In the mdx mouse the proximal limb muscles undergo a process of degeneration which affects 100% of the skeletal muscle fibres in some muscles (Tanabe et al., 1986), the fibres then undergo a process of regeneration and repair. During this period there is a striking change in the morphology of the regenerated mdx EDL fibres; up until 15 weeks they are normal cylindrical shaped with no splits or deformities, by 17 weeks 30% of fibres have simple splits and relatively mild deformities, while by 40 weeks in excess of 90% of the EDL fibres have multiple splits and quite striking gross abnormalities (Head et al., 1992). We are hypothesizing that these split, morphologically abnormal fibres are weaker and more susceptible to damage by lengthening contractions of a moderate strain than age matched morphologically normal fibres. We used male mdx and male littermate controls from our new line of mdx mice (N1F1 mdx mice). This provides us with dystrophin-positive controls on an identical genetic background to the dystrophin-deficient animals. This is important because the majority of studies on mdx mice use a separate wild type colony as a control and it has been shown that there is a remarkable degree of genetic variation between recently divergent mouse strains (Adams et al., 2005). Animals were killed by an overdose of Halothane (ethics approval granted by UNSW). The EDL muscles were attached at one end to a force transducer and to a servo controlled linear tissue puller at the other end. The muscles were then placed in an organ bath, superfused with oxygenated Krebs and externally stimulated via two platinum plates attached to a current amplifier driven by an AM-systems stimulator. The muscles were set to optimal length Lo. The muscles were tetanically stimulated at 100hz, and once the force reached its maximal plateau the lengthening contraction (12, 15 or 20% plus Lo) was given as a ramp, hold and release. The ramp speed was 1mm/sec and the total duration of stimulation was 5 seconds. This protocol was repeated twice at 5 minute intervals to allow recovery from fatigue. The isometric force was recorded after a 20 minute recovery. The muscles were then removed, weighed and single fibres enzymatically (collagenase 1) isolated and viewed on a confocal microscope in order to count the number of split fibres. In aged (45-52 weeks) mdx animals, 100% of fibres were split and there was an irreversible 35% ± 9.8 (n=6) drop in isometric force as a result of our lengthening contraction protocol. In contrast, there were no split or deformed fibres in age matched controls, and isometric force was largely unaffected (force drop 3.8%± 4.8% n=5) by this relatively mild eccentric contraction protocol. Numerous previous studies (e.g. Yeung et al., 2003) have demonstrated that dystrophin-deficient fast-twitch muscles are damaged by eccentric contractions; we hypothesise that the extent of the eccentric damage is significantly increased by the presence of deformed fibres. Adams, D.J., Dermitzakis, E.T., Cox, T., Smith, J., Davies, R., Banerjee, R., Bonfield, J., Mullikin, J.C., Chung, Y.J., Rogers, J. & Bradley A. (2005) Nature Genetics 37, 532-6. Head, S.I., Williams, D.A. & Stephenson, D.G. (1992) Proceedings of the Royal Society (London) B 248, 163-169. Tanabe, Y., Esaki, K. & Numura, T. (1986) Acta Neuropathologica 69, 91-95. Yeung, E., Head, S.I. & Allen, D.G. (2003) Journal of Physiology (London) 552(2), 449-458.

Proceedings of the Australian Physiological Society

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Phosphorylation of CSQ affects Ca2+ binding and interactions with anchoring protein junctin N.A. Beard1, S. Cheung1, L. Wei1, M. Varsànyi2 and A.F. Dulhunty1, 1John Curtin School of Medical Research, ANU, Canberra, ACT 0200, Australia and 2Institut für Physiologische Chemie, Ruhr Universität, Bochum, Germany. Depolarisation of the sarcolemma triggers Ca2+ release through ryanodine receptor (RyR) calcium release channels in the sarcoplasmic reticulum (SR) of skeletal muscle. Calsequestrin (CSQ) is the major Ca2+ binding protein found within the SR, and binds Ca2+ with a high capacity and moderate affinity. Recent studies have shown that CSQ also regulates RyRs. The best studied mechanism of CSQ-RyR interaction is indirect, thought to be mediated by anchoring proteins triadin and junctin and results in RyR inhibition (Szegedi et al., 1999; Beard et al., 2002). The relative importance of triadin and junctin in facilitating the interaction with the RyR is not clear, nor is the role in vivo of CSQ phosphorylation on the interaction between triadin, junctin and the RyR. Given that CSQ is isolated in both a phosphorylated and dephosphorylated form, it is conceivable that a CSQ phosphorylation/dephosphorylation cycle is important in regulating the RyR. Our hypothesis is that as the site of CSQ phosphorylation is believed to be close to the putative Ca2+ binding site, and triadin and junctin binding sites, that changes in phosphorylation may modify Ca2+ binding capacity and the ability of CSQ to interact with triadin and/or junctin. We have investigated the effects of CSQ phosphorylation on CSQs role as a Ca2+ binding protein and regulator of the native RyR. Rabbit skeletal CSQ cDNA was subcloned into a pGex 5X-1 vector (containing a glutathione-S-transferase tag), transformed and expressed in Escherichia coli BL21(DE3) cells. CSQ was phosphorylated and dephosphorylated according to established methods (Cala & Jones, 1991). We found that the Ca2+ binding capacity was significantly reduced in dephosphorylated CSQ (deP-CSQ), compared with phosphorylated CSQ (P-CSQ) over a range of [Ca2+] from 100 nm – 5 mM. This was most evident at the physiological free [Ca2+] (1 mM) and at 5 mM Ca2+ (a concentration shown to dissociate CSQ from the native RyR). Despite these changes in binding capacity, both P-CSQ and deP-CSQ caused similar significant inhibition of the native RyR (at 1 mM luminal Ca2+; Beard et al., 2005). As the putative major Ca2+ binding region and site of CSQs interaction with triadin and junctin are presumed identical (residues 354-367), we investigated what effects P-CSQ/deP-CSQ had on its interactions with triadin and junctin. Using CSQ-GST fusion protein affinity chromatography, we found that under close to physiological conditions (150 mM NaCl, 1 mM Ca2+), both P-CSQ and deP-CSQ bound triadin and junctin. Not surprisingly, under conditions known to completely dissociate CSQ from the native RyR (5 mM Ca2+), neither P-CSQ nor deP-CSQ interacted with a significant amount of triadin or junctin. Curiously, at low luminal Ca2+ (100 nM), CSQ binding to these two anchoring proteins was phosphorylation-dependent. P-CSQ bound significant amounts of both triadin and junctin, whilst deP-CSQ was found only to interact with triadin, but not with junctin. In experiments where 100 nM luminal Ca2+ was used to depolymerise P-CSQ and deP-CSQ from a solubilized SR sample, a significant proportion of both forms of CSQ remained tethered close to the RyR/triadin/junctin complex. The combination of these results suggests that an interaction with junctin is not required to tether CSQ close to the native RyR. These results show firstly, that CSQ dephosphorylation reduces the ability of CSQ to bind Ca2+. Although this does not affect overall CSQ regulation of the RyR, subtle effects of altering CSQs phosphorylation status on channel gating, or CSQs ability to act as a luminal Ca2+ sensor remain to be investigated. Secondly, these results illustrate that triadin, but not junctin, is essential for association of CSQ with the native RyR. Further investigation on whether the alteration in junctin binding results in altered regulatory effects on the native RyR may elucidate a specific role of junctin and the potential P-CSQ/deP-CSQ cycle on SR Ca2+ release. Thirdly, the results show that conformational changes that alter Ca2+ binding capacity do not necessarily alter CSQ binding to triadin and junctin and therefore the specific residues which comprise both the major Ca2+ binding site and triadin/junctin binding sites are not identical. Beard, N.A., Casarotto, M.G., Wei, L., Varsányi, M., Laver, D.R. & Dulhunty, A.F. (2005) Biophysical Journal 88, 3444-3454. Beard, N.A., Sakowska, M.M., Dulhunty, A.F. & Laver, D.R. (2002) Biophysical Journal 82, 310-320. Cala, S.E. & Jones, L.R. (1991) Journal of Biological Chemistry 266, 391-398. Szegedi, C., Sarkozi, S., Herzog, A., Jona, I. & Varsanyi, M. (1999) Biochemical Journal 337, 19-22.

Proceedings of the Australian Physiological Society

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A calsequestrin polymer is necessary for the Ca2+ binding protein to regulate RyR channels L. Wei, N.A. Beard and A.F. Dulhunty, John Curtin School of Medical Research, Australian National University, Canberra, ACT 0200, Australia. Calsequestrin (CSQ) forms a complex with the Ca2+ release channel ryanodine receptor (RyR) and anchoring proteins, triadin (Tri), and junctin (Jun) in the lumen of the sarcoplasmic reticulum (SR) of skeletal and cardiac muscles. CSQ acts to both regulate the RyR, and buffer the [Ca2+]free inside the SR at 1 mM (Beard et al., 2004). CSQ requires a compact structural conformation to achieve high capacity Ca2+ binding (He et al., 1993). CSQ is thought to undergo a self-polymerisation as local [Ca2+] increases, and is believed to form a polymer at the physiological [Ca2+] (1 mM) in the SR. Our aim was to determine whether the CSQ polymer and its regulatory interaction within the complex would be disrupted and whether CSQ would be dissociated from the quaternary complex, when the intraluminal [Ca2+] falls to a low level at which CSQ is thought to be depolymerised. To achieve this, we examined the effects of low luminal Ca2+ on CSQs ability to regulate the RyR and correlated these effects with known Ca2+-dependent changes in CSQ structure. New Zealand male white rabbit were euthanized by a captive bolt and back and leg muscle used to prepare heavy SR vesicles. Heavy SR vesicles were reconstituted into artificial planar lipid bilayers, which separate two chambers, cis (cytoplasmic) and trans (luminal). Trans [Ca2+] was kept at 1 mM during incorporation and then lowered to 100 nM by the addition of BAPTA or EGTA. Channel activity was recorded at positive and negative potentials. To determine the effect of [Ca2+] on CSQ association with the RyR/Tri/Jun complex, SR vesicles were solubilised in 0.5% triton X-100, followed by an ultracentrifugation and resuspended in a solution containing 1 mM Ca2+. The suspension was divided to three fractions and incubated at 4°C for 1 h in 1 mM, 1 m M and 100 nM Ca2+ after adjustment of [Ca2+] by BAPTA, prior to a second centrifugation. Resultant pellets and supernatants were subjected to SDS-PAGE and immunoblot analysis. In single channel studies, a sudden delayed increase in activity was observed after the native RyR was exposed to low luminal Ca2+ (100 nM) for ∼3 min. Returning trans [Ca2+] to 1 mM did not fully reverse the increase in activity to control levels, but addition of 16 µg/ml of exogenous CSQ to the trans chamber completely restored control channel activity. These changes were similar to those seen with dissociation of CSQ from the RyR/Tri/Jun, with high luminal ionic strength or Ca2+ (Beard et al., 2004), but a longer exposure time was required before the sudden increase in activity was observed. The levels of CSQ in the membrane pellet of solubilised junctional face membrane (containing the RyR/Tri/Jun/CSQ complex) exposed to different [Ca2+], were compared with levels found in the original membrane fractions, to determine if CSQ was dissociated from the membrane. Increasing amounts of CSQ were dissociated from the membrane pellet as [Ca2+] fell, with 8.6%, 35.8% and 63% of the total CSQ dissociated by 1 mM, 1µM and 100 nM Ca2+, respectively. This is in contrast to ∼100% dissociation with high ionic strength or high Ca2+. Additional single channel experiments showed that the response of RyRs associated with depolymerised CSQ to changes in luminal Ca2+ between 100 nM to 1 mM was typical of that seen in channels in which CSQ was fully dissociated. Since CSQ is depolymerised with low [Ca2+] but retains its ability to bind to triadin and junctin (Shin et al., 2000), we suggest that the CSQ remaining associated with the junctional face membrane was bound to triadin and junctin and that the dissociated CSQ was depolymerised and dissociated from the residual bound CSQ. Channel data suggests that the delayed RyR activation reflects depolymerisation and dissociation of CSQ not bound to triadin and junctin. The longer delay before the sudden increase in channel activity after exposure to low Ca2+ suggest a different physical process to the dissociation from triadin and junctin seen with high ionic strength and high Ca2+. The results further suggest that the residual CSQ remaining associated with the RyR/Tri/Jun complex after depolymerisation was unable to regulate RyR activity in the normal manner until it formed a polymer with the excess exogenous CSQ. Therefore we concluded that a CSQ polymer is required for the calcium binding protein to regulate the RyR via triadin and junctin. Beard, N.A., Laver D.R. & Dulhunty, A.F. (2004) Progress in Biophysics and Molecular Biology 155, 33-69. He, Z., Dunker, A.K., Wesson C.R. & Trumble, W.R. (1993) Journal of Biological Chemistry 268, 24635-24641. Shin, D.W., Ma, J. & Kim D.H. (2000) FEBS Letters 486, 178-182.

Proceedings of the Australian Physiological Society

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Digoxin and exercise effects on Na+,K+-pump activity, content, isoform gene and protein expression in human skeletal muscle X. Gong1, A. Petersen1, S. Sostaric1, C. Goodman1, D. Cameron-Smith2, R. Snow2, K. Murphy1, K. Carey2, J. Aw3, H. Krum3 and M. McKenna1, 1School of Human Movement, Recreation and Performance, Centre for Ageing, Rehabilitation, Exercise and Sport, Victoria University, Melbourne, VIC 8001, Australia, 2School of Exercise Science and Nutrition, Deakin University, Melbourne, VIC 3125, Australia and 3Department of Epidemiology and Preventive Medicine, Monash University, Alfred Hospital, Melbourne, VIC, Australia. Digoxin is a specific inhibitor of the Na+,K+-pump and is used to treat patients with severe heart failure. In these patients, digoxin binds and blocks ∼13% of Na+,K+-pumps in skeletal muscle and exacerbates muscle K+ loss during exercise. Furthermore in heart failure patients there is no compensatory upregulation of Na+,K+-pump with chronic digitalisation. We have shown that exercise impairs Na+,K+-pump activity, whilst in isolated rat muscles, Na+,K+-pump inhibition leads to early muscle fatigue (Clausen, 2003). Hence, Na+,K+-pump function is likely to be important for skeletal muscle performance. However, the effects of digoxin on Na+,K+-pump content, activity, protein abundance or isoform expression in skeletal muscle of healthy individuals are unknown and were investigated here. Ten active, but not well-trained healthy volunteers (9 M, 1 F) gave written informed consent. Exercise tests were performed after taking digoxin (DIG, 0.25 mg.d-1) or a placebo (CON) for 14 d, in a randomised, counterbalanced, cross-over, double blind design, with trials separated by 4 weeks. Subjects performed incremental cycle ergometer exercise to measure VO2peak and to determine 33, 67 and 90% VO2peak work rates. On d 14 subjects completed 10 min cycling at each of 33% and 67% VO2peak, then to fatigue at 90% VO2peak. Muscle biopsies taken at rest, after 67%, 90%VO2 peak and 3 h recovery were analysed for Na+,K+-pump content (3H-ouabain binding site), maximal activity (3-O-methyfluorescein phosphatase, 3-O-MFPase), isoform protein abundance and mRNA expression. The Na+, K+-pump isoform (α1-α3, b 1-b 3) protein contents were measured on muscle extracts, using specific antibodies and western blotting, with isoform mRNA expression determined with real-time RT-PCR analysis. Serum digoxin was 0.7±0.2 nM at d 13 and 0.8±0.2 nM at d 14 (Mean±SD). Despite this, muscle maximal + Na ,K+-pump activity was unchanged by digoxin. However, Na+,K+-pump activity was decreased after exercise, by 13% and 11% at fatigue and 3 h post-exercise, compared to rest, respectively (P<0.05). Furthermore, there was no change in the Na+,K+-pump content with either digoxin or exercise (Rest Digoxin 373±95, Rest Placebo 368±75 pmol.g wet weight-1). No significant change occurred with digoxin for mRNA expression of any of the α1, α2, α3, b 1, or b 3 isoforms. However, digoxin increased the mRNA expression of the total α mRNA (sum of α1, α2, α3) and the total b mRNA (sum of b 1 and b 3) at rest by 1.9- and 1.8-fold, respectively (P<0.05), suggesting an effect of digoxin on Na+,K+-pump gene expression. An exercise effect was observed on α3 mRNA expression, being 2.1-and 2.4-fold higher at 3 h post-exercise than during exercise at 67% VO2peak and fatigue, respectively (P<0.05). Similarly, b 3 mRNA expression at 3 h post-exercise was increased by 1.8-, 1.4and 1.6-fold, compared to rest, 67% VO2peak exercise and fatigue, respectively (P<0.05). Digoxin did not alter the protein abundance of any isoform in resting muscle. However, at 3 h post-exercise, the protein abundance was greater with digoxin than in placebo for both α2 and b 3 (P<0.05). The b 1 protein expression was increased at 3 h post-exercise by 2.2-and 1.5-fold compared to during exercise at 67% VO2peak and fatigue, respectively (P<0.05). Similarly, b 3 protein expression was increased at 67% VO2peak and 3 h post-exercise compared to rest, by 1.5-and 1.6-fold, respectively (P<0.05). In summary, digoxin treatment had only minimal effects on muscle Na+,K+-pumps in healthy individuals. Whilst Na+,K+-pump content, activity or isoform protein expression at rest were unchanged, the subunit total mRNA expression was increased with digoxin and a greater post-exercise protein abundance was found with digoxin for α2 and b 3. The lack of reduction in pumps with digitalisation in healthy muscles suggests either that pumps were upregulated and/or that digoxin dissociation was increased. Clausen, T. (2003) Physiolgical Reviews 83, 1269-324. Funded by NH&MRC

Proceedings of the Australian Physiological Society

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Reduced long-term depression is recovered in aging mdx cerebellar Purkinje cells J.L. Anderson, S.I. Head and J.W. Morley, School of Medical Sciences, UNSW, NSW 2052, Australia. Duchenne muscular dystrophy (DMD) is characterized primarily by a loss of skeletal muscle and marked CNS and cognitive defects (Anderson et al,, 2002). It is known that DMD is due to the mutation of a gene which produces the protein dystrophin, of which there are seven identified isoforms, expressed in a range of tissues including brain. In the cerebellum an isoform of dystrophin is found exclusively in Purkinje neurons and is selectively localised in post-synaptic regions of GABAergic synapses. We have previously demonstrated (Anderson et al,, 2004) a deficit in long-term depression (LTD) in cerebellar Purkinje cells in the mdx mouse (an animal model of DMD) compared to controls at 3 months of age. mdx mice. --> In the present study we investigated LTD and short-term plasticity at the parallel fibre to Purkinje cell synapse in cerebellar brain slices from ageing (6-12 months) control and mdx mice. Mice were anaesthetised with halothane, decapitated, cerebellum removed, bisected, placed in ice-cold aCSF and sagittal slices (250m M) cut. Individual Purkinje cells were visualised using a 40´ immersion lens and IR-DIC optics. Intracellular electrodes (∼120MW ) filled with potassium acetate were used and a stimulating electrode was placed in the molecular layer of the slice (<250m M from the cell under study). We found that the deficit in LTD which we reported (Anderson et al,, 2004) in mdx mice at 3 months of age was no longer evident in aging mdx mice, and that these cells showed a long lasting and robust LTD. In addition, there were no differences in short-term plasticity between the ageing control and mdx mice. Anderson, J.L., Head, S.I., Rae, C. & Morley, J.W. (2002) Brain 125, 4-13. Anderson, J.L., Head. S.I. & Morley, J.W. (2004) Brain Research 1019, 289-92.

Proceedings of the Australian Physiological Society

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Differential action of w -conotoxins CVID and CVIB on voltage-gated calcium channels in rat sensory neurons L.M. Motin, R.J. Lewis and D.J. Adams, School of Biomedical Sciences, University of Queensland, Brisbane, QLD 4072, Australia. Selective antagonists of voltage-gated calcium channels (VGCCs) are of considerable interest both as research tools and potential therapeutic agents. The selectivity of VGCC antagonists is essential for dissecting the various Ca2+ channel types underlying the whole-cell Ca2+ current whereas potency and reversibility play an important role in the use of a VGCC antagonist as a pharmaceutical agent. w -Conotoxins GVIA, MVIIA and MVIIC have been used routinely as selective blockers of N- and P/Q-types of VGCCs in excitable cells. However, the newly discovered w -conotoxins from Conus catus, CVID has been shown to have the highest selectivity for N-type over P/Q-type VGCCs among the other N-type selective VGCC antagonists (Lewis et al., 2000). The present study investigated the selectivity, potency and reversibility of action of w -conotoxins CVID and CVIB in isolated sensory neurons dissociated from rat dorsal root ganglia (DRG) and on recombinant VGCCs expressed in Xenopus oocytes. Bath application of either CVID or CVIB inhibited depolarizationactivated whole cell Ba2+ currents in DRG neurons with pIC50 values of -8.12 ± 0.05 and -7.64 ± 0.08, respectively. The block of Ba2+ currents in DRG neurons by CVID appeared to be irreversible after >30 min washout whereas Ba2+ currents exhibited rapid recovery from block by CVIB (>80% within 3 min). w -Conotoxin CVIB inhibited more of the whole-cell Ba2+ current in DRG neurons than CVID and the recoverable component of the Ba2+ current inhibited by CVIB was mediated by the N-type VGCC. The potency of CVID and CVIB block of N- and P/Q-type VGCCs was compared with the w -conotoxins, GVIA, MVIIA and MVIIC. w -Conotoxins GVIA and MVIIA inhibited Ba2+ currents in DRG neurons to a similar degree as CVID. The residual current amplitude obtained in the presence of maximally effective concentrations of the w -conotoxins was: GVIA, 54 ± 0.2%; MVIIA, 41 ± 0.04% and CVID, 34 ± 1%. The residual current after block by CVIB was 3 ± 5% of control level reflecting non-selective N- and P/Q- action of the toxin. w -Conotoxin CVIB reversibly inhibited Ba2+ currents mediated by N- (Cav2.2) and P/Q- (Cav2.1) type VGCCs expressed in Xenopus oocytes. The α2d 1 auxiliary subunit coexpressed with Can 2.2 and Can 2.1 reduced the potency of CVIB as reported previously for CVID at recombinant N-type VGCCs (Mould et al., 2004). The present study demonstrates that w -conotoxins CVID and CVIB can be successfully used for pharmacological isolation of N- and P/Q- components of the Ca2+ conductance in rat DRG neurons. CVID selectively and irreversibly blocked the N-type component of the whole cell Ba2+ current in DRG neurons but blocked reversibly the recombinant N-type (Cav2.2) VGCC. In contrast, CVIB blocked reversibly the N-type component and blocked irreversibly P/Q- component of the whole cell Ba2+ current in DRG neurons. w -Conotoxins CVID and CVIB may be useful as antagonists of N- and P/Q-type VGCCs in sensory neurons involved in processing primary nociceptive information. Lewis, R.J., Nielsen, K.J., Craik, D.J., Loughnan, M.L., Adams, D.A., Sharpe, I.A., Luchian T., Adams, D.J., Bond, T., Thomas, L., Jones, A., Matheson, J-L., Drinkwater, R., Andrews, P.R. & Alewood, P.F. (2000) Journal of Biological Chemistry 275, 35335–35344. Mould, J., Yasuda, T., Schroeder, C.I., Beedle, A.M., Doering, C.J., Zamponi, G.W., Adams, D.J. and Lewis, R.J. (2004) Journal of Biological Chemistry 279, 34705-34714.

Proceedings of the Australian Physiological Society

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Evidence from collision experiments that onset chopper neurons in the guinea pig cochlear nucleus receive excitatory input from centrifugal collaterals D. Robertson and W.H.A.M. Mulders, The Auditory Laboratory, Discipline of Physiology, School of Biomedical Biomolecular and Chemical Sciences, The University of Western Australia, Crawley, Western Australia 6009, Australia. In mammals, centrifugal pathways from the superior olivary complex to the cochlea, send collateral projections to the first brainstem nucleus, the cochlear nucleus. The action of these collateral pathways needs to be taken into account in any attempt to understand the role of the centrifugal olivocochlear system in auditory processing. To date however there is only incomplete understanding of the neuronal targets of the collaterals and of their synaptic effects (Benson & Brown, 1990; Mulders et al., 2003; Mulders et al., 2002). We have observed in guinea pigs that electrical stimulation at the floor of the IVth ventricle (the site of passage of the centrifugal axons as they ascend from the superior olivary complex), gives rise to short-latency action potentials in well-characterized onset chopper neurons. The nature of these electrically-evoked spikes however, is unclear, since they show an ability to follow quite high rates of electrical stimulation, perhaps consistent not with an excitatory synaptic drive from centrifugal collaterals, but rather, antidromic spikes initiated in the dorsallyascending axons of the onset chopper neurons. We set out to distinguish between these possibilities by using classical collision techniques to distinguish antidromic from synaptically-driven action potentials. The experiments were performed in guinea pigs anaesthetized with intraperitoneal sodium pentobarbitone (30mg/kg) and 0.15ml intramuscular Hypnorm (fluanisone, 10mg/ml and fentanyl citrate, 0.315mg/ml). During electrical stimulation, the animals were paralyzed by intramuscular administration of Pancuronium. Heart rate was continuusly monitored and regular supplementary doses of both anaesthetics were given throughout the experiment. Data from a small number of well-characterized onset chopper neurons, showed that action potential collision only occurred at delays that were either the same as, or shorter than the delay between shocks and shock-evoked spikes in the same neurons. This result is inconsistent with the electrically-evoked spikes being antidromic and strongly suggests that the axon collaterals of olivocochlear neurons exert excitatory synaptic effects on onset chopper neurons. Furthermore, the robust nature of the spiking to high rates of shocks suggests that the synaptic connection is a powerful one, giving rise to strong and comparatively secure excitation of this class of cochlear nucleus neurons. Benson, T.E. & Brown M.C. (1990) Journal of Comparative Neurology 295: 52-70. Mulders, W.H., Paolini, A.G., Needham, K. & Robertson, D. (2003) Hearing Research 176: 113-121. Mulders, W.H., Winter I.M. & Robertson, D. (2002) Hearing Research 174: 264-280.

Proceedings of the Australian Physiological Society

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P2Y receptor activation inhibits the formation and proliferation of primary mouse subventricular-derived neurospheres M.R. Stafford, P.F. Bartlett and D.J. Adams, School of Biomedical Sciences and The Queensland Brain Institute, University of Queensland, Brisbane QLD 4072 Australia. Purinergic receptors mediate a variety of biological effects in response to extracellular nucleotides. In the brain, astrocytes and neurovascular endothelial cells release nucleotides such as ATP and may have regulatory roles in the stem cell micro-environment. Extracellular ATP also mediates Ca2+ wave propagation between astrocytes. Further, a correlation between Ca2+ wave intensity and cortical neuronal production in the ventricular zone of the embryo suggests a potential role for extracellular ATP and Ca2+ waves in early neurogenesis. In the present study we show that (i) sub-ventricular zone (SVZ) stem cells express purinergic receptors, (ii) ATP evokes intracellular Ca2+ transients in SVZ-derived neurospheres, and (iii) purinergic agonists can affect primary neurosphere formation and proliferation. HSAloPNAlo stem cells were purified from the SVZ of adult mice using flow cytometry. The stem cell population expressed mRNA for P2Y1, 2, 6, 12 and 14 receptor subtypes. In primary neurospheres loaded with Fura-2AM, ATPg S (1-30 m M) and ADPb S (1-30 m M) evoked Ca2+ transients in the presence and absence of external Ca2+. Transients were reversibly attenuated by PPADS (20 m M) and completely abolished by the P2Y1 antagonist MRS2179 (30 m M), suggesting the presence of functional metabotropic P2Y1 receptors. To study purinergic effects on sphere formation, single cell suspensions derived from primary SVZ tissue were treated with purinergic agonists/antagonists and grown under sphere forming conditions. ATPg S and ADPb S (10-30 m M) but not UTP, UDP, UDP-glucose or αb methylene ATP (100 m M) reduced both the size and frequency of primary neurospheres. This inhibitory effect was partially antagonized by MRS2179 (30 m M) and completely reversed by the P2Y12 antagonist MRS2395 (10 m M). Taken together, these data demonstrate that ATP and ADP evoke P2Y1 mediated Ca2+ transients in SVZ-derived neurospheres and that the inhibitory effect of adenine nucleotides on neurosphere formation and proliferation involves P2Y1 and P2Y12 receptor activation. Modulation of either stem cell proliferation or differentiation by purinergic receptor mediated G-protein signalling pathways may therefore represent a potential modulatory mechanism within the stem cell niche.

Proceedings of the Australian Physiological Society

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Protein kinase A inhibits cell growth induced by overexpression of IK channels C.J. Fowler, K. Ngui, B. Hunne, D. Poole, J.B. Furness and C.B. Neylon, Department of Anatomy and Cell Biology, The University of Melbourne, VIC 3010, Australia. Intermediate-conductance (IK) potassium channels have been shown to play a key role in the proliferation of different cell types. Blockers of IK channels are effective in inhibiting the growth of lymphocytes, proliferative smooth muscle cells and various cancer cells. We have shown previously that the IK channel is regulated by cAMP-dependent protein kinase (PKA). As the PKA pathway is generally thought to be growth inhibitory, we wished to examine whether PKA can modulate the influence of IK channel activity on cell growth. An IK-expressing stable cell line was generated in HEK293 cells. The V5 antibody epitope was engineered into the rat IK cDNA, which was transfected into HEK293 cells and cultured in the presence of G418. Western blotting of cell extracts revealed an anti-V5 immunoreactive band at 43kDa which was not present in untransfected HEK293 cells. This band corresponds to the recombinant IK channel subunit. For proliferation assays, cells were seeded at 50,000 cells per 35mm diameter dish and cultured up to 5 days in DMEM containing 10% foetal bovine serum. Cells were counted in duplicate using a haemocytometer. The IK-expressing stable cell line proliferated at a significantly faster rate compared to the untransfected control HEK293 cells. This enhanced cell growth was completely inhibited by the IK channel antagonist, clotrimazole (10µM). To determine the effect of PKA, cells were exposed to the adenylate cyclase activator forskolin (10µM) during the rapid growth phase. Forskolin prevented the enhanced growth of IK channeloverexpressing cells but had no effect on proliferation of untransfected HEK293 cells. Serine 332 on the IK channel is a strong PKA consensus site (Neylon et al., 2004). To determine whether the inhibition of cell growth by PKA was due to direct phosphorylation of S332, we generated a stable cell line overexpressing IK channels containing the S332A mutation. Cells overexpressing the S332A mutant grew at a similar rate to those overexpressing wild type IK channels. However, the inhibition of cell proliferation by forskolin was attenuated. Forskolin produced only 30% inhibition of the enhanced growth of cells overexpressing S332A-IK channels, compared to 100% for wild type IK channels. We conclude that PKA can prevent the influence of IK channels on cell growth, and this effect is mediated partially through direct phosphorylation of S332 on the IK channel. Neylon, C.B., D’Souza, T., & Reinhart, P.H. (2004) Pflügers Archiv 448, 613-620.

Proceedings of the Australian Physiological Society

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Post-transcriptional regulation of CFTR protein expression by 5¢ untranslated region encoded regulatory elements S-J. Conroy, W.L Davies and A.E.O. Trezise, School of Biomedical Science, The University of Queensland, QLD 4072, Australia. Cystic fibrosis is a common, fatal genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). CFTR encodes for a cAMP regulated chloride channel present in epithelial, cardiac and neuronal tissues, the loss of which impairs electrolyte transport across epithelial cells resulting in cystic fibrosis (CF) disease. CFTR expression is tightly controlled by a combination of transcriptional, post-transcriptional, translational and post-translational regulatory mechanisms, resulting in complex spatial, temporal, and pathological expression patterns. However, the regulatory mechanisms controlling CFTR expression in vivo are not well understood. Despite the importance of the transcriptional regulation of CFTR, we have recently shown that CFTR is subject to post-transcriptional regulation through the action of upstream open reading frames (uORFs) encoded within the CFTR 5¢ untranslated region (5¢ UTR) (Davies et al., 2004). We have investigated a highly conserved uORF present in the 5¢ UTR of the predominant epithelial of CFTR mRNA isoform. Evidence for uORF involvement in post- transcriptional regulation has been found in many eukaryotic genes, and has been attributed to disruption of ribosome scanning during translation, thereby modulating translation initiation at the main coding region. We investigated the functional importance and mechanism of action of the 5¢ UTR in CFTR posttranscriptional regulation. We generated a series of expression constructs linking wildtype and mutant rabbit CFTR 5¢ UTR sequences to the firefly luciferase reporter gene. Following transfection of HT29 and CHO cells, measurement of luciferase activity indicated the effect of CFTR 5¢ UTR encoded regulatory elements on translation of the main coding region. For each construct, triplicate independent transfections were performed and co-transfection with renilla luciferase controlled for minor differences in transfection efficiency. It was found that the wildtype CFTR 5¢ UTR (uORF present) supported translation of the main coding region at just 50% of the level produced by the positive control (beta-globin) 5¢ UTR, suggesting the presence of negative regulatory elements. Mutations that increase translation of the uORF result in a further 50-70% decrease in translation of the main coding region. In contrast, the elimination of the uORF translation, by truncation of the 5¢ UTR or direct mutation of the uORF, produces a 50% increase in translation of the main coding region compared to the wildtype CFTR 5¢ UTR. Overall the effect of each 5¢ UTR construct was very similar in both CFTR expressing (HT29) and non-expressing (CHO) cell lines. However, some small differences are indicative of tissue-specific trans-acting modulatory factors. These results identify a new mechanism of CFTR regulation, confirming the importance of 5¢ UTR regulatory elements in modulation of CFTR expression. Davies, W.L., Vandenberg, J.I., Sayeed, R.A. & Trezise, A.E. (2004) Biochemical and Biophysical Research Communications 319, 410-418.

Proceedings of the Australian Physiological Society

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Nedd4-2, ClC-5 and albumin endocytosis in the proximal tubule: a role for SGK-1? D.H. Hryciw1, J. Ekberg1, A. Lee1, I.L. Lensink2, S. Kumar2, W.B. Guggino3, D.I. Cook4, C.A. Pollock5 and P. Poronnik1, 1School of Biomedical Sciences, University of QLD, Brisbane, QLD 4072, Australia, 2Hanson Institute, IMVS, Adelaide, SA 5000, Australia, 3Department of Physiology, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA, 4Department of Physiology, University of Sydney, NSW 2006 Australia, and 5Kolling Institute, RNSH, University of Sydney, NSW 2065, Australia. Retrieval of urinary albumin by the proximal tubule is achieved by receptor-mediated endocytosis that involves a macromolecular complex that includes megalin/cubulin receptor, the Cl- channel ClC-5, Na-H exchanger isoform 3 (NHE3) and v-H+-ATPase. Defects in this uptake pathway result in increased albumin excretion, albuminuria and eventually nephropathy. Genetic defects in ClC-5 in patients with Dents disease lead to persistent proteinuria. ClC-5 knockout mice also have proteinuria, demonstrating a key role for ClC-5 in this process. ClC-5 is expressed at the apical cell membrane of the proximal tubule and we have been investigating the molecular basis for the role of ClC-5 in albumin uptake. ClC-5 has a large intracellular C-terminus that can potentially act with numerous regulatory proteins. Previously it has been demonstrated that the cell surface expression of ClC-5 can be regulated by an ubiquitin ligase, WWP2. In the current study we showed that Nedd4-2 interacts with ClC-5 and that this interaction is an essential component of constitutive albumin uptake (Hryciw et al., 2004). We also investigated whether serum- and glucocorticoid-inducible kinase (SGK-1) plays a role in this endocytic process. We first used Glutathione S transferase (GST) fusion pulldowns to show that C-terminus of ClC-5 bound both Nedd4 and Nedd4-2. The Xenopus oocyte expression was then used to show that Nedd4-2 but not Nedd4 reduced ClC-5 currents in a manner that was dependent on an intact proline rich motif containing a tyrosine (PY) in ClC-5. Using luminescence detection of an influenza hemagglutinin-HA-epitope-tagged ClC-5, the decrease in ClC-5 currents was confirmed to be due to a reduction in cell surface levels of ClC-5. Acute exposure of opossum kidney (OK) cells to albumin resulted in a rapid increase in the protein levels of both ClC5 and Nedd4-2 and an increase in proteasome activity. Conversely, inhibition of the proteasome or silencing of endogenous Nedd4-2 in OK cells caused significant decreases in albumin endocytosis. These data indicate that constitutive albumin uptake involves the upregulation of the ubiquitin/proteasome system In the cortical collecting duct, it is hypothesized that SGK inhibits the action of Nedd4-2 on ENaC. We therefore investigated the role of SGK-1 on albumin uptake in OK cells. Overexpression of wildtype SGK-1 resulted in a significant increase albumin endocytosis 114 ± 3.5%; n = 4; P Our data clearly demonstrate that, similar to the reabsorption of Na+ by the cortical collecting duct, that the constitutive uptake of albumin involves ubiquitin ligases, the proteasome and SGK-1. However, it appears that the effects of SGK-1 do not involve either Nedd4-2 or ClC-5 in OK cells. These data highlight the complex nature of the endocytic process that mediates the retrieval of albumin from the glomerular filtrate. Hryciw DH, Ekberg J, Lee A, et al. (2004) Journal of Biological Chemistry, 279:54996-5007.

Proceedings of the Australian Physiological Society

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Na+ H+ exchanger regulatory factor 2 (NHERF-2) is a scaffold for the plasma membrane Ca2+ ATPase (PMCA) W.A. Kruger1, G.R. Monteith2, L. Tongpao1 and P. Poronnik1, 1School of Biomedical Sciences, The University of Queensland, St Lucia, QLD 4072, Australia and 2School of Pharmacy, The University of Queensland, St Lucia, QLD 4072, Australia. Resting cytosolic Ca2+ levels are maintained at nanomolar levels by the sequestration of Ca2+ into intracellular stores or the extrusion of Ca2+ across the plasma membrane by the PMCA. Despite the ubiquitous distribution of PMCA and its pivotal role in Ca2+ signalling, little is known about how PMCA activity is regulated during G protein coupled receptor signalling. There are 4 isoforms of PMCA (1-4) and many splice variants of all isoforms have been identified and all PMCA-b splice variants have a consensus class 1 PSD-95/Dlg/Zo-1 (PDZ) binding motif (Strehler et al., 2001). Protein-protein interactions mediated by PDZ modules are now recognized as playing a key role in spatially constraining many ion channels and transporters into signalling complexes in membrane microdomains (Pawson et al., 1997). Previously, PMCA 2b has been reported to interact with NHERF-2 in a heterologous expression system (DeMarco et al., 2003). This study investigated whether PMCA interacts with NHERF-2 in a native epithelial cell and the physiological significance of this interaction in terms of G-protein mediated Ca2+ signalling via the muscarinic M3 receptor. This study used the polarised epithelial HT29 cell line which expresses only the M3 isoform of the muscarinic receptor. RT-PCR and Western blotting were used to confirm the presence of both PMCA and NHERF-2 in these cells. Cell surface biotinylations were performed to investigate the changes in levels of PMCA at the plasma membrane following activation of M3 receptor by acetylcholine (ACh). NHERF-2 contains 3 binding domains, PDZ-1, PDZ-2 and the C-terminus. We used GST-fusions of these domains as well as full length NHERF-2 to characterise the interaction between NHERF-2 and PMCA. These interactions were validated in vivo using co-immunoprecipitation with a polyclonal NHERF-2 antibody and subsequent Western blotting with a pan-PMCA antibody. To examine the functional role of NHERF-2, endogenous protein was knocked down by transfecting siRNA plasmids. Changes in intracellular Ca2+ were measured using FURA-2 in a microplate assay. RT-PCR and Western blots confirmed that HT29 cells expressed both NHERF-2 and PMCA isoform 1 and 4 (n = 3). Importantly, we found that the levels of PMCA at the plasma membrane increased by 62 ± 12% (n = 3) within 1 min of exposure to ACh and returned to control levels within 3 min. GST-pulldown experiments in HT29 cell lysates clearly showed that PMCA interacted with the second PDZ module of NHERF-2 (n=4). Coimmunoprecipitation experiments using HT29 cell lysates confirmed the interaction between NHERF-2 and PMCA occurred under in vivo conditions (n=3). Silencing of NHERF-2 reduced the levels of endogenous NHERF-2 by a 68 ± 10% (n = 3). When we examined the Ca2+ response to ACh in the cells where NHERF-2 had been silenced we observed that the rate of recovery from the peak Ca2+ transient was 50 ± 10% (n = 3; P < 0.05) faster than in control cells. These data reveal for the first time that the increase in intracellular Ca2+ in response to M3 receptor activation is accompanied by a rapid increase in PMCA at the plasma membrane, presumably due to translocation from subplasmalemmal stores. The functional interaction between NHERF-2 and PMCA may underlie the changes in recovery rate of Ca2+ following exposure to ACh. Further studies will provide new insights into how scaffold proteins may confer specificity in terms of G-protein mediated Ca2+ signalling. Demarco, S., Chicka, M. & Strehler, E. (2003) Journal of Biological Chemistry 277, 10506-11. Pawson, T. & Scott, J. (1997) Science 278, 2075-80. Strehler, E. & Zacharias, D. (2001) Physiological Reviews 81, 21-50.

Proceedings of the Australian Physiological Society

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NHERF1 - a novel scaffold protein for the astroglial glutamate transporter GLAST A. Rayfield1, A. Lee1, D. Pow2, D. Hryciw1, T.A. Ma1, S. Broer3, C. Yun4 and P. Poronnik1, 1School of Biomedical Sciences, University of Queensland, Brisbane, QLD 4072, Australia,2 Department of Anatomy, University of Newcastle, NSW 2308, Australia,3 Division of Biochemistry and Molecular Biology, Faculty of Science, Australian National University, Canberra, ACT 0200, Australia and 4Department of Medicine, Emory University, Atlanta, Georgia 30322, USA. The Na+/H+ exchanger regulatory factor (NHERF) proteins, NHERF1 and NHERF2 are renal epithelial PSD95/Dlg/ZO-1 (PDZ) domain containing proteins (Weinman et al., 1995; Yun et al., 1997). NHERF proteins contain two tandem PDZ domains (PDZ1 and PDZ2), which associate with specific C-terminal motifs of target peptides. NHERF proteins also contain a C-terminal region able to interact with the Ezrin-Radixin-Moesin (ERM) proteins which provides a link to the actin cytoskeleton. While NHERF proteins have been extensively characterised in kidney and other tissues, very little is known regarding NHERF expression in the central nervous system (CNS) and its possible-binding partners in the CNS. Tissues used for immunohistochemical and biochemical analyses were isolated from euthanised adult Wistar rats, following procedures approved by the University of Queensland Animal Ethics Committee. In this study, we performed immunohistochemical characterisation of the cellular distribution of NHERF1 and NHERF2 in the adult rat brain. Immunohistochemistry was performed on adult rat brain sections using polyclonal antibodies specific for NHERF1 and NHERF2. Expression of NHERF1 was shown to be widespread in brain, most prominently in hippocampus, thalamus, choroid plexus and cerebellum. In these different regions of the brain, NHERF1 was primarily restricted to astrocytes. NHERF2 expression was primarily restricted to endothelial cells of blood vessels and capillaries. The distribution in adult rat brain was similar to that of GLAST, an astroglial glutamate transporter that also contains a potential PDZ binding consensus sequence (ETKM) in its COOH-terminus (Lehre, et al., 1995). Double immunofluorescence labelling studies were performed using antibodies specific for GLAST and NHERF1 and imaged by confocal microscopy, colocalisation of GLAST and NHERF1 was detected in astroglial cells. Using solubilised adult rat brain lysate, coimmunoprecipitation experiments demonstrated that GLAST, NHERF1 and ezrin co-associate in vivo. To determine which domains of NHERF1 and GLAST interact we performed pull-down assays using solubilised adult rat brain lysate and GST-fusion proteins of the GLAST COOH-terminus, full-length NHERF1 and various domains of NHERF1 (PDZ1, PDZ2 and ERM domain). These experiments revealed that the GLAST-NHERF1 interaction requires the COOH-terminal ETKM sequence of GLAST and utilises the PDZ1 domain of NHERF1. Therefore we have demonstrated that NHERF1 links GLAST to the actin cytoskeleton through ezrin, leading to the formation of a multi-protein complex. Linkage of GLAST to NHERF1 may serve as an important mechanism for localising the GLAST transporter to specialised membrane sites in astrocytes or for regulating transport activity. Lehre, K P., Levy, L.M., Ottersen, O.P., Storm-Mathisen, J. & Danbolt, C.N. (1995) Journal of Neuroscience 15, 1835-1853. Weinman, E.J., Steplock, D., Wang, Y. & Shenolikar, S. (1995) Journal of Clinical Investigation 95, 2143-2149. Yun, C.H., Oh, S., Zizak, M., Steplock, D., Tsao, S., Tse, C.M., Weinman, E.J. & Donowitz, M. (1997) Proceedings of the National Academy of Sciences of the United States of America 94, 3010-3015.

Proceedings of the Australian Physiological Society

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Molecular cloning and characterisation of the mouse ‘system IMINO’ transporter S. Kowalczuk1, A. Bröer1, M. Munzinger1, N. Tietze1, K. Klingel2 and S. Bröer1, 1School of Biochemistry & Molecular Biology, Australian National University, Canberra, ACT 0200, Australia and 2Department of Molecular Pathology, University of Tübingen, 72076 Tübingen, Germany. The SLC6 family consists of transporters for amino acids, neurotransmitters and osmolytes. These transporters play an important role in the removal of neurotransmitters in brain tissue and in amino acid transport in epithelial cells. The SLC6 family also contains a number of orphan transporters. Recently we identified a new member of the SLC6 family (B0AT1 or SLC6A19), which is closely related to the orphan transporters and transports neutral amino acids. We hypothesized that other orphan transporters may be amino acid transporters as well. To test this hypothesis we studied the mouse slc6a20 gene. The mouse has two homologues that correspond to single the human SLC6A20 gene, which are known as XT3 and XT3s1. RT-PCR analysis revealed expression of XT3s1 in the brain, kidney, small intestine, thymus, spleen and lung, while expression of XT3 was restricted to kidney and lung. Subsequently, we isolated full-length cDNA clones of XT3s1 and XT3 from brain and kidney, respectively. In situ hybridisation showed strong expression of XT3/XT3s1 in the proximal tubules of kidney cortex, in intestinal villi and in the brain. Expression of mouse XT3s1, but not XT3, in Xenopus laevis oocytes induced Na+- and Cl--dependent transport of proline, hydroxyproline, glycinebetaine, MeAIB and pipecolic acid. Activation analysis suggests a 1Na+/1Cl-/proline cotransport, which would be electroneutral. However, uptake experiments under voltageclamp conditions suggest translocation of 1 charge per proline molecule. This apparent discrepancy can be explained by the very high affinity of the chloride binding site - chloride transport is likely to occur by way of an exchange process and thus will not affect the electrogenicity of the transporter. The substrate specificity and mechanism of transport by XT3s1 fits well with the properties of the classical ‘system IMINO’, one of the major proline resorption systems of the intestine and kidney (Stevens & Wright, 1985). Together, the expression pattern and functional characteristics of SLC6A20 suggest a possible involvement in the inherited aminoaciduria iminoglycinuria. Stevens, B.R. & Wright, E.M. (1985) Journal of Membrane Biology 87, 27-34.

Proceedings of the Australian Physiological Society

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Increased acetaminophen hepatotoxicity in the NaS1 sulphate transporter null mouse S. Lee, P.A. Dawson and D. Markovich, School of Biomedical Sciences, University of Queensland, Brisbane, QLD 4072, Australia. Sulphate (SO42-) plays an important role in the detoxification of numerous xenobiotics, including the widely used analgesic drug, acetaminophen (APAP) (Cole & Evrovski, 2000). The Na+-SO42- cotransporter, NaS1 is expressed in the kidney, where it maintains blood sulphate levels (Markovich, 2001). NaS1 knock-out (Nas1-/-) mice exhibit hyposulphataemia and hypersulphaturia (Dawson et al. 2003). This project assessed the molecular, biochemical and physiological consequences of APAP challenge. Male Nas1+/+ and Nas1-/- mice aged 1-4 months (n= 5-7 mice), were injected with 125-, 250- or 500-mg/kg of APAP i.p. The animals were sacrificed at various times (0, 2, 4, 5, 6 and 12 hours) after APAP administration. Serum alanine aminotransferase (ALT) levels in Nas1+/+ and Nas1-/- mice were measured as an indicator of APAP-induced liver injury. ALT levels were 3-fold higher in Nas1-/- mice when compared to Nas1+/+ mice at 12 hours after APAP treatment (250-mg/kg). This supports our histological findings of increased cellular damage in Nas1-/- mice. Extensive haemorrhaging was observed in lobular areas of Nas1-/- mice (500-mg/kg APAP, t=5 hours postinjection) but not in Nas1+/+ mice. Hepatic glutathione (GSH) depletion was greater in Nas1-/- mice (87% reduction), compared to Nas1+/+ mice (63% reduction) at 250-mg/kg dosage regime (t=2 hours post-injection) whereas repletion of GSH showed no significant differences between and Nas1+/+ and Nas1-/-mice. The GSTpi mRNA levels were significantly induced (2-fold) in Nas1-/- mice, when compared to Nas1+/+ mice. The induction of GSTpi mRNA levels in APAP-treated Nas1-/- mice, could be a compensatory response to the GSH depletion. The mRNA levels of CYP3A11, which are responsible for the production of reactive metabolite, Nacetyl-p-benzoquinone imine (NAPQI) (Zhang et al., 2002), were significantly increased (1.5-fold) in Nas1-/mice when compared to Nas1+/+ mice (250-mg/kg APAP, t=2 hours post-injection). In summary, we have identified increased APAP-induced hepatotoxicity and more rapid GSH depletion in the hyposulphataemic Nas1-/- mice and this may be due to low blood sulphate levels, which limits APAP sulphonation. This study suggests the potential role of NaS1 in the modulation of APAP-induced hepatotoxicity. Cole, D.E. & Evrovski, J. (2000) Critical Reviews in Clinical Laboratory Sciences 37, 299-344. Dawson, P.A., Beck, L. & Markovich, D. (2003) Proceedings of the National Academy of Science U.S.A. 100, 13704-13709. Markovich, D (2001) Physiological Reviews 81, 1499-1534. Zhang, J., Huang, W., Chua, S.S., Wei, P. & Moore, D.D. (2002) Science 298, 422-424.

Proceedings of the Australian Physiological Society

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The influence of dietary fish oil and exercise upon oxidative status biomarkers in a rat model R. Henry, A.J. Owen and P.L. McLennan, Smart Foods Centre, Department of Biomedical Science, University of Wollongong, NSW 2522, Australia. Introduction. Oxidative stress is implicated in cardiovascular and many other diseases, as well as in normal aging. Highly unsaturated omega-3 fatty acids are very susceptible to oxidation and the generation of reactive oxygen species while intense exercise promotes an environment for increased oxidation. Paradoxically, both chronic dietary fish oil consumption and chronic exercise are associated with reduced cardiovascular disease morbidity and mortality. Objective. This study aimed to determine the effect of dietary fish oil and exercise training on membrane fatty acid composition and biomarkers of oxidative stress in tissues representative of different levels of oxidation. Methods. Male Wistar rats, fed either saturated fat (SF) or fish oil (FO) diets for 6 weeks were exercise trained (weighted swimming, 1h/d, 5d/w with 2% body weight on tail) or remained sedentary. Rats rested for 2 days and fasted overnight were anaesthetised (pentobarbitone sodium 60mg/kg i.p) and killed by rapid exsanguination. Liver and skeletal muscles (diaphragm, abdominal sheath and white vastus lateralis) were analysed for membrane fatty acids, lipid peroxidation products and endogenous antioxidants: glutathione peroxidase and superoxide dismuatse. Outcomes. In all tissues FO feeding increased EPA, DHA, total n-3 PUFA and the unsaturation index and decreased arachidonic acid, total n-6 PUFA and n-6/n-3 ratio (p<0.05). Exercise training increased membrane arachidonic acid (p=0.023) in the FO liver but decreased DHA(p=0.002) and total n-3 PUFA (p=0.017) compared to FO sedentary. The liver compared to muscle tissue and diaphragm compared to other muscles had higher membrane arachidonic acid (AA) and lower DHA content. Lipid hydroperoxidation varied according to tissue phospholipid unsaturation but there was no additional effect of fish oil consumption or exercise training. Activities of glutathione peroxidase and superoxide dismutase varied according to tissue metabolic activity (liver >> muscle (diaphragm > abdominal muscle)) with no additional effect of fish oil consumption or exercise training. Conclusion. Fatty acid composition and antioxidant enzyme activity may be related to the oxidative functions of different tissues. Despite incorporation of n-3 PUFA into cell membranes, fish oil feeding did not increase tissue oxidative stress measured at rest and exercise training was not associated with altered oxidative stress biomarkers at rest.

Proceedings of the Australian Physiological Society

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Simulation of visual processing in retinal ganglion cells M. Watson,1 G. Holmes,1 T. Byrne2 and S. Cornford,1 1Department of Biological & Physical Sciences, Faculty of Sciences, 2Faculty of Engineering and Surveying, University of Southern Queensland, Toowoomba, QLD 4350, Australia. The retina contains photoreceptors for light detection as well as bipolar, horizontal, amacrine and ganglion cells. These form a neural network where synaptic convergence, divergence and integration take place. It serves as a simple network to simulate and thus can be used as a learning tool to demonstrate neural processing, desensitization, experimental design and protocol. The simulation is designed for students to facilitate inquiry based learning. A given retinal ganglion cell responds to light directed to a specific area of the retina. This area is called the ‘receptive field’. Ganglion cell receptive fields have a ‘centre’ and an antagonistic ‘surround’ and can be classified as ‘On Centre’ or ‘Off Centre’. On Centre are activated by light in the centre of the receptive field and inhibited by light in the surrounding receptive field. Off Centre are inhibited by light in the centre of the receptive field and activated by light in the surrounding receptive field. The model simulates the receptive fields of four ganglion cells. The four receptive fields are arranged into 4 arrays with each array containing 64 Light Dependent Resistors (LDR’s; EG&G Vactec). These simulate the photoreceptors contained within each receptive field. The LDR’s are connected via the Multiplexor (Temic) and through the programming of a HC12 microprocessor (Motorolla) simulate the ‘On Centre’ and ‘Off Centre’ receptive fields. Students shine a variable point of light onto one of the four array’s and will see either an increase or decrease in the clock rate output of the HC12. This represents a change in the discharge of the ganglion cell. The clock rate output of the HC12 is visualised by means of an oscilloscope. The clock rate output will vary depending on whether they are activating ‘On’ or ‘Off’ LDR’s. Prolonged stimulation of the LDR’s reduces the clock rate output to pre- stimulus levels to simulate desensitization. Students are required to design an experimental protocol that determines an optimal standard stimulus, a systematic protocol for testing the different receptive fields and a protocol for gathering and analysing the data. The simulation challenges students to determine: (i) the optimum size, strength and duration of a stimulus that is sufficient to stimulate the receptive field; (ii) the receptive field characteristics of retinal ganglion cells; (iii) how such receptive fields can be formed on the basis of the cell types and connections found in the retina.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/83P

Excitation-contraction coupling from 1969 to 2005 A.F. Dulhunty, Division of Molecular Bioscience, John Curtin School of Medical Research, Building 54, Off Mills Rd, Australian National University, ACT 0200, Australia. The past 40 years have seen an explosion of information about the molecular components of many cell processes including the excitation-contraction (EC) coupling which controls Ca2+ release and triggers contraction in muscle. In the 1960’s it was understood that “a switch” allowed the action potential that travelled along the transverse (T-) tubular invaginations of the surface membrane to release Ca2+ from the sarcoplasmic reticulum (SR). Nothing was known of the molecules or signalling systems involved. There was hot debate about the nature of the switch, whether it was chemical, mechanical or electrical. The early 1970’s saw the discovery of a tiny electrical “charge movement” which reflected the movement of a dipole in the T-tubule membrane that was linked to, and preceded, Ca2+ release. The charge movement was likened to a lever that pulled a plug from the terminal cisternae to dump Ca into the myoplasm. The molecule that generated the charge movement was (i) before action potential (ii) after action potential thought to be the dihydropyridine receptor (DHPR) L-type action potential Ca2+ channel. The >2 million dalton ryanodine receptor transverse-tubule surface membrane (RyR) Ca2+ release channel was identified in the 1980’s. Sarcoplasmic reticulum Expression of recombinant proteins in DHPR- or RyR-null terminal cisternae cells in the late 1980’s and 1990’s confirmed that the α1 subunit of DHPR and the RyR were essential for EC Ca coupling. In the following decade, several interactions Ca Ca between the proteins have been defined and the very important role of associated proteins recognised. It is no Actin/myosin longer thought that the DHPR and RyR transiently connect Ca after an action potential. Rather, a tightly coupled relaxed muscle contraction macromolecular complex is thought to respond to changes in surface membrane potential in a manner that is highly regulated by cytoplasmic factors and by the Ca2+ load in the SR. The molecular complex extends from the extracellular space into the lumen of the SR, spanning the T-tubule homer ( ) and SR membranes and the junctional gap between dihyropyridine receptor Glycolytic enzymes ( ) calmodulin ( ) voltage sensor ( ) and them. The RyR is coupled to the II-III and III-IV RyR calcium release channel ( ). cytoplasmic linker loops and C-terminal tail of the α1 FKBP12 ( ) subunit of the DHPR and to the soluble b subunit. Ca Anchored kinase ( ) Among many proteins that associate with the Ca cytoplasmic domain of the RyR and regulate its junctin ( ) triadin ( ) activity are the critically important FK506 binding calsequestrin ( ) HRP ( ) proteins, anchored kinases, calmodulin, Homer and members of the glutathione transferase (GST) Actin/myosin GSTs ( ) Ca structural family. Glycolytic enzymes are abundant SOCE-like channel ( ) contraction around complex. Within the SR lumen, the RyR communicates with the calcium binding protein, calsequestrin (CSQ), with the CSQ anchoring proteins triadin and junctin, with a histidine rich protein (HRP) and with GSTs. The activity of the channel is modulated by phosphorylation and oxidation. The complex not only regulates Ca2+ release from the SR, but also Ca2+ influx from the extracellular environment through the DHPR and store-operated calcium entry (SOCE)-like channels. Despite the extent of current knowledge, there is much to learn - we remain ignorant about the atomic structures of the proteins, the molecular nature of the interactions between them and indeed the residues involved in most of the interactions. 2+

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Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/84P

AuPS/ASB Meeting - Canberra 2005 Symposium 3: Physiology Teaching in the 21st Century: Trends, Challenges and Innovations Thursday 29 September 2005 Chair: Kay Colthorpe and Hardy Ernst

Challenges facing physiology educators in the 21st century Ann Sefton, Faculty of Medicine, University of Sydney, NSW 2006, Australia. Physiology is a core subject in health-related curricula and in medical science programs; aspects are often incorporated into other courses. Broadly, the challenges facing those who teach physiology include issues relating to the increasing amount and complexity of subject knowledge, the different destinations and expectations of the students, the changing nature of those student cohorts, the costs and difficulties of providing modern experimental work, and the need to adopt appropriate, evidence-based educational practices. Issues include the need for clarity in the specific and generic goals or outcomes, whether for a single unit or an entire program. Designing an explicit progression in knowledge as well as in generic and specific skills supports students. Setting limits provides clarity and avoids unnecessary duplication. Students learn in many different ways; providing a range of learning experiences helps to support them effectively. Active learning is a core aim. Practicals pose challenges, but offer valuable opportunities for interactive group work. Skills gained include designing an experiment, obtaining, recording, analysing and presenting data in a range of different ways. Problem- or case-based studies can be used effectively within physiology, and physiologists contribute to integrated problem-based medical and other health science programs. Well-designed information technology offers on-line learning resources, simulations and the means to design and record results for a range of experiments. IT can provide flexibility for students who increasingly need to work. Formative assessment supports learning. Well-designed summative assessment, matched to the goals of the program, evaluates achievement in a range of essential knowledge and skills. Targeted evaluation provides helpful feedback to staff.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/85P

Research led teaching and learning in physiology R.E. Kemm, Department of Physiology, University of Melbourne, Parkville, VIC 3010, Australia. ‘Research Led Teaching’ (RLT) and its effective implementation and evaluation are the focus of my interests in improving Science Physiology teaching. My aim is to describe some findings during a recent overseas study tour. Since The Boyer Commission Report on Educating Undergraduates in Research Universities (1988), there has been a strong move by research universities to include RLT in their mission statements. The problem is how to marry the great success of research universities and the commitment of their researcher/academics to research with the effective sharing of their philosophy and enthusiasm with undergraduate students in a RLT philosophy. Projects in various universities address how to introduce this institutional ethos of RLT in curriculum design, such as Warwick University and others, and these can provide useful guides to best practice. Few research universities have published detailed management plans to implement their RLT policies, fewer still have definitive curriculum changes that reflect their mission statements and even fewer have yet provided evidence of changed student learning practices or learning outcomes. It has been difficult to find good examples of coherent and extensive use of RLT in biomedical science curricula, let alone in science physiology. However on my recent study tour I found good, but often isolated, examples of RLT in biomedical science courses as well as interest in other institutions in moving towards formally incorporating RLT practices into their curricula. In biomedical sciences, students usually undertake courses with lectures, laboratory classes and varying amounts of tutorials or e-Learning activities. There may be some problem-based learning elements introduced into science curricula, but funding limits for staff have restricted such approaches to medical courses. In trying to design appropriate science curricula within a research university, the limitations/advantages of each of these modes of delivery/interaction needs to be considered. Another important issue is how to provide central support to academic staff to helps them initiate changes in their approaches to teaching to match the institutional philosophy. An overall objective for graduates in Science physiology would be for them to have the skills of experimental investigators so that they can continue with a professional career in science or have a thorough appreciation of evidence based research to support other careers. Many of these skills are generic and apply widely across the spectrum of University graduates, whereas others more specific to biomedical sciences need to be added. These common generic skills are usually well identified within an institution’s guidelines for teaching and learning. A major task is to design a curriculum that help the students understand that the challenge for them is to become independent learners during their undergraduate courses so that they are able to be effective life long learners adapting to a rapidly changing world of science. Students are often driven by assessment and many will take a surface, rather than deep approach to learning if that is what is rewarded. It is essential that there is a constructive alignment of assessment that rewards a deep understanding of the subject, rather than using traditional examination processes that reward a detailed knowledge base in the discipline. It is also imperative that students have formative assessment of their progress with such skills, such as critical reviewing of literature and problem solving, rather than be assessed only in end of semester examinations. Traditional mentoring, with few students per academic, achieved this in the past. Increasing pressure of student numbers means this is no longer a viable option and new ways, supported by e-Learning, need to be incorporated to assist in the progressive development of many generic skills. Lecturers are slower to introduce RLT components into the earlier years of physiology, but many more elaborate on their own research in final years of physiology. Enquiry based learning is often practiced in student-centred laboratory classes, but formal structuring of hypothesis testing and experimental design is not often undertaken. There are some examples of on-line e-Learning to reduce staff workloads with new initiatives. The final challenge is to evaluate whether or not RLT incorporated throughout a curriculum, has resulted in useful learning outcomes. Valid statistical comparisons of different curricula are always difficult in education, so it maybe more useful to see if the curricula goals are met. Student engagement questionnaires can help determine if the students have appreciated the RLT approach and have adopted a deeper learning style. However, new instruments need to be designed to see if students have the desired skills on graduation, such as hypothesis formation and testing, analytical and interpretive skills etc. Some such instruments are available, but more are needed. The Boyer Commission on Educating Undergraduates in the Research University (1998) Reinventing undergraduate education: A blueprint for America’s research universities. http://naples.cc.sunysb.edu/Pres/boyer.nsf/

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/86P

Problem-based learning (PBL): A novel and effective approach for teaching research skills by addressing contemporary research problems in physiology J. Schwartz1 and P. Buckley1,2, 1Discipline of Physiology, School of Molecular and Biomedical Science, University of Adelaide, Adelaide SA, Australia and 2School of Pharmacy & Medical Sciences, University of South Australia, Adelaide SA, Australia. (Introduced by Michael Roberts) The biomedical science degree at Adelaide is oriented to research graduates, so we introduced a new unit in Physiology III. A unique component of that unit is a PBL course, the aims of which are to expose students to the science of addressing unresolved research issues, thereby developing their skills of scientific thinking and hypothesis setting and testing. We base the full-year course on a series of 7-8 contemporary research questions, ranging from basic cellular physiology to medicine. Students are also challenged to address ethical issues incorporated into the PBL research cases. Students work together in small-groups on assignments, researching literature, and participate in regular group discussions. Assessment in this course tests the process of scientific thinking and teamwork, as well as content knowledge. The PBL course prepares students for a research career by providing as much of a real research team experience as can be engineered into a single component of the year 3 curriculum. Students learn to appreciate the value in advancing science of teamwork, brainstorming in groups to produce thought-provoking discussions and other virtues normally acquired only in laboratory-based research training schemes. PBL cases begin with the introduction of a contemporary research issue by having the students interpret a series of key observations or results. Subsequent discussion is directed at achieving a basic level of background understanding through marshalling the students¿ own knowledge and on-the-spot research of relevant literature. Subsequently, students do further literature research, identify gaps and discrepancies in the current understanding of the scientific problem and frame hypotheses and design strategies to address the gaps/discrepancies. The students discuss matters as though members of a research team, and individuals report to the PBL group on material in which they develop specific expertise. We assessed the impact of the PBL course by determining the extent to which attitudes toward researchoriented skills are changed. In 2001-2004, students were asked at the beginning and end of Physiology III to rate the importance of individual aspects of the course on a scale from 0 to 10 (least to most). Surveys were anonymous, but coded to match individuals¿ responses. Students had no access to their earlier responses when completing the second survey. The familiar and conventional educational category, Importance of Informational Content and Facts, was considered as a standard aspect of all courses, whereas the others are more related to the PBL course; these were: A. stimulation of new thoughts through discussion (8.28±1.39 beginning; 8.96±0.98 end) B. capacity to provoke new thoughts by others (7.55±1.65; 8.38±1.23) C. introduction of new ideas (8.29±1.2; 8.29±1.03) D. experience working in teams (8.37±1.53; 8.52±1.25) E. discussion of current questions in science (8.31±1.44; 8.70±1.28) F. discussion of ethical issues in science (7.77±1.70; 7.84±1.66) G. self-directed learning in research. (7.77±1.70; 7.84±1.66) 48 students completed the survey. Responses were analysed by paired T-test and two-way ANOVA and Tukey’s test, as appropriate. Aspects A, B, E and G were significantly more important aspects to the students at the end of the year than at the beginning. There was also a significant effect of the year¿s experience. At the beginning of the year, there was no significant difference among the importance attached to the various aspects. By the end of the year, the students considered A, D, E, and G more important than informational content and facts (8.00±1.58 beginning; 7.68±1.38 end). In summary, we introduced a novel PBL system for educating students in the science of research through discussion of contemporary research issues. The program apparently has been effective in shaping the students’ thinking about physiology in terms of research.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/87P

Improving learning outcomes for students in Clinical Physiology C.R Dallemagne, School of Life Sciences, Queensland University of Technology, GPO Box 2434, Brisbane, Queensland 4001, Australia. Principles of effective teaching and good practice have been explored by several authors, and is the corner stone of learning (Crickering & Zelda, 1987; Ramsden, 2003). Two are encouraging active learning and emphasizing ‘time on task’. Application of these principles can be facilitated by the various media modes available. Interactive media form gives investigative and exploration experiences and are facilitated by web resources (amongst others) (Laurillard, 2002). Learning facts does not necessarily increase understanding and critical thinking. Self confidence in one’s ability is needed to engage in critical thinking activities (Van Wiegel, 2005). A lack of such confidence leads students to rely on a surface approach of learning facts and figures as this seems safer. Learning and thinking skills are distinct entities but necessarily complement each other (Marton & Ramsden, 1988). Similarly, reflection on learning in specific content domains is preferable to learning “metacognitive skills” (ibid.). Dispositional behaviour modulates students’ approach to their learning of specific topics. The Project Zero “patterns of thinking” project has identified three distinct components necessary for a favourable dispositional behaviour towards critical and creative thinking, namely ability, inclination and sensitivity (Harvard Graduate School of Education, Project Zero). Not all students may possess all three, some may lack the discipline-sensitive imagination to develop their own activities for their learning: technology helps us devise well-designed exercises. Clinical Physiology is an advanced unit, designed for students in Biological Sciences and Nutrition. It builds on understanding and knowledge of basic Anatomy and of 2nd level Physiology. The teaching strategies assist students in understanding the rationale for development of the pathophysiology, diagnostic investigations and treatment of major disorders of the human body. Designing activities that students enjoy interacting with, will encourage them to spend more time with the material, hence increasing ‘time on task’ in a productive way. Developed on line resources were analysed by the Flashlight Evaluation model consisting of a triangulation between technology, activity and learning outcome (Ehrmann 1998). The methodology for the study has been described elsewhere (Goss et al., 2003). Results showed that access and navigation were satisfactory, but there was poor use of interactive activities. Students’ answers in examination reflected mostly a surface approach to learning: facts, but no development of reasoning in the answers. The results of the evaluation of the on line site, and the examination answers motivated us to further develop the on line site with the aim of encouraging active learning and time-on-task. Using interactive activities that are interesting and enjoyable can help students develop inclination and sensitivity in the thinking of the discipline. The topics were clearly identified and within each topic case histories were or are being developed with a common structure. Active learning will be fostered by generating discussion using several on line strategies including a general discussion forum and a chat room. Critical thinking will be developed by some of the elements of the site as students reflect on their conceptualisation of case studies. Time-on-task is the third element we wish to address. As the activities are more enjoyable, and the students perceive the benefit of engaging with them for their studies, they will increase time spent on the subject matter. Feedback given via the on line teaching, in lectures and tutorials, should encourage students’ participation. Chickering, A.W. & Zelda F.G. (1987) American Association Higher Education Bulletin, 39, 3-7. Ehrman S. (1998) TLT Group, http://www.tltgroup.org/about/ehrmann.html. Goss H., Winslett G., Matt I. & Rowe J. (2003) Flashlight - an evaluation model with enlightening outcomes. Brisbane: Effective Teaching and Learning Conference Proceedings 2003. Harvard Graduate School of Education Project Zero (2001). Dispositional characteristics. http://www.pz.harvard.edu/Research/PatThk.htm. Laurillard D. (2002) Rethinking University Teaching. A conversational framework for the effective use of learning technologies. 2nd ed. London: RoutledgeFalmer. Marton, F. & Ramsden P. (1988) What does it take to improve learning? Chapter 14 In P. Ramsden (Ed), Improving learning : new perspectives. London: Kogan Page. Ramsden P. (2003) Learning to teach in higher education. 2nd ed. London: RoutledgeFalmer. Van Wiegel (2005) From Course Management to Curricular Capabilities. Chapter 12 In P. McGee Course Management Systems for Learning: Beyond Accidental Pedagogy, Patricia McGee, Ali Jafari & Colleen Carmean (eds). Idea Group Inc. http://www.idea-group.com.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/88P

AuPS/ASB Meeting - Canberra 2005 Symposium 4: Membrane Protein Structure and Interactions Thursday 29th September 2005 Chair: Frances Separovic

Seeing spots: miscibility transitions in lipid/cholesterol membranes S.L. Keller and S.L. Veatch, Departments of Chemistry and Physics, University of Washington, Seattle, WA 98195-1700, U.S.A. (Introduced by Frances Separovic) Mammalian cells are surrounded by an outer wall or "plasma membrane" of proteins and lipids arranged in opposing leaflets of a bilayer. There is growing evidence that this membrane is not uniform, but instead laterally phase separates into "raft" domains rich in particular lipids and proteins. We study a simpler physical model of cell membranes, giant unilamellar vesicles (GUVs). Using fluorescence microscopy, we can directly observe liquid domains in free-floating vesicles containing three components: a lipid with high melting temperature (e.g. a saturated lipid), a lipid with low melting temperature (e.g. an unsaturated lipid), and a “membrane active” sterol (e.g. cholesterol). Liquid domains in vesicles exhibit interesting behavior. They collide and coalesce until only one bright domain and one dark domain remain on each vesicle. Domains also finger into stripes near the critical point, and can bulge out of or into the vesicle (Veatch & Keller, 2003, Veatch & Keller, 2005).

By recording miscibility transition temperatures for many lipid compositions, we have mapped ternary phase diagrams. Our fluorescence microscopy studies give us qualitative tie-lines across the phase diagram. These tie-lines run from a region that is rich in the unsaturated lipid to a region rich in the saturated lipid, with little change in cholesterol. Applying this statement to the figure above, the bright domains are rich in unsaturated lipid, and the dark domains are rich in the saturated lipid, and only to a lesser extent in cholesterol. Using NMR (in collaboration with Klaus Gawrisch’s laboratory at the National Institutes of Health, Bethedsa, MD, USA), we have quantitatively verified the direction of the tie-lines, and have then estimated free energies to transfer lipids between phases, which are at most a few kBTs (Veatch et al., 2004). In other studies, we have captured domains in lipid layers on glass substrates, and found that they assume static, noncircular shapes. We have substituted different sterols for cholesterol, and found that those which are structurally similar to cholesterol produce coexisting liquid domains in vesicles, just as cholesterol does (Beattie et al., 2005). Finally, we have compared the phase diagrams of bilayer systems to monolayer systems and found them very different (Stottrup et al., 2005). Beattie, M.E., Veatch, S.L., Stottrup, B.L. & Keller, S.L. (2005) Biophysical Journal in press. Stottrup, B.L., Stevens, D.S. & Keller, S.L. (2005) Biophysical Journal 88, 269-276. Veatch, S.L. & Keller, S.L. (2003) Biophysical Journal 85, 3074-3083. Veatch, S.L. & Keller, S.L. (2005) Biochimica et Biophysica Acta in press. Veatch, S.L., Polozov, I.V., Gawrisch, K. & Keller, S.L. (2004) Biophysical Journal 86, 2910-2922.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/89P

Role of the plasma membrane in amyloid formation and toxicity M.I. Aguilar1, X. Hou1, S. Subasinghe1, A. Mechler2, K. Hall1, L. Martin2 and D.H. Small1, 1Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC 3800, Australia and 2School of Chemistry, Monash University, Clayton, VIC 3800, Australia. A number of neurodegenerative diseases are caused by the aggregation and deposition of amyloid in the nervous system. For example, the deposition of b -amyloid peptide (Ab ) is considered to be a key event in the pathogenesis of the Alzheimer’s disease (AD), which is the prototypic amyloidosis. Another example is Transthyretin (TTR), a plasma protein produced in the liver and the choroid plexus that can form amyloid. TTR is the predominant component of the amyloid fibrils in familial amyloidotic polyneuropathy (FAP), a hereditary disorder characterized by systemic extracellular deposition of amyloid fibrils, mainly in the peripheral nervous system. Native TTR consists of four identical subunits that form an extensive b -sheet structure, which is prone to misfolding. So far nearly eighty mutations have been identified in TTR, most of which are amyloidogenic. It is believed that structural modifications by these mutations destabilize the native tetrameric conformation and favour its dissociation into monomeric structure, which is the building block of TTR amyloid fibrils. While the mechanism by which amyloidogenic proteins cause neurotoxicity is unclear, it is now emerging that the cytotoxicity of amyloids is a direct consequence of binding to the plasma membrane. We have therefore used surface plasmon resonance (SPR) for the study of Ab - and TTR-membrane interactions to determine whether this binding could explain the toxic effects of Ab and TTR seen in cell culture. Our results show that Ab and TTR bind to the lipids of the plasma membrane through electrostatic interactions and that the amount of binding is increased upon aggregation. We also show that the amount of Ab and TTR binding to the plasma membrane correlates with the degree of cytotoxicity observed in cell culture. Finally, we demonstrate that binding of Ab and TTR amyloid to the plasma membrane alters membrane fluidity, providing a possible explanation for the cytotoxic effect. Overall, the results strongly support the view that both Ab and TTR toxicity is a direct consequence of binding to lipids in the membrane. However, there are specific differences in the factors that affect this interaction. In particular, binding of Ab was strongly influenced by the concentration of cholesterol in the membrane but did not affect TTR binding. This presentation demonstrates the application of SPR to the study of the molecular interactions associated with AD and FAP and how this information enhances our molecular understanding of neurodegenerative diseases

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/90P

Are chloride intracellular ion channel proteins (CLICs) really channels? Exploring their membrane structure L.J. Brown1, D.R. Littler2, A. Mynott2, S.J. Harrop2, S.N. Breit3, M. Mazzanti4 and P.M.G. Curmi2, 1Department of Chemistry and Biomolecular Sciences, Macquarie University, NSW 2109, Australia, 2School of Physics, University of New South Wales, NSW 2052, Australia, 3Centre for Immunology, St Vincent’s Hospital, Sydney NSW 2010, and 4University or Rome, La Sapienza, Roma, Italy. Most proteins adopt a well-defined three-dimensional structure, however, it is increasingly recognized that some proteins can exist with at least two or more stable conformations. Recently, a class of Chloride Intracellular ion Channel proteins (CLICs) has been shown to exist in both soluble and integral membrane forms. Members of this class of ion channels have a CLIC domain of approximately 240 amino acids in length and vary widely in their cellular and sub-cellular distribution. They are associated with a variety of intracellular membranes and are involved in numerous diverse physiological processes including cell cycle regulation, bone re-absorption, tubular formation and apoptosis. However, the function of CLICs as ion channels is still controversial because of their unusual dual-environmental nature and because none of the family members show a clear identifiable transmembrane domain. Our group has now determined the structure of the soluble conformation/s for several members of this family. Despite this knowledge and because of their auto-inserting nature, the membrane channel structure is still proving difficult to determine using traditional atomic resolution structural techniques. It is therefore necessary to establish how these ubiquitous, soluble proteins can unfold, insert into membrane bilayers and refold to form ion channels. Furthermore, the processes that control this mechanism in the cell also require clarification but may include regulation by oxidation and disulphide bond formation, as in the case of CLIC1. The CLIC family members may also be modulated by pH, alteration in lipid composition, divalent cations, phosphorylation and interaction with other proteins. A combination of structural studies including Electron Paramagnetic Resonance (EPR) and fluorescence spectroscopy, with functional studies performed in parallel, was used to investigate the insertion of CLIC1 into the membrane bilayer. The results demonstrate a role for the conserved cysteine residue at position 24 for insertion of CLIC1 into the membrane and subsequent chloride channel activity. The transmembrane region has been confirmed as comprising residues 24-46 in the N-domain of CLIC1. EPR experiments also show that insertion is likely to involve a large conformational re-arrangement of the C-terminal domain of CLIC to allow the N-domain to span the membrane bilayer.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/91P

Structure and dynamics of cellular components using fluorescence and X-ray diffraction techniques Leann Tilley and Nick Klonis, Department of Biochemistry, La Trobe University, Bundoora, VIC 3083, Australia. The techniques of Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Correlation Spectroscopy (FCS) can be employed using the optics of a confocal microscope to examine the organization and dynamics of different components in cellular systems. For these studies the protein of interest is generated as a chimera with the green fluorescent protein (GFP) and expressed in transfected live cells. We have prepared constructs encoding GFP appended to N-terminal fragments of a series of exported malaria parasite proteins, including the major cytoadherence antigen, PfEMP1. We have used FRAP techniques to examine PfEMP1-GFP dynamics in live cells and have found that the chimera exhibits a half-time for fluorescence recovery of a few seconds indicating that it is trafficked to the host cell membrane as a protein complex. These measurements are at the limit of the accessible time domain using FRAP analysis, therefore we have explored the use of FCS as a means of monitoring the rapid motion of GFP-labelled proteins. For FCS measurements, fluctuations in fluorescence intensity are measured as molecules move in and out of a small illuminated region (volume ∼0.5 fL). Analysis of the fluctuations as a function of time provides information about the diffusion of the labelled species and has enabled us to measure the diffusion coefficient diffusion of GFP in the cytoplasm of the malaria parasite. A new Centre of Excellence in Coherent X-ray Science has been established to develop techniques for imaging cellular architecture with greatly increased resolution and to develop methods for determining the structures of membrane protein without the need for crystallisation. The methods employ a highly coherent curved beam and imaging in the far field with iterative Fourier transformation protocols to extract phase image information. Soft X-rays have wavelengths of about 1-10 nm; these wavelengths allow imaging at high spatial resolution and will be used to study the intracellular structures of P. falciparum-infected erythrocytes with a resolution of down to 10 nm.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/92P

AuPS/ASB Meeting - Canberra 2005 Symposium 5: Regulation of Membrane Transport Thursday 29th September 2005 Chair: David Adams

The canonical transient receptor potential channel 1 is an essential structural component of the mechanosensitive calcium permeable channel in vertebrate cells O.P. Hamill1, R. Maroto1, A. Kurosky2, T.G. Wood2, A. Raso3 and B. Martinac3, 1Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, Texas, U.S.A., 2Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas, U.S.A. and 3Department of Pharmacology, University of Western Australia, Crawley, WA, Australia. The mechanosensitive cation channel (MscCa) transduces membrane stretch into cation (Na+, K+, Ca2+ and Mg2+) flux across the cell membrane, and is implicated in cell volume regulation, cell locomotion, muscle dystrophy and cardiac arrhythmias (Hamill & Martinac, 2001). However, the membrane protein(s) forming the MscCa in vertebrates remains unknown. Here we use an identification strategy based on detergent-solubilizing of frog oocyte membrane proteins followed by liposome reconstitution and evaluation by patch-clamp (Sukharev et al., 1993; Maroto et al., 2005). The oocyte was chosen because it expresses the prototypical MscCa (³ 107 MscCa/oocyte) that is preserved in cytoskeleton-deficient membrane vesicles (Zhang et al., 2000). We identified a membrane protein fraction that reconstituted high MscCa activity and showed an abundance of an 80 kDa protein identified immunologically as the canonical transient receptor potential channel 1 (TRPC1) (Wes et al., 1995; Brereton et al., 2000). Heterologous expression of the human TRPC1 resulted in a > 1000% increase in MscCa patch density, whereas injection of a TRPC1-specific antisense RNA abolished endogenous MscCa activity. hTRPC1 transfection of CHO-K1 cells also significantly increased MscCa expression. These observations indicate that TRPC1 is a component of the vertebrate MscCa, which like various prokaryotic Ms channels (Martinac & Kloda, 2004), is gated by tension developed in the lipid bilayer. Brereton, H.M., Harland, M.L., Auld, A.M. & Barritt, G.J. (2000) Molecular and Cellular Biochemistry 214, 63-74 (2000). Hamill, O.P. & Martinac, B. (2001) Physiological Reviews 81, 685-740. Maroto, R., Raso, A., Wood, T.G., Kurosky, A., Martinac, B. & Hamill, O.P. (2005) Nature Cell Biology 7, 1443-1446. Martinac, B. & Kloda, A. (2003) Progress in Biophysics and Molecular Biology 82, 11-24. Sukharev, S.I., Martinac, B., Arshavsky, V.Y. & Kung, C. (1993) Biophysical Journal 65, 177-183. Wes, P.D., Chesvesich, J., Jeromin, A., Rosenberg, C., Stetten, G., & Montell, C. (1995) Proceedings of the National Academy of Sciences of the United States of America 92, 9652-9656. Zhang, Y., Gao, F., Popov, V.L., Wen, J.W. & Hamill, O.P. (2000) Journal Physiology 523, 117-130.

Proceedings of the Australian Physiological Society

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Sensing pressure with K2P channels

Eric Honore, Institut de Pharmacologie Moléculaire et Cellulaire, CNRS-UMR 6097, Institut Paul Hamel, 660 Route des Lucioles, Sophia Antipolis, 06560 Valbonne, France. (Introduced by Boris Martinac) The K2P channels are highly conserved from C. elegans to humans. They are structurally distinct from other K+ channel family members, with four transmembrane segments and 2P domains in tandem. K2P channels are homo- or hetero-dimers that play a dominant role in cell electrogenesis, controlling the resting membrane potential and the action potential duration. The K2P channel TREK-1 is predominantly expressed in the central and peripheral nervous system, with a particularly strong expression during early development. TREK-1 is activated by membrane stretch as well as cell swelling. Mechanical force is transmitted directly to the channel via the lipid bilayer. Moreover, intracellular acidosis strongly sensitizes TREK-1 to membrane stretch, leading to channel opening at atmospheric pressure. TREK-1 is reversibly opened by polyunsaturated fatty acids, including arachidonic acid (AA). Activation of TREK-1 by AA in the excised patch configuration indicates that the effect is direct by interacting either with the channel protein or by partitioning into the lipid bilayer. Additionally, TREK-1 channel activity is reversibly stimulated by volatile general anaesthetics including halothane. The recent invalidation of TREK-1 in a mouse model demonstrates that this K+ channel is important for neuroprotection against epilepsy and ischemia. Furthermore, TREK-1 -/- mice are also more resistant to volatile general anaesthetics, indicating a key role for TREK-1 in the mechanism of general anaesthesia. Mutagenesis studies have demonstrated that the cytosolic carboxy terminal domain of TREK-1 plays a key role in TREK-1 gating. Protonation of a key residue in this region, E306, leads to channel activation. Interaction of the carboxy terminal domain of TREK-1 with the inner leaflet phospholipids including PIP2 is critical for channel activity and is controlled by a cluster of cationic residues. Conversely, down-modulation of TREK-1 is achieved by receptor- coupled protein kinase A phosphorylation of residue S333. In conclusion, the TREK channels are polymodal K+ channels that integrate multiple physical and chemical stimuli.

Proceedings of the Australian Physiological Society

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Varieties of mechanotransduction: the cytoskeletal stress fibre as a force transmitter and a mechanosensor Masahiro Sokabe1, Kimihide Hayakawa2 and Hitoshi Tatsumi1, 1Department of Physiology, Nagoya University Graduate School of Medicine, Nagoya, 464-8558, Japan and 2ICORP/SORST Cell Mechanosensing, JST, Nagoya, 464-8550, Japan. It has been established that bacterial mechanosensitive (MS) channels are activated directly by stress in the membrane. However, whether eukaryote MS channels need some other accessory proteins, typically cytoskeletons, for their activation has been a pending problem. Theoretically the cytoskeleton would be a more efficient force transmitter than the membrane owing to its larger elastic modulus. It is likely that higher organisms have utilized such a device to increase the sensitivity of their MS channels. However, no direct evidence has been provided to show the role of cytoskeleton in MS channel activation. We have developed two methods by which we can stretch the actin-based cytoskeleton (stress fibres) while monitoring MS channel activities either by Ca2+ influx or whole cell currents measurements in cultured endothelial cells (HUVECs). In one method a fibronectin coated glass bead, which is attached on the apical cell surface and connected to the basal focal adhesions via stress fibres, was mechanically moved to stretch the attached stress fibres. In the other method, phalloidin coated beads microinjected into the cell where they attached to stess fibres. One of these was pulled with laser tweezers to stretch the attached stress fibre. In either way, we could consistently record stretch activated currents and Ca2+ transients that originated from the activation of cation selective MS channels carried by HUVECs. The force required for a single MS channel activation was estimated as low as 1-2 pN. Ultra fast near field Ca2+ imaging resolved the Ca2+ influx spots across individual MS channels near basal focal adhesions. Simultaneous imaging of integrin molecules indicated that MS channels are located near integrin molecules as close as a few hundred nm. Forces originally created by membrane deformation and transmitted through a stress fibre/integrin complex seem to activate MS channels. MS channels serve as a typical fast mechanosensor, however, they are not the only mechanosensor in the cell. Turning to slowly going mechanotransduction, we can see another world of mechanosensors. Endothelial cells in situ exhibit a spindle-like shape, aligning their long axis running along the vessel. They lose this characteristic shape when cultured in dish. However, they recover their original shape and alignment when subjected to uniaxial cyclic stretch (20% at 1Hz) that mimics circumferential cyclic stretch in the vessel. In other words, cells can detect the direction of applied forces and convert this information into their morphology. We found that Ca2+ influx via MS channels was indispensable for this mechanically induced cell shape change, but that the Ca2+ signal by itself could not indicate the direction of the force to the cell. As the stress fibres in the stretch axis were preferentially disrupted by cyclic stretch in a few minutes and then reorganized perpendicular to the stretch axis, we suspected that the stress fibre, particularly its major component actin fibre, might be a force direction sensor. To test this hypothesis in a more direct way, we examined the dynamics of single actin fibres in response to mechanical stretch. Surprisingly, relaxing but not stretching an actin fibre caused its rapid depolymerization with an aid of cofilin, a soluble actin-depolymerizing factor. In living cells disruption of stress fibres caused by actin fibre depolymerization activates several downstream signal molecules around the focal adhesion, which eventually leads to a cell shape change. In this sense, the stress fibre (actin fibre) is eligible as a mechanosensor. In conclusion, the stress fibre serves as a force transmitter in fast mechnotransduction, and acts as a mechanosensor with direction sensitivity in slow mechanotransduction.

Proceedings of the Australian Physiological Society

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Role of tryptophan residues in ion channel function Amitabha Chattopadhyay, Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India. (Introduced by David Adams) The linear peptide gramicidin forms prototypical ion channels specific for monovalent cations and has been extensively used to study the organization, dynamics and function of membrane spanning channels. The role of tryptophan residues is a key issue in ion permeation by gramicidin (Becker et al., 1991). We have monitored the organization and dynamics of gramicidin tryptophans in various types of microheterogenous molecular assemblies using wavelength-selective fluorescence and other approaches (Rawat et al., 2004; Kelkar & Chattopadhyay, 2005). Taken together, these results provide comprehensive information on the dynamics of the functionally important tryptophan residues of gramicidin. Experiments using synthetic analogues of gramicidin containing single tryptophan residues further help to delineate the crucial role of tryptophan in maintaining the ion conducting conformation of gramicidin. Becker, M.D., Greathouse, D.V., Koeppe, R.E. 2nd & Andersen, O.S. (1991) Biochemistry 30, 8830-8839. Kelkar, D.A. & Chattopadhyay, A. (2005) Biophysical Journal 88, 1070-1080). Rawat, S.S., Kelkar, D.A. & Chattopadhyay, A. (2004) Biophysical Journal 87, 831-843. Author e-mail: [email protected]

Proceedings of the Australian Physiological Society

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AuPS/ASB Meeting - Canberra 2005 Symposium 6: Membrane Associated Proteins that Regulate Muscle Contraction Thursday 29th September 2005 Chair: Angela Dulhunty

From DHPR to RyR and back again: What lies along the way? Kurt Beam, Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado, U.S.A. In skeletal muscle, excitation-contraction coupling depends on a bi-directional interaction between the dihydropyridine receptor (DHPR), a voltage-gated calcium channel in the plasma membrane, and the type 1 ryanodine receptor (RyR1), a homo-tetrameric calcium release channel in the sarcoplasmic reticulum (SR). As a consequence of this bi-directional interaction: (i) the DHPR, in response to depolarization of the plasma membrane, elicits Ca2+ release via RyR1 without an intervening second messenger, (ii) RyR1 increases the amplitude of Ca2+ currents via the DHPR, and (iii) DHPRs within the plasma membrane are organized into groups of four (tetrads) such that each DHPR is apposed to a subunit of RyR1. A number of approaches have been used to probe the protein-protein interactions that link the DHPR and RyR1, including expression of cDNAs in muscle cells null for DHPR subunits or for RyR1, biochemical analyses of binding, and application of peptides to isolated RyR1. However, these have not yet produced a consistent picture. We have been examining several alternative approaches for establishing the spatial interrelationships between DHPRs and RyR1. To determine the orientation of DHPRs within tetrads, the fluorescent proteins ECFP or EYFP were fused to sites of α1S or b 1a. Between N- and C-terminals, fluorescence resonance energy transfer (FRET) occurred between α1S subunits adjacent within tetrads, but not between adjacent b 1a subunits, consistent with the idea that the Nand C-terminals are oriented towards, and away from, the center of tetrads for α1S and b 1a, respectively. As a second approach, we have been determining which sites of the DHPR may be in close proximity to RyR1. This is accomplished by attachment of an ECFP-EYFP tandem (“CY”, 23 residue linker) or a biotin acceptor domain (BAD: 70 or 97 residues) to DHPR sites. For CY-b 1a and α1S-CY, FRET efficiency increased after expression in dyspedic myotubes (no RyR1) compared to dysgenic myotubes, suggesting that RyR1 may closely appose the b 1a N-terminal and α1S C-terminal. In the case of the BAD fusions, expressing myotubes were fixed and permeabilized and exposed to fluorescently labeled NeutrAvidin (∼60 kDa). NeutrAvidin had access to BAD at the N- and C-terminals of b 1a and to the α1S N-terminal and II-III loop (“peptide A” region). NeutrAvidin did not have access to α1S-BAD in dysgenic myotubes, but did have access to α1S-BAD in dyspedic myotubes. Thus, two independent approaches suggest that the C-terminal of α1S may be closely apposed to RyR1.

Proceedings of the Australian Physiological Society

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Regulation of ryanodine receptors from skeletal and cardiac muscle by components of the cytoplasm and lumen D.R. Laver, School of Biomedical Sciences, University of Newcastle, and Hunter Medical Research Institute, Callaghan, NSW 2308, Australia. Contraction in skeletal and cardiac muscle occurs when Ca2+ is released from the sarcoplasmic reticulum (SR) through ryanodine receptor (RyR) Ca2+ release channels. Ca2+, Mg2+ and ATP are key regulators of RyRs. Skeletal (RyR-1) and cardiac (RyR-2) RyRs are modulated differently by these ligands and these differences may underlie the different characteristics of excitation-contraction (EC) coupling in skeletal and cardiac muscle. RyRs are regulated by two Ca2+/Mg2+-dependent mechanisms. They are activated at ∼1 m mol/l [Ca2+] and inhibited at mmol/l [Ca2+] in the cytoplasm. Mg2+ can inhibit RyRs by binding at the Ca2+ activation and inhibition sites. ATP strongly activates RyR-1 in the virtual absence of cytoplasmic Ca2+ while in RyR-2, ATP primarily enhances Ca2+ activation. The Ca2+ load of the SR is an important stimulator of Ca2+ release in skeletal and cardiac muscle. It is known that luminal Ca2+ stimulates RyRs but the mechanisms for this are not understood. In cardiac muscle, the release of Ca2+ from the SR strongly reinforces RyR activation, a process called Ca2+-induced Ca2+ release (CICR). Although CICR should provide an explosive, positive feedback in Ca2+ release, the quantity of Ca2+ released from the SR has a graded, stable dependence on the magnitude of the Ca2+ inflow through the DHPRs. In order to understand the mechanisms controlling Ca2+ release in skeletal and cardiac muscle, single RyRs and RyR arrays were incorporated into artificial lipid bilayers. SR vesicles were prepared from the back and leg muscles of New Zealand rabbits and from sheep hearts. Animals were killed by barbiturate overdose prior to muscle removal. SR vesicles containing RyRs were incorporated into artificial planar lipid bilayers which separated baths corresponding to the cytoplasm and SR lumen. The baths contained 30-230 mmol/l CsCH3O3S, 20 mmol/l CsCl, 10 mmol/l TES (pH 7.4) plus various amounts of Ca2+, Mg2+ and ATP. Channel activity was recorded using Cs+ as the current carrier. Several proteins influence the way RyRs are regulated by luminal Ca2+. The luminal proteins, calsequestrin (CSQ), triadin and junctin are associated with RyRs. By dissociating CSQ from RyR-1 it was shown that CSQ inhibits RyRs and can enhance the activating effect of luminal Ca2+. In addition, CSQ dissociates from RyRs when luminal Ca2+ exceeds 4 mmol/l. These observations reveal several possible mechanisms by which CSQ can act as a sensor for luminal [Ca2+]. The action of luminal Ca2+ on RyR-1 and RyR-2 was strongest in the absence of cytosolic Ca2+ and the potency of the luminal Ca2+ was enhanced by membrane potentials favouring Ca2+ flow from lumen to cytoplasm. At these voltages, RyR-1 activity rose ∼5-fold by raising luminal [Ca2+] from zero to ∼100 m mol/l while a further increase to mmol/l levels caused ∼30% fall from peak activity. RyR-2 had a more exaggerated Ca2+ dependence than RyR-1. RyR-2 activity increased ∼100 fold between zero and 100 Ca2+ and decreased by 90% from peak activity at 1 mmol/l Ca2+. Luminal Mg2+ inhibited RyRs by competing with luminal Ca2+ for both activating and inhibiting luminal sites. Thus Ca2+ and Mg2+ regulated RyR activity in a very similar way from both the luminal and cytoplasmic sides. RyRs showed coupled gating when conditions favoured Ca2+ flow from the luminal to cytoplasmic baths. The rate constant for channel opening was increased by the opening of other RyRs in the bilayer. This indicates that the close packed RyR arrays seen in muscle are retained during isolation and bilayer incorporation. In these arrays, luminal Ca2+ can permeate an open channel to activate neighbouring RyRs. Coupled openings were followed by a rapid and complete closure of all the channels that occurred within 10 ms. This may be the first inactivation phenomena demonstrated in vitro that could possibly explain the rapid termination of Ca2+ sparks and the graded control of Ca2+ release in cardiac EC coupling.

Proceedings of the Australian Physiological Society

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Structural and functional characterisation of the interaction of the dihydropyridine receptor IIIII loop with the ryanodine receptor M.G. Casarotto, Y. Cui, Y. Karunasekara, P.J. Harvey, N. Norris, P.G. Board and A.F Dulhunty, Division of Molecular Bioscience, The John Curtin School of Medical Research, The Australian National University, ACT 0200, Australia. In skeletal muscle, the dihydropyridine and ryanodine receptors (DHPR & RyR) are two membrane proteins that play a central role in excitation-contraction coupling. It is now widely accepted that an interaction between these two proteins is involved in triggering the release of calcium via the RyR into the SR. Recent attention has focused on the exact site of interaction and the loop between the second and third repeats of the skeletal DHPR α1 subunit (II-III loop) has been shown to be a critical region interaction site. In an attempt to correlate the structure of this loop with its function our group has previously determined the structure of several functionally active peptides derived from the II-III loop, however structural data for the whole II-III loop at a molecular level has remained elusive. In this study we focus on the structure/function relationship of the full DHPR II-III loop. This protein has been fully expressed, purified and fully assigned by multidimensional NMR techniques. The conformation of the protein exists as a series of helical elements and turns arranged in an open type structure. The location of the binding site on the RyR has been identified and this fragment has been expressed, purified and refolded. We show by fluorescence experiments that these proteins interact with micromolar affinity. We highlight the regions of the II-III loop that are important for interaction with the RyR.

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ClC-1 chloride channel - matching its properties to a role in skeletal muscle G. Rychkov, School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, SA 5005, Australia. ClC-1 is a member of a large family of Cl- channels. It is primarily expressed in skeletal muscle, and is essential for maintaining normal electrical excitability of the muscle. Mutations in the gene encoding ClC-1 have been shown to cause myotonia, an impairment of skeletal muscle relaxation after voluntary contraction. Myotonia results from an increase in muscle excitability that can be detected in electromyograms in the form of myotonic runs. In humans, there are two forms of this disease: autosomal recessive Becker-type myotonia congenita, and autosomal-dominant myotonia or Thomsen disease. ClC-1, as the other members of this family, is a dimeric, double pored channel, with each monomer forming an individual conduction pathway. ClC-1, which has been studied extensively using electrophysiological techniques, shows a complex gating behaviour. It displays two types of gating — a faster gating process that opens and closes each protopore independently (the ‘fast’ or ‘single pore’ gates), and a slower gating process that closes both protopores simultaneously (the ‘slow’ or ‘common’ gate). Both types of gating depend on permeating anions, and intracellular and extracellular pH. Recent results show that gating of ClC-1 is also regulated by intracellular nucleotides. Dependence of ClC-1 on pH and ATP makes it a likely contributor to a complex mechanism that regulates muscle contractility in exercise and fatigue. The exact role of ClC-1 in muscle physiology, however, is yet to be established.

Proceedings of the Australian Physiological Society

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Pores, channels and transporters: computational studies of membrane transport Peter Tieleman, Department of Biological Sciences, University of Calgary, 2500 Unniveristy Drive NW, Calgary AB, T2N 1N4, Canada. A key function of biological membranes is to provide mechanisms for controlled transport of ions, nutrients, metabolites, peptides and proteins between a cell and its environment. We are using computer simulations to study several processes involved in transport. In model membranes, the distribution of small molecules can now be accurately calculated; we are making progress toward understanding the factors that determine the partitioning behavior in the inhomogeneous lipid environment, with implications for, e.g.. the energetics of arginine-lipid interactions in voltage-gated potassium channels. Computer simulations of complex membrane proteins such as potassium channels and ABC-transporters can give detailed information about the atomistic dynamics that forms the basis of ion transport, selectivity, conformational change, and the molecular mechanism of ATP-driven transport. I will illustrate this with recent simulation studies of the voltage-gated potassium channel KvAP and the ABC-transporter BtuCD.

Proceedings of the Australian Physiological Society

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AuPS/ASB Meeting - Canberra 2005 Free Communications 5: Cardiac muscle Thursday 29th September 2005 Chair: Lea Delbridge

Does lignocaine increase the chance of survival from massive heart attack? S.M. Weiss and P.W. Gage, John Curtin School of Medical Research, Australian National University, ACT 0200, Australia. Introduction: Lignocaine (lidocaine) blocks voltage-activated sodium channels and has been used extensively since the 1960s in patients presenting with suspected acute myocardial infarction (AMI). In a review of many clinical trials and publications, Yadav & Zipes (2004) concluded that although prophylactic lignocaine administered after a suspected AMI appeared to reduce the incidence of primary ventricular fibrillation (VF, the fastest and most lethal tachyarrhythmia) by as much as 33%, lignocaine was also associated with an increased incidence of bradycardia (slow heart rate), asystole (no heart beat), and subsequent mortality. Because of this, Yadav & Zipes recommended that on the basis of contemporary information, prophylactic lidocaine should not be used in the management of patients with proved or suspected AMI. Aim: To determine in an animal model of AMI whether lignocaine reduces the incidence of tachyarrhythmias (VF and/or haemodynamically compromising ventricular tachycardia (VT)) when administered prior to a coronary artery occlusion sufficient to produce an AMI. Methods: 21 pigs (M+F, 20-35 kg) were sedated with stresnil (1-2 mg/kg im), anaesthetised with thiopentone sodium (10-15 mg/kg iv) and maintained under general anaesthesia with a mixture of isoflurane (0.5 – 2%) in oxygen. Artificial ventilation was maintained at a volume of 15 ml/kg and a rate of 12 breaths per minute. An intravenous saline drip was maintained for intra-operative hydration or lignocaine administration. Blood pressure (BP) and a lead II electrocardiogram (ECG) were monitored, digitised and recorded. Lignocaine (2.5 – 12 mg/kg bolus plus 0.05 – 0.24 mg/kg/min iv continuous infusion) was administered to 11 of the pigs. Following a mid-sternotomy and dissection of the pericardium, the left anterior descending coronary artery (LAD) was ligated 40 min (39 +/- 13 min sd) after the commencement of lignocaine or saline administration mid-way along its length. Results: The results in Table column 1 refer to the number of animals; the remaining columns refer to all animals. Sustained is defined as lasting longer than 15s and likely to be fatal if not externally reverted.

Control (n=10) Lignocaine (n=11)

animals developing sustained arrhythmia 10 6

sustained VTs in 1st 2 h

sustained VFs in 1st 2 h

13 3

43 10

non-sustained arrhythmias between 1 and 15s in 1st 2 h 91 70

total arrhythmias in 3rd hour

88 2

Discussion and Conclusion: The results clearly showed that when lignocaine was administered prior to a coronary artery occlusion it significantly reduced the number of animals which developed a haemodynamically compromising tachyarrhythmia and the number of sustained and non-sustained tachyarrhythmias for all animals.So why then do Yadav & Zipes recommend that lignocaine not be used? Consider the following 2 points: a) After a coronary artery occlusion, the distal tissue becomes ischaemic, hypoxic, and ultimately infarcted. Between the ischaemic region and the surrounding perfused region there is a border zone which receives limited perfusion. Clearly then, iv lignocaine administered after a coronary artery occlusion can not have a pharmaceutical effect on the ischaemic region other than at the border zone. b) Tachyarrhythmias can develop from AMIs in which the occlusion remains intact as well as from AMIs in which the occlusion dissipates and the tissue becomes reperfused. From these 2 points, we can develop 3 scenarios: 1) that an ischaemic region can become reperfused subsequent to an occlusion if the occlusion dissipates, 2) that an occlusion can remain intact but the ischaemic region can be small either because the occlusion is in a small artery or because the border zone is wide as a result of extensive collateral circulation, and 3) that an occlusion can remain intact and produce a large ischaemic region with a narrow border zone. In light of our results and the reduction in incidence in arrhythmias quoted by Yadav & Zipes, we suggest that lignocaine would likely reduce the incidence of arrhythmias in the first 2 scenarios wherein iv lignocaine could perfuse a large portion of the ischaemic region. In contrast, we suggest that in the 3rd scenario, iv lignocaine would never reach the ischaemic region and subsequently it would have no effect on that tissue, irrespective of the dose administered. Finally, we suggest that the lignocaine-related bradycardic and asystolic deaths referred to by Yadav & Zipes may have resulted from overdosing of lignocaine in a setting where it was showing no effect as in scenario 3. In this instance lignocaine, being a sodium channel blocker, would be shutting-down cell conduction. Because of these considerations, we argue with Yadav & Zipes’ recommendation and suggest that lignocaine is beneficial in reducing the incidence of tachyarrhythmia in those AMIs where it can be delivered to the ischaemic tissue but the serum levels need.to be kept low so action potentials are not blocked. Yadav, A.V. & Zipes, D.P. (2004) The Americal Journal of Cardiology 94, 606-608.

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Acute application of n-3 polyunsaturated fatty acids modify calcium sparks in permeabilised rat cardiac myocytes Bonny Honen, Rebecca Dalton, Dirk van Helden and Derek Laver, School of Biomedical Sciences, University of Newcastle and Hunter Medical Research Institute, Callaghan, NSW 2308, Australia. Animal studies have demonstrated that acute administration of n-3 polyunsaturated fatty acids (PUFAs) prevent ischemia-induced arrhythmias (Billman, et al., 1997). During and following ischemia, the sarcoplasmic reticulum (SR) becomes overloaded with Ca2+ and spontaneous release events occur (Daniels et al., 1991). The subsequent rise in cytosolic [Ca2+] activates sarcolemmal current which can in turn produce after-depolarisations and arrhythmias. PUFAs can reduce the ionic currents responsible for the cardiac action potential and this is believed to be the mechanism for their cardio-protective effects (Xiao et al., 1997). Studies on the effects of PUFAs on Ca2+ handling have shown that 10 m mol/l of eicosapentaenoic acid (EPA) resulted in a 15% reduction in the amplitude of spontaneous Ca2+ waves (Negretti et al., 2000). Also 15 m mol/l EPA was found to reduce both the width and duration of Ca2+ sparks by ∼25% (Honen et al., 2003). When PUFAs were applied directly to the SR Ca2+ release channel (Ryanodine receptor, RyR) in artificial bilayers, 30-50 m mol/l caused a 50-80% decrease channel activity. It is not clear if the action of PUFA’s on cell Ca2+ handing is mediated primarily by the sarcolemma or SR. This study aimed to determine if PUFAs could directly affect the Ca2+ release properties of the intact SR. This was done by measuring the properties of Ca2+ sparks in permeabilised cardiac myocytes in which sarcolemmal ion currents did not contribute to Ca2+ release within the cell. Sprague-Dawley rats were anesthetized with sodium pentobarbitone (1ml/kg), the hearts were removed and the cardiac ventricular myocytes were isolated by enzymatic digestion. Following isolation, the myocytes were treated with saponin to permeablise the sarcolemma. Ca2+ sparks were viewed using confocal microscopy in line scan mode using the Ca2+ indicator fluo-3. Fatty acids tested were oleic acid (OA), arachidonic acid (AA), EPA and docosahexaenoic acid (DHA). Images of Ca2+ sparks were collected prior to the addition of fatty acids and at 2 min and 5 min following their addition. Sham experiments were performed to ensure SR Ca2+ rundown did not occur. Spark properties did not vary during experiments in both sham and OA (mono unsaturated fatty acid) treated cells indicating that rundown did not occur. However, PUFA’s did affect some spark properties. AA at 50 m mol/l, significantly reduced (10%) spark width within 2 min of exposure. EPA at 50 m mol/l significantly reduced spark intensity (21%) within 5 min. Exposure to 50 m mol/l DHA for 2 min reduced intensity by ∼25% and spark mean rate of rise by ∼20%. Following exposure for 5 min, spark frequency reduced by ∼30% and spark width reduced by ∼7%. Even at 30 m mol/l DHA was observed to significantly alter spark properties within 2 min. The actions of fatty acids on Ca2+ sparks in this study were similar to those seen on the open probability of RyRs (Honen et al., 2003). During ischemia, fatty acids are released within the cell by PLA2. Fatty acid concentrations (all species) up to 0.73 mmol/l have been measured in rat aortic plasma during transient ischemia (Chen et al., 2001). Previously we have shown that 10-20% of membrane fatty acids can release n-3 PUFAs and so it is quite possible for free PUFA levels to reach 70 m mol/l. Therefore under physiological ischemic conditions it is likely that PUFAs could play an important role in protecting myocardium from ischemia by modulating Ca2+ handling. Billman, G.E., Kang, J.X. & Leaf, A. (1997) Lipids 32, 1161-1168. Chen, L.G., Nohara, R., Hirai, T., Li, X.H., Kataoka, K., Hosokawa, R., Masuda, D., Fujita, M., Taguchi, S. & Sasayama, S. (2001) Japanese Circulation Journal 65, 550-555. Daniels, M.C.G., Fedida, D., Lamont, C. & ter Keurs, H.E. (1991) Circulation Research 68, 1408-1421. Honen, B.N., Saint, D.A. & Laver D.R. (2003) Journal of Membrane Biology 196, 95-103. Negretti, N., Perez, M.R., Walker, D. & O’Neill, S.C. (2000) Joournal of Physiology 523, 367-375. Xiao, Y.F., Gomez, A.M., Morgan, J.P., Lederer, W.J. & Leaf, A. (1997) Proceedings of the National Academy of Science USA 94, 4182-4187.

Proceedings of the Australian Physiological Society

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Abnormal calcium transients and calcium handling protein expression in cardiomyocytes from mdx (dystrophic) mice I.A. Williams and D.G. Allen, Institute for Biomedical Research, School of Medical Sciences, University of Sydney F13, NSW 2006, Australia. Duchenne muscular dystrophy (DMD) is a fatal X-linked genetic disorder caused by deficiency of the cytoskeletal protein dystrophin. DMD patients have extensive skeletal muscle degeneration and a dilated cardiomyopathy (DCM). The mdx mouse also lacks dystrophin and in skeletal muscle it exhibits membrane damage and an abnormal influx of Ca2+. The present study was aimed at characterising ventricular performance which may contribute to DCM in mdx mice. Ventricular myocytes were isolated from 8-week old wild-type and mdx mice and intracellular Ca2+ measured with the fluorescent indicator fluo-4 during electrical and caffeine (10 mM) induced stimulation. Protein expression of the ryanodine receptor (RyR), the sarco endoplasmic reticulum calcium ATPase (SERCA) and phospholamban were analysed using immunoblotting techniques. The peak of the electrically stimulated Ca2+ transient was significantly greater in mdx mice, but the time to peak was significantly shorter. These findings were not the result of increased sarcoplasmic reticulum (SR) Ca2+ loading as the caffeine-induced Ca2+ transient peak was unchanged in mdx mice. The increase in peak calcium transient and the decreased time to peak could be due to the significantly increased levels of RyR protein expression (4-fold), allowing more rapid Ca2+ release from the SR during excitation. However, this is not usually found in established DCM (Kubo et al., 2001). It was found that the rate of decline of the electrically stimulated Ca2+ transient was significantly slower in mdx mice, but the rate of decline of the caffeine-induced Ca2+ transient was unchanged, suggesting that the slower removal of Ca2+ from the intracellular milieu was a result of decreased SERCA activity, and not decreased sodium-calcium exchanger activity. SERCA protein levels were unchanged, but phospholamban levels were increased significantly (2-fold). The slower rate of decline of the Ca2+ transient in mdx mice is therefore possibly a result of increased inhibition of SERCA by phospholamban, a finding which is consistent with other studies of DCM (Meyer et al., 1995). This is further supported by the finding that the SERCA/phospholamban ratio was significantly smaller in mdx mice. It is concluded that dystrophin deficiency causes impairment in the Ca2+ handling properties of mdx ventricular myocytes, which may play a role in the development of DCM. Future work will test whether the increase in peak Ca2+ transients in the mdx mouse is an early compensatory mechanism that reverses as the cardiomyopathy progresses, by investigating myocyte Ca2+ handling in older mice. Kubo, H., Margulies, K.B., Piacentino, V 3rd., Gaughan, J.P. & Houser, S.R. (2001) Circulation 104:1012-8. Meyer, M., Schillinger, W., Pieske, B., Holubarsch, C., Heilmann, C., Posival, H., Kuwajima, G., Mikoshiba, K., Just, H. & Hasenfuss, G. (1995) Circulation 92, 778-784. This work was supported by a Northcote Graduate Scholarship to IAW and by the NHMRC.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/104P

Mechanisms underlying the stretch-dependent slow inotropic response in isolated mouse myocardium M.L. Ward1 and D.G. Allen2, 1Department of Physiology, University of Auckland, New Zealand and 2School of Medical Sciences, University of Sydney F13, NSW 2006, Australia. When cardiac muscle is subjected to stretch the force of contraction increases, allowing the intact heart to adjust its output to the body’s demand (Allen & Kentish, 1985). This increase in contractility has been shown in vivo to occur in two distinct phases. Initially there is an abrupt increase in force that coincides with the stretch, and secondly there is a slower response that develops over a period of a few minutes (the ‘‘slow force response’’). The first of these responses is largely due to a change in the sensitivity of the contractile proteins to Ca2+, whereas the slow force response is accompanied by a concomitant increase in the magnitude of the intracellular Ca2+ transient (the event that initiates contraction). It has been proposed that stretch-activated channels contribute to Ca2+ entry after stretch (Calaghan & White, 2004). The aim of the present study was to reinvestigate the mechanisms underlying the slow force response of cardiac muscle. Mice were euthanased and cardiac trabeculae or papillary muscles (< 1 mm in length, and 0.1 - 0.3 mm in diameter), dissected from the right ventricle of mouse hearts, were mounted in a muscle chamber between a hook attached to a force transducer and a lever connected to a motor capable of making precise changes in muscle length. Each preparation was then subjected to a step increase in length for 2 minutes whilst isometric force was recorded.

Response of a representative mouse papillary muscle subjected to step increases in length before, and during application of GdCl3. One minute after the initial length change, active force increased by 77 ± 17% of the force immediately following the stretch (n = 16). Subsequent application of either 400 µM streptomycin, or 20 µM GdCl3 (blockers of stretch-activated channels) reduced the slow force response (p £ 0.01) for identical step increases in length (streptomycin: from 86 ± 25% to 38 ± 14% (n=9), or GdCl3: from 65 ± 21% to 12 ± 7%, n=7), suggesting a possible role for stretch-activated channels in the slow force response. Allen, D.G. & Kentish, J.C. (1985) Journal of Molecular and Cellular Cardiology 17, 821-840. Calaghan, S. & White, E. (2004) Journal of Physiology 559, 205-214.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/105P

Salutary effects of pyruvate are more evident in female than male glut4-deficient mouse hearts C.E. Huggins1, J. Favaloro2, J. Proietto2, S. Pepe3 and L.M.D. Delbridge1, 1Department of Physiology, University of Melbourne, VIC 3010, Australia, 2Department of Medicine, University of Melbourne, Austin and Repatriation Medical Centre, Heidelberg, VIC 3084, Australia and 3Alfred Hospital and Baker Heart research Institue, Prahran, VIC 3181, Australia. The mechanisms involved in diabetic cardiac pathology are not well understood. Insulin resistance, defined as a decrease in the ability of insulin to stimulate cellular glucose uptake is often termed the “prediabetic” state. Insulin-stimulated glucose uptake in the heart is mediated by the glut4 transporter. It is known that alterations in substrate availability are associated with cardiac hypertrophy, reduced energy production and subsequent cardiac contractile dysfunction (Taegtmeyer et al., 2002). There is some epidemiologic evidence indicating that diabetes has greater negative impact on cardiovascular morbidity and mortality in women than men (Sowers, 1998). The goal of this study was to investigate the metabolic basis for this sex-specific vulnerability in the heart. The inotropic actions of pyruvate, a metabolic product of glycolysis and an oxidizable fuel in the heart, were investigated in a genetic animal model of insulin resistance. Hearts of age-matched female and male mice (22 week) from three genetic groups were evaluated: wildtype (WT), ‘knock-down’ (KD, 15% WT glut4) and ‘knock-out’ (KO, £ 5% WT glut4). Mice were anaesthetised with pentobarbitone sodium (70mg/kg, ip), and hearts excised and arrested in iced KrebsHenseleit buffer. Hearts were perfused (Langendorff-mode) in normoxic conditions with Krebs-Henseleit bicarbonate buffer (37ºC). Left ventricular function was measured using a fluid-filled balloon interfaced to a pressure transducer (MLT884). 5mM glucose with 100uU/ml insulin was provided as the substrate for basal measurements. The perfusate was then supplemented with 5mM pyruvate. Under basal conditions hearts of female and male glut4-KO mice exhibited significantly reduced developed pressure relative to WT. Hearts of female glut4-KD mice were significantly more functionally impaired than hearts of male glut4-KD mice relative to WT. Pyruvate supplementation significantly improved developed pressure in female and male glut4-KD & glut4-KO hearts. Interestingly, female glut4-KO hearts were most responsive to pyruvate supplementation. Developed Pressure

Genotype

Basal (mmHg)#

With 5 mM pyruvate (mmHg)# WT 137.9 ± 12.9 126.5 ± 11.6 Female KD 106.2 ± 13.1 * 139.7 ± 13.3 KO 108.4 ± 5.7 * 182.7 ± 7.6 WT 151.2 ± 13.3 147.2 ± 6.7 † Male KD 145.8 ± 6.2 175.9 ± 6.3 KO 91.7 ± 10.4 * 157.4 ± 18.9 # p<0.05 sex ´ genotype * p<0.05 vs WT, †p<0.05 vs KO The acute improvement in isolated function of glut4-deficient hearts after pyruvate supplementation suggests that substrate limitation is a major cause of contractile dysfunction. The responsiveness of glut4-deficient hearts to pyruvate indicates that adaptive metabolic remodelling may have occurred early in development to preserve metabolic ‘flexibility’. This adaptation may be accentuated in insulin resistant hearts of females. Sowers, J.R. (1998) Archives of Internal Medicine 158, 617-321. Taegtmeyer, H., McNulty, P. & Young M. (2002) Circulation 105, 1727-1733.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/106P

The angiotensin type 2 receptor prevents cell death in neonatal cardiomyocytes of the hypertrophic heart rat E.R. Porrello1,2, A. D’Amore2, C.L. Curl1, S.B. Harrap1, W.G. Thomas2 and L.M.D. Delbridge1, 1Department of Physiology, The University of Melbourne, VIC 3010, Australia and 2Baker Heart Research Institute, Prahran, VIC 3004, Australia. The Hypertrophic Heart Rat (HHR) displays cardiomyocyte hypertrophy in Association with an apparent reduction in myocyte number in adulthood (Harrap et al.). This suggests the possibility of reduced hyperplasia or increased apoptosis during very early cardiac development. The angiotensin AT1 and AT2 receptor subtypes have been implicated in both cellular growth and apoptosis, although the precise mechanisms are unclear. Cardiac AT2 receptor expression is high during early development (Bastien et al.), and it has been suggested that AT2 receptor-mediated actions counterbalance those of the AT1 receptor. Specifically, it has been proposed that the AT2 receptor inhibits growth and promotes apoptosis, but data from transgenic and knock-out experiments do not support this hypothesis. The aim of this study was to determine the relationship between cardiac AngII receptor expression levels and neonatal cardiomyocyte growth and apoptotic responses in the HHR compared with their Normal Heart Rat (NHR) control strain. Cardiac ventricles were freshly harvested from HHR and NHR neonates at post-natal day 2. Tissue AT1A and AT2 mRNA expression levels were quantified by real-time RT-PCR. Relative to NHR, HHR neonatal hearts exhibited significantly higher AT2 and lower AT1A receptor expression levels (4.6-fold higher AT2/AT1 ratio in HHR compared with NHR). Neonatal cardiomyocytes were isolated by enzymatic digestion and plated at high density (1250 cells/mm2). Adenoviruses containing constructs for either the AT1A or AT2 receptors were created. After 48 hours, myocytes were infected with either AT1A and/or AT2 receptors to achieve a physiological level of receptor expression (150 fmol receptor protein/mg total cell protein). In addition, to mirror receptor expression in neonatal HHR hearts, cells were infected with AT1A and AT2 receptors in a 4:1 ratio. Adenoviruses also coexpressed green fluorescent protein (GFP), making possible to identify and morphologically assess infected cells. To assess myocyte apoptosis counts were performed (5 fields from each triplicate well in n = 4 experiments) of infected HHR and NHR cells that displayed vacuolisation. The incidence of apoptosis was studied after 72 hours exposure to 0.1 m M AngII. When infected with the AT1A receptor alone, HHR myocytes showed significantly higher proportions of apoptotic cells than NHR (22.7%, SE 4.1 vs 1.1%, SE 0.6, P < 0.001). With the addition of the AT1A receptor antagonist candesartan (1 m M), the proportion of apoptotic cells in HHR with the AT1A receptor alone fell to levels similar to those seen in NHR (1.8%, SE 0.8). A similar suppression of apoptosis was observed (2.0%, SE 0.9) when the PKC signal transduction pathways that mediate AT1A receptor signalling were inhibited with BIM (1 m M). When cells were infected with both the AT1A and AT2 receptors, evidence of apoptosis in HHR cells virtually disappeared (0.4%, SE 0.1). In HHR neonatal cardiomyocytes, intrinsic (presumably genetic) differences seem to predispose to significantly increased AngII-induced apoptosis when the AT1A receptor is expressed in isolation. Coexpression of the AT1A and AT2 receptors rescues the cells from apoptosis. These findings suggest novel protective physiological mechanisms for the AT2 receptor in early cardiac growth. Bastien, N.R., Ciuffo, G.M., Saavedra, J.M. & Lambert, C. (1996) Regulatory Peptides, 63, 9-16. Harrap, S.B., Danes, V.R., Ellis, J.A., Griffiths, C.D., Jones, E.F. & Delbridge, L.M.D. (2002) Physiological Genomics, 9, 43-48.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/107P

AuPS/ASB Meeting - Canberra 2005 Free Communications 6: Cellular signaling Thursday 29th September 2005 Chair: Craig Neylon

Analyses of the actin cytoskeleton using fluorescence resonance energy transfer (FRET) C.G. dos Remedios1, D. Chhabra1, I. Dedova1, D. Safer2 and E DeLaCruz3, 1Institute for Biomedical Research F13, University of Sydney, NSW 2006, Australia, 2Department of Physiology, University of Pennsylvania, School of Medicine, Philadelphia, PA, 19104, USA, and 3Department of Molecular Biophysics & Biochemistry, Yale University, 266 Whitney Ave, PO Box 208114, New Haven, CT, 06520-8114, USA. Actin is the principal component of microfilaments whose assembly/disassembly is essential for cell motility. It is present in the nucleus, where it may regulate gene expression. Cofilin is the principal regulator of actin assembly in cells. It can bind actin and translocate it into the nucleus during times of stress. We used fluorescence resonance energy transfer (FRET) and confocal microscopy to analyse the interactions of cofilin and G-actin in the nucleus and cytoplasm. By measuring the rate of photobleaching of fluorescein-labeled actin ± Cy5-labeled cofilin, we show that most of the nuclear G-actin is bound to cofilin, but only half is bound in the cytoplasm. A significant proportion of cofilin in the nucleus and cytoplasm binds added TMR-labeled G-actin. These data suggest there is significantly more cofilin-G-actin complex and less free cofilin in the nucleus. The actin cytoskeleton can also be probed in solution using FRET spectroscopy. This method can not only detect binding events but it can also detect structural changes in these proteins. We recently demonstrated that thymosin b 4 (tb 4) binding induces spatial rearrangements within subdomains 1 and 2 of G-actin. Tb 4 binding increases the distance between Gln-41 and Cys-374 of actin by 2 Å and decreases the distance between bound ATP (b ATP at the NUC site) and Lys-61 by 1.9 Å. The distance between Cys-374 and Lys-61 is minimally affected. Our results favour a model where tb 4 changes the orientation of actin subdomain 2. This conformational change presumably accounts for the reduced rate of nucleotide and amide hydrogen exchange from actin monomers.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/108P

Profilin binding to sub-micellar concentration of polyphosphoinositides PI(4,5)P2 and PI(3,4,5)P3 P.D.J. Moens, School of Biological, Biomedical and Molecular Sciences, The University of New England, Armidale, NSW 2351, Australia.

Profilin is a small (12-14 kDa) actin binding protein which promotes filament turnover. Profilin is also involved in the signalling pathway linking the receptors in the cell membrane to the microfilament system within the cell. Profilin is thought to play critical roles in this signalling pathway through its interaction with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] (Lu et al., 1996). So far, profilin’s interaction with polyphosphoinositides (PPI) has only been studied in micelles or small vesicles. Profilin binds with high affinity to small clusters of PI(4,5)P2 molecules. The binding stoichiometry of PI(4,5)P2 to profilin ranges from 5:1 to 10:1 (Goldschmidt-Clermont et al., 1991). In the cell, PPI lipids are not structured as they are in micelles or small vesicles, therefore their interaction with profilin might be quite different. In this work, we investigated the interactions of profilin with sub-micellar concentrations of PI(4,5)P2 and PI(3,4,5)P3. We determined the relevant association/dissociation constant by fluorescence anisotropy when sub-micellar concentrations of fluorescently labelled PPI lipids bind to profilin. We show that the association/dissociation constant of profilin with sub-micellar concentrations of PPI lipids is significantly different to that of profilin with micelles or small vesicles. We also show that profilin binds more strongly to sub-micellar concentrations of PI(3,4,5)P3 than to sub-micellar concentrations of PI(4,5)P2. Goldschmidt-Clermont, P.J., Kim, J.W., Machesky, L.M., Rhee, S.G. & Pollard, T.D. (1991) Science 251, 1231-3. Lu, P.J., Shieh, W.R., Rhee, S.G., Yin, H.L. & Chen, C.S. (1996) Biochemistry 35, 14027-34.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/109P

Phospholipase Cg is essential for activation of store-operated Ca2+ channels in liver cells T. Litjens1, T. Nguyen1, E. Aromataris1, M. Roberts1, G. Barritt2 and G. Rychkov 1, 1School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, SA 5005 and 2School of Medicine, Flinders University of South Australia, G.P.O. Box 2100, Adelaide, SA 5001, Australia. Release of Ca2+ from intracellular stores in non-excitable cells results in activation of Ca2+ influx through so-called store-operated Ca2+ channels (SOCs) on the plasma membrane (Putney et al., 2001). Activation of these channels occurs in response to a decrease in the concentration of Ca2+ in the lumen of the endoplasmic reticulum, and it does not depend on how this decrease in [Ca2+] is initiated. The molecular mechanism that underlies this phenomenon is poorly defined. Phospholipase Cg (PLCg ) has been previously shown to be either directly involved in activation of SOCs or to modulate their activity through the production of additional IP3 in a number of cell lines (Patterson et al., 2002). The identity of the SOCs regulated by PLCg , however, has not been established. In this work we used short interfering RNA (siRNA) to specifically reduce the expression of the genes encoding PLCg 1 and PLCg 2 and whole cell patch clamping technique to measure activation of store-operated Ca2+ current (ISOC) in H4IIE liver cells. Immunofluorescence and Western blotting were employed to verify the effectiveness of siRNA and the time course of the knock down of PLCg . We have found that transfection of H4IIE liver cells with siRNA against PLCg 1 results in time dependent reduction of PLCg 1 protein with maximal effect apparent at 72-96 h. At the same time the amplitude of the ISOC developed in response to intracellular perfusion with IP3 in cells transfected with siRNA against either PLCg 1 or 2 has decreased. The average maximal amplitude of ISOC decreased from -3.3±0.2 pA/pF (n=23) in control cells to -2.3±0.3 pA/pF (n=15) in cells transfected with siRNA against PLCg 1 and to -1.5±0.25 pA/pF (n=13) in cells transfected with siRNA against PLCg 2. Co-transfection with two siRNAs against PLCg 1 and PLCg 2 together resulted in further reduction of the current to -0.65±0.17 pA/pF (n=14). Similar results were obtained when thapsigargin was used to activate ISOC instead of IP3. It is concluded that PLCg is required for activation of ISOC in liver cells, however, the catalytic activity of PLCg in this process in not essential. Putney, J.W., Jr., Broad, L.M., Braun, F.J., Lievremont, J.P. & Bird, G.S. (2001) 114, 2223-2229. Patterson, R.L., van Rossum, D.B., Ford, D.L., Hurt, K.J., Bae, S.S., Suh, P.G., Kurosaki, T., Snyder, S.H. & Gill, D.L. (2002) Cell 111, 529-541.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/110P

Distinct characteristics of exocytosis in mouse pancreatic acinar cells Peter Thorn1, Olga Larina1 and Ian Parker2, 1Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1PD and 2Department of Neurobiology and Behavior, University of California Irvine, CA 92697, USA. Exocytosis, the fusion of a vesicle with the plasma membrane is the principal way a cell can release lipophobic substances to the outside environment. It is probable that the basic machinery of exocytosis is similar across different cell types. But recent studies have shown the process of exocytosis may be differently regulated in different cells. Here we describe novel characteristics of the prolonged (many minutes) exocytotic events in exocrine cells of the mouse pancreas. Mice were humanely killed (in accord with local guidelines) and the pancreas gland removed. The gland was then incubated in collagenase (Worthington CLSPA) for 5-10 minutes at 37°C. The tissue was then resuspended in extracellular solution (containing [mM] NaCl 145, KCl 5, MgCl2 1, CaCl2 1, HEPES 10 – pH 7.4 NaOH) and gently triturated to produce a preparation of large clusters of acinar cells. The clusters were then placed on Poly-l-lysine coated coverslips. The cell clusters were imaged using a custom-built 2-photon microscope. Images were then processed using Metamorph software (Universal Imaging). We imaged lobules and smaller fragments of mouse pancreatic tissue that retained the typical morphology of the intact exocrine glands. Inclusion of a fluorescent probe (Sulphorhodamine B or Oregon Green, Molecular Probes) in the extracellular bathing medium labelled acinar ducts and the extracellular space between cells, but dyes were excluded from the cell interior. Addition of ACh or the uncaging of caged CCh, with a flash of UV light, rapidly evoked fluorescence spots in the cell. These fluorescent spots were exclusively observed in the apical regions of cells, had the same diameter as secretory granules and had similar kinetics to the release of digestive enzymes. Our observations are therefore consistent with fluorescence labelling of zymogen granules. Using fluorescence recovery after photobleaching (FRAP) techniques we show that the fusion pore remains open for protracted periods of time (minutes) to allow free exchange between the aqueous granule lumen and the outside. Although, at later times, we show that granules do not take up extracellular dye indicating that the fusion pore can close. Finally, using lipophillic dyes, we show no evidence for interchange of lipid between the plasma membrane and the vesicle membrane during the lifetime of the vesicle. We propose that these distinct characteristic of exocytosis in exocrine glands may represent adaptations to the characteristic physiological responses of these cells.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/111P

Synchronization of Ca2+ oscillations through interaction of intracellular Ca2+ stores and L-type Ca2+ channels M.S. Imtiaz, J. Zhao, K. Hosaka and D.F. van Helden, The Neuroscience Group, School of Biomedical Sciences, Faculty of Health and Medical Sciences, The University of Newcastle, NSW 2308, Australia. Many lymphatic and blood vessels undergo spontaneous constriction-dilation cycle known as vasomotion. It has been shown that cyclical Ca2+ release from inositol 1,4,5-trisphosphate (IP3) operated intracellular Ca2+ stores and influx of Ca2+ through L-Ca2+ channels underlie lymphatic vasomotion (Zhao & van Helden, 2003). Experimental observations show that blocking L-Ca2+ channels abolishes synchronous Ca2+ oscillations, leaving only asynchronous oscillations. Based on such experimental observations and theoretical studies, we have previously shown that L-Ca2+ channels form a long-range coupling link between oscillatory Ca2+ stores, and are essential for synchronization of store Ca2+ release (Imtiaz et al., 2002; Zhao et al., 2002). The present study examines this L-Ca2+ channel-mediated long-range coupling mechanism. Synchronization of Ca2+ oscillations can occur through diffusion of Ca2+ or IP3 through gap junctions. In the present study we investigate Ca2+ store entrainment through voltage dependent L-Ca2+ channel-mediated store Ca2+ release for a cell pair. Such a coupling mechanism is significantly more effective than the chemical coupling-based class of models, as membrane potential has a coupling effect over distances several orders of magnitude greater than either diffusion of Ca2+ or IP3 through gap junctions (Imtiaz et al., 2002). We encapsulate experimental observations in a model where; 1) each local oscillator is composed of a cytosolic-store Ca2+ excitable system, 2) local Ca2+ oscillations are coupled to membrane potential, and, 3) membrane potential exerts a positive feedback on the local Ca2+ oscillator through Ca2+ influx through L-Ca2+ channels. We construct a coupled cell pair according to the schema outlined above. We study the synchronization properties of the above cell pair system. It is shown that even weak electrical coupling is sufficient to synchronize heterogeneous cell pairs. A comparison is made between electrical and chemical coupling through diffusion of Ca2+ or IP3. It is shown that chemical coupling is not effective when cells are weakly coupled and have different intrinsic frequencies. This is consistent with experimental observations where only asynchronous oscillations are observed during blockade of L-Ca2+ channels. The result of this study show that electrical coupling acting through L-Ca2+-mediated modulation of store Ca2+ release is able to synchronize oscillations of cells even when cells are weakly coupled (or widely separated) and/or have different intrinsic frequencies of oscillation. Imtiaz, M., Zhao, J., & van-Helden, D.F. (2002) Proceedings of the Australian Physiological and Pharmacological Society http://www.aups.org.au/Meetings/200211/abstracts/1148.html Zhao, J., Imtiaz, M., and van Helden, D. (2002) Proceedings of the Australian Physiological and Pharmacological Society http://www.aups.org.au/Meetings/200211/abstracts/1149.html Zhao, J. & van Helden, D.F. (2003) British Journal of Pharmacology 140, 1399-1413.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/112P

Characterization of the of the electrical activity underlying spontaneous contractions in the mouse ureteropelvic junction R.J. Lang, B. Zoltkowoski, J. Hammer, W. Meeker, I. Wendt and H. Parkington, Department of Physiology, Monash University, Clayton, Vic 3800, Australia. The unique role of the upper urinary tract is to propel urine from the kidneys to the bladder for storage until micturition. The decreasing presence of ‘atypical’ smooth muscle cells (SMC) with distance from the renal fornix has long been correlated with a decreasing gradient in contraction frequency to suggest that these atypical SMC are the primary pacemaker cells underlying pyeloureteric motility. However, we have previously described the properties of a population of electrically active cells, with many of the morphological features of interstitial cells of Cajal (ICC) the pacemaker cells of the intestine, in the spontaneously active renal pelvis of the guinea pig which was absent in the electrically-quiescent ureter (Klemm et al., 1999). These ICC-like cells were not immuno-reactive to c-Kit, but c-Kit positive cells have recently been described in the upper urinary tract of mouse, pig and human (Metzger et al., 2005). We have investigated the possible function of c-Kit positive cells in the urinary tract in portions of the mid renal pelvis from humanely killed mice using intracellular microelectrodes containing Lucifer Yellow and in single cells freshly dispersed from the ureteropelvic junction and proximal ureter, using conventional whole cell and single channel patch clamp techniques. In approximately 60% of microelectrode recordings (at 33°C), muscle contractions (3.2±0.6 min-1, n=9) were directly correlated in time with the discharge of an action potential which consisted of an initial spike followed by a prolonged plateau (1.2±0.3 s, n=9). These action potentials were blocked by nifedipine (1 µM) and the cells identified, via Lucifer Yellow filling, as spindle-shaped smooth muscle cells when viewed under a fluorescent microscope. The remaining recordings consisted of higher frequency (33±8 min-1, n=9) electrical discharges which were not directly coupled to muscle contraction. These electrical discharges were recorded in both spindle-shaped smooth muscle cells as well as stellate-shaped cells as revealed by Lucifer Yellow. These high frequency electrical events were reduced only in amplitude and duration by nifedipine (1 µM). Membrane depolarizing steps to potentials positive to -40 mV applied to single ‘spindle-shaped’ myocytes under voltage clamp (at 22°C) evoked a Ca2+ current upon which was superimposed a transient outward current (IKto), and a slowly developing outward current which inactivated little over 200 ms. IKto was 50% inactivated at a holding potential of -84.3 ±2.9 (n=3) mV and selectively blocked by 4-aminopyridine (1-3 mM). The littleinactivating outward current was blocked by tetraethylammonium (TEA 3 mM) or iberiotoxin (100 nM) suggesting that this current arose from the activation of large conductance Ca2+-activated K+ (BKCa) channels, which were readily recorded and characterized in excised membrane patches. In contrast, depolarization of stellate- or ‘staghorn’-shaped cells to potentials positive to -40 mV evoked only a slowly-developing/decaying outward current that was partially blocked by TEA (2-20 mM). Some of these cells also displayed spontaneous transient inward currents which reversed near -10 mV. We postulate that contractions of the mouse ureteropelvic junction arise from nifedipine-sensitive action potential discharge in smooth muscle cell bundles. In addition these action potentials could well be driven by pacemaker potentials generated in neighbouring c-Kit positive ICC-like cells observed under the fluorescent microscope. Klemm, M.F., Exintaris, B. & Lang, R.J. (1999) Journal of Physiology 519, 867-884 Metzger, R., Schuster, T., Till, H., Franke, F.-E. & Dietz, H.G. (2005) Pediatric Surgery International 21, 169-174.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/113P

AuPS/ASB Meeting - Canberra 2005 Free Communications 7: Muscle physiology Thursday 29th September 2005 Chair: Derek Laver

Active metabolism of mouse papillary muscle C. Widén and C.J. Barclay, Muscle Energetics Laboratory, Heart Foundation Research Centre, School of Physiotherapy & Exercise Science, Griffith University, PMB50 Gold Coast Mail Centre, Gold Coast, QLD 9726, Australia. With the development of genetically modified mice, there is need for a cardiac muscle model for determining the physiological and functional consequences of the various genetic manipulations. There have been no measurements of energy use or work capacity of the isolated mouse papillary muscles and the aim of this study was to characterise the mechanical and energetic properties of these preparations. Papillary muscles were dissected from the left ventricle of hearts from 6- to 12-week old male Swiss mice. The mice were rendered unconscious by inhalation of 80% CO2-20% O2 gas mixture and killed by cervical dislocation. All animal-handling procedures were approved by the Griffith University Animal Ethics Committee. Active metabolism of left ventricular papillary muscles was measured in vitro (27°C) using the myothermic technique (see Figure). Muscles were bathed in aerated (95% O2-5% CO2) Krebs solution with glucose provided as metabolic substrate. The energy output of the mouse papillary muscles performing isometric contractions was measured at contraction frequencies 1 – 4 Hz. The mean absolute heat output was 6.8 ± 1.1 mJ g-1 twitch-1 (mean ± SEM; n = 11) at 1 Hz and decreased with increasing contraction frequency. Tension-independent heat, an index of metabolism primarily associated with calcium cycling, was also measured. The tension-independent heat accounted for 18.9 ± 2.6 % (n = 6) of the total metabolism. In a more realistic contraction protocol (Mellors & Barclay, 2001), designed to closely simulate the reported changes in muscle shortening (Semafuko & Bowie, 1975) work output and enthalpy output were measured and resulted in a maximum net mechanical efficiency of 17 % (n = 10). The model is now well established and will be used to study energetic aspects of cardiac pathologies and heartfocussed genetic changes. Mellors, L.J. & Barclay, C.J. (2001) Journal of Experimental Biology 204, 3765-3777. Semafuko, W.E. & Bowie, W.C. (1975) American Journal of Physiology 228, 1800-1807.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/114P

Functional and electrophoretic identification of two Troponin C isoforms in toad skeletal muscle fibres B. O’Connell, R. Blazev and G.M.M. Stephenson, School of Biomedical Sciences, Victoria University, Melbourne, VIC 3011, Australia. Activation of contraction in striated muscle of vertebrates is regulated by the binding of Ca2+ to the myofibrillar protein Troponin C (TnC). In mammals, TnC is known to exist as two isoforms, one found in fasttwitch skeletal muscle (TnC-f), the other found in both slow-twitch skeletal and in cardiac muscle (TnC-s/c) (Gomes et al., 2002). These isoforms confer to fibres in which they are expressed different contractile activation characteristics with respect to Ca2+ and Sr2+ (for example, see O’Connell et al., 2004b). So far only one TnC isoform from anuran muscle, similar in structure and Ca2+ -binding properties to the rabbit TnC-f, has been purified and sequenced. However, single fibre studies have shown inter-fibre differences with respect to contractile activation characteristics, which suggests that anuran striated muscle expresses more than one TnC isoform. Thus, the main aims of the present study were (i) to definitively establish whether anuran striated muscle expresses more than one TnC isoform, and if so (ii) to examine the relationship between the myosin heavy chain (MHC) and TnC isoform expression in anuran muscle fibres and (iii) to characterise the anuran TnC isoforms according to the Sr2+- and Ca2+-activation properties conferred to the single fibres in which they are found. Adult (body weight 250-380 g) cane toads (Bufo marinus) were killed by double pithing in accordance with procedures approved by Victoria University AEEC. The TnC isoform composition of cardiac muscle and of 198 single fibres from the rectus abdominis muscle was investigated using a recently developed method for the unequivocal identification of TnC isoforms on SDS-polyacrylamide gels (O’Connell et al., 2004a). The same single fibres were also analysed for their MHC isoform content using the alanine-SDS-polyacrylamide gel electrophoresis protocol of Goodman et al. (2003). For a subpopulation of 15 fibres, the Sr2+ - and Ca2+ -activation characteristics were measured and related to the TnC isoform present. Our results show that like mammalian striated muscle, the anuran striated muscle expresses two TnC isoforms which can be distinguished electrophoretically. The slowest migrating TnC isoform (TnC-t) was detected in all fibres displaying only twitch MHC isoforms, regardless of their number or identity; the other (TnC-T/c) was detected in fibres displaying the slow-tonic MHC isoform and in cardiac muscle. Fibres containing the TnC-T/c isoform were found to be ∼47 times more sensitive to Sr2+ and ∼3 times more sensitive to Ca2+ than fibres containing the TnC-t isoform. From these data we conclude that both anuran and mammalian striated muscle contain two TnC isoforms that play an important role in determining the contractile activation characteristics of the fibres in which they are expressed. Gomes A.V., Potter J.D. & Szczesna-Cordary D. (2002) IUBMB Life, 54, 323-333. Goodman C., Patterson M. & Stephenson G. (2003) American Journal of Physiology, 284, C1448-C1459. O’Connell, B., Nguyen, L.T. & Stephenson, G.M.M. (2004a) Biochemical Journal, 378, 269-274. O’Connell, B., Stephenson G., Blazev, R. & Stephenson, G.M.M. (2004b) American Journal of Physiology, 287, C79-C87. This work is supported by the Australian Research Council.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/115P

X-ray diffraction analysis of the effects of myosin chain-2 phosphorylation on the structure of fast skeletal muscle fibres Joseph F.Y. Hoh1, Maki Yamaguchi2, Masako Kimura2, Shigeru Takemori2 and Naoto Yagi3, 1Department of Physiology and Institute for Biomedical Research, The University of Sydney, NSW 2006, Australia, 2Department of Physiology, The Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan and 3Japan Synchrotron Radiation Research Institute (JASRI), Sayo-gun 679-5198, Japan. The isometric twitch tension of a fast skeletal muscle is enhanced by a factor of about 2 following a brief tetanic stimulation (Close & Hoh, 1968). This phenomenon, known as post-tetanic potentiation (PTP), is currently thought to be due to the phosphorylation of the fast myosin light chain-2 (MLC2) by the enzyme myosin light chain kinase (MLCK), which is activated by Ca/calmodulin during the tetanus. Phosphorylation of MLC2 in permeabilized fibres enhances their Ca sensitivity, producing more force during submaximal Ca activation. Phosphorylation of MLC2 in isolated thick filaments causes the loss of the regular helical arrangement of myosin heads characteristic of normal relaxed filaments (Levine et al., 1996). It was postulated that MLC2 phosphorylation increases the mobility of myosin heads, which spend more time in proximity to thin filaments, leading to force enhancement. In this work, we test this hypothesis by using X-ray diffraction to detect structural changes in muscle fibres following MLC2 phosphorylation. The experiments were done on glycerinated rabbit psoas fibres. Muscle bundles were isolated from animals killed by stunning and exsanguination. Bundles containing 10 glycerinated fibres were prepared for Xray diffraction after exposure to: 1) relaxing solution containing 10 mM 2,3-butanedione monoxime to dephosphorylate endogenously phosphorylated MLC2, 2) subthreshold Ca solution (pCa 6.8), 3) phosphorylating solution containing 2mM calmodulin, 0.15mM MLCK (pCa 6.8) and 10mM tautomycin to inhibit endogenous phosphatase, 4) calmodulin/MLCK solution without Ca. X-ray diffraction analyses were carried out on beam line BL45XU at the SPring-8 synchrotron facility. Equatorial reflections 1,1 and 1,0 are due to longitudinally oriented planes in the muscle filament lattice that pass through thick and thin filaments (1,1) and thick filaments only (1,0). The 1,1/1,0 intensity ratio gives information about distribution of mass around the filaments. In the presence of relaxing solution, 1,1/1,0 ratio was low, indicating that myosin heads were mostly located near thick filaments. When fibres were exposed to pCa 6.8, the ratio was nearly doubled, indicating a movement of the myosin heads towards thin filaments even with no force development. After exposing fibres to phosphorylating solution for 20 minutes, the ratio significantly increased further. At 2 minutes after the enzyme was washed out in low Ca solution, the ratio decreased to control level. Prolonging the wash out time did not change the ratio significantly. Incubating fibres in enzyme without Ca produced no change in ratio. MLC2 phosphorylation and dephosphorylation under our experimental conditions were verified using two-dimensional polyacrylamide gel electrophoresis. Lattice spacings decreased slightly on exposure to low Ca, but no significant change was observed following phosphorylation. However, reducing the lattice spacing by increasing sarcomere length dramatically reduced the change in 1,1/1,0 ratio with phosphorylation. The present results provide structural evidence for a movement of cross-bridges towards the thin filaments following MLC2 phosphorylation, thereby strongly supporting this as the molecular mechanism for PTP. Sarcomere length dependence of the effects of phosphorylation correlates well with earlier work showing that the phosphorylation-induced increase in Ca sensitivity was similarly reduced by increased sarcomere length, as well as by osmotic compression (Levine et al., 1996). These procedures enhance Ca sensitivity in their own right by bringing cross-bridges closer to thin filaments. Thus, at long sarcomere lengths, the cross-bridges are already close to thin filaments, and phosphorylation has little further effect. We predict that in intact fibres, post-tetanic potentiation should decrease with sarcomere length. The increased 1,1/1,0 ratio at pCa 6.8 suggests that elevation of baseline Ca following a tetanus may contribute to twitch potentiation early in PTP. Close R. & Hoh J.F.Y. (1968) Journal of Physiology 197, 461-477. Levine R.J., Kensler R.W., Yang Z., Stull J.T. & Sweeney H.L. (1996) Biophysical Journal 71, 898-907.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/116P

Calpain-1 and calpain-3 are not autolysed with exhaustive exercise in humans R.M. Murphy1, R.J. Snow2 and G.D. Lamb1, 1Department of Zoology, La Trobe University, VIC 3086, Australia and 2School of Exercise and Nutrition Sciences, Deakin University, Burwood, VIC 3125, Australia. Calpain-1 and calpain-3 are Ca2+-dependent proteases found in skeletal muscle. Autolysis of the calpains is observed by Western blotting as the cleaving of the full-length proteins to shorter products (see the Figure, A and B), which results in their activation. Biochemical assays suggest that calpain-1 becomes proteolytically active in the presence of 3-200 µM Ca2+. Although calpain-3 is poorly understood, its activation is proposed to be much more Ca2+-sensitive (∼1 µM) than calpain-1. Adult Long Evans hooded rats were killed by an overdose of halothane, as approved by the Animal Ethics Committee at La Trobe University and the extensor digitorum longus (EDL) muscles were removed. Human muscle samples were obtained from the vastus lateralis using the needle biopsy technique. These samples were left over from a completed study which was approved by the Deakin University Human Ethics Committee. As shown in the Figure (A and B), we characterised the Ca2+-dependence of autolysis of the calpains in human muscle samples and rat EDL muscle samples homogenised in solutions mimicking the intracellular environment at various [Ca2+] (0, 2.5, 10 and 25 µM).

Autolysis of calpain-3 was found to occur over a similar [Ca2+] range as that for calpain-1, and both calpains displayed a seemingly higher Ca2+-sensitivity in human compared to rat muscle homogenates, with ∼15 % autolysis observed following 1 min exposure to 2.5 µM Ca2+ in human muscle and almost none following 1-2 min exposure to the same [Ca2+] in rat muscle. Since intracellular [Ca2+] may transiently peak in the range found to activate calpain-1 and calpain-3, we examined the effect of two types of exhaustive cycling exercise (30 s "all-out", n=8 and 70 % VO2 peak until fatigue, n=3) on the amount of autolyzed calpain-1 or calpain-3 in human muscle. Following the sprint exercise, the percent decline in peak power was 45 ± 11 % (mean ± sd). In the endurance exercise trials, subjects cycled for 107 ± 27 min. Despite the exhaustive nature of the exercise, autolysis of calpain-1 or calpain-3 did not occur due to the exercise (Figure, C and D). These findings show that the time- and concentration-dependent changes in cytoplasmic [Ca2+] occurring during concentric exercise fall near, but below that necessary to activate calpains in vivo.

Proceedings of the Australian Physiological Society

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Increased fatigue resistance in EDL muscle of the obese mouse is associated with an increase in the proportion of hybrid IIB+IID fibres R. Blazev1, J.G. Kemp1,2, D.G. Stephenson3 and G.M.M. Stephenson1, 1School of Biomedical Sciences, Victoria University, VIC 3011, Australia, 2School of Exercise Science, Australian Catholic University, VIC 3065, Australia and 3Department of Zoology, La Trobe University, VIC 3083, Australia. Fatigue resistance is an important indicator of the functional status of a muscle. Current data on the fatigue characteristics of the extensor digitorum longus (EDL) muscle from the genetically obese (ob/ob) mouse, a commonly used animal model of type 2 diabetes, are limited and inconsistent. Of the two studies carried out to date on this muscle, one shows an increased fatigue resistance in the obese animal (Warmington et al., 2000) while the other shows no difference between the obese animal and its lean control (Bruton et al., 2002). Therefore, in the present study we re-examined the fatigue characteristics of EDL muscles from ob/ob and lean mice. We also determined, using a single fibre approach, the fibre type composition of the two muscles as this parameter is closely related to muscle fatigability. Male ob/ob and lean mice (18-22 weeks, C57BL strain) were killed by halothane overdose in accordance with Victoria University AEEC procedures, and muscle dissection was carried out as described in Bortolotto et al. (2000). Isometric contractions in EDL muscle were elicited at optimal length via supramaximal pulses (13 V cm-1; 0.2 ms duration) in carbogen bubbled Krebs solution (Pedersen et al., 2003) with 10 mmol l-1 glucose and 10 m mol l-1 tubocurarin, at 25 ± 1°C. Force-frequency responses were determined using stimulation trains of 500 ms and train frequencies of 1-110 Hz, with a 3 min rest period between stimuli. Fatigue resistance was evaluated using a fatigue protocol similar to that described in Chin & Allen (1997), and consisted of repeated maximum tetanic stimulation (110 Hz, 350 ms train duration) at decreasing time intervals (4 s, 3 s, 2.5 s; each for total 2 min) until the force declined to 30% of the initial force (P0). This protocol was repeated following a 60 min rest period. Contralateral EDL muscles were employed for electrophoretic analyses of myosin heavy chain isoform (MHCi) composition in whole muscle homogenates and single muscle fibres using a modified version of the Talmadge & Roy (1993) SDS-PAGE protocol. In comparison to EDL muscle from lean mice (n=8), EDL muscle from ob/ob mice (n=8) displayed an increased resistance to the first fatigue bout (time to 30% P0: 164.4 ± 6.2 s vs 146.1 ± 2.8 s; P<0.05) and greater recovery of peak force between fatigue bouts. Type IIB was the predominant fibre type in randomly dissected single fibres from EDL muscle of ob/ob (78.9%, n=57) and lean (95.1%, n=61) mice. However, the fibre population from ob/ob mice contained a greater proportion of hybrid fibres (21.1% vs 4.9%) co-expressing MHCIIb and MHCIId isoforms (i.e. hybrid IIB+IID fibres). Consistent with this result, EDL muscle (n=6) from ob/ob mice contained a smaller proportion of MHCIIb (52.4% vs 65.7%) and larger proportions of MHCIId (31.9% vs 25.7%) and MHCIIa (15.7% vs 8.6%) isoforms. This shift in the MHCi composition of EDL muscle from ob/ob mice towards a slower profile was also reflected in the force-frequency relationship at suboptimal frequencies (greater % force relative to maximum force at 30 Hz and 50 Hz in obese muscle) and a prolonged twitch half-relaxation rate (72.4 ± 6.0 ms in obese vs 49.2 ± 3.4 ms in lean; P<0.05). The shift towards slower fibre types and the increased fatigue resistance observed in the present study for EDL muscle from the ob/ob mouse may be part of an adaptive response to the obese/diabetic condition, whereby the physiological role of the EDL muscle changes from a muscle enabling rapid movement to a muscle enabling better maintenance of posture under conditions of increased body weight. Bortolotto, S.K., Cellini, M., Stephenson, D.G. & Stephenson, G.M.M. (2000) American Journal of Physiology, 279, C1564-C1577. Bruton, J.D., Katz, A., Lännergren, J., Abbate, F. & Westerblad, H. (2002) Pflügers Archiv, 444, 692-699. Chin, E.R. & Allen, D.G. (1997) Journal of Physiology, 498, 17-29. Pedersen, T.H., Clausen, T. & Nielsen, O.B. (2003) Journal of Physiology, 551, 277-286. Warmington, S.A., Tolan, R. & McBennett, S. (2000) International Journal of Obesity, 24, 1040-1050. Talmadge, R.J. & Roy, R.R. (1993) Journal of Applied Physiology, 75, 2337-2340. This work is supported by the NHMRC (Australia).

Proceedings of the Australian Physiological Society

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Insulin-like growth factor-I gene transfer by electroporation enhances skeletal muscle regeneration and function after injury J.D. Schertzer and G.S. Lynch, Basic and Clinical Myology Laboratory, Department of Physiology, The University of Melbourne, Victoria 3010, Australia. Although skeletal muscle has the ability to regenerate after injury, functional repair can be slow, inefficient, and is often incomplete. In addition to the tightly controlled induction of myogenic regulatory factors and other muscle specific genes, muscle damage and subsequent repair processes induce the release of various biologically active molecules which are critical for regeneration. Insulin-like growth factor-I (IGF-I) is particularly relevant given that levels are elevated after injury during the formation of new fibres or the growth of existing fibres. Given that several studies have demonstrated that IGF-I enhances various aspects of skeletal muscle regeneration, a basis exists for the administration of IGF-I to enhance muscle regeneration and to promote functional recovery after injury (Rabinovsky et al., 2003; Takahashi et al., 2003). However, a comparison of various delivery methods on the efficacy of IGF-I during skeletal muscle regeneration has not been performed. The purpose of this study was to compare the time course of muscle regeneration following delivery of IGF-I to injured muscles via non-viral gene transfer or systemic protein administration. We assessed the time course of functional recovery during muscle regeneration following systemic administration of IGF-I protein via mini-osmotic pump (1 mg/kg/day) or electroporation-assisted plasmid-based gene transfer. Twelve to fourteen-week-old male C57/BL10 mice were anaesthetised deeply (pentobarbitone sodium, 60 mg/kg) and tibialis anterior (TA) muscles were injured by an intramuscular injection of the myotoxic agent, notexin, which causes complete destruction of injected muscle fibres but does not damage muscle precursor cells that are activated for subsequent regeneration. Contractile properties of the TA muscle were measured in situ (with an intact nerve and blood supply) at 7, 14, 21 and 28 days post injury and the mice were killed by cardiac excision whilst anaesthetised. At 14 days post injury, tetanic force was 36% greater following electroporation-assisted IGF-I gene transfer compared to control (P < 0.05), whereas systemic IGF-I protein administration had no effect on tetanic force at this time. At 21 days post injury, tetanic force was 31% greater following electroporation-assisted IGF-I gene transfer and 35% greater following IGF-I protein delivery compared to controls (P < 0.05). Our results show that IGF-I enhanced muscle regeneration and functional restoration after injury, regardless of the route of administration. However, electroporation-assisted plasmid delivery promoted functional recovery earlier than systemic IGF-I protein administration. The findings highlight the potential of IGF-I to minimise functional disability after injury and demonstrate that non-viral plasmid based gene transfer can be superior to continuous systemic protein administration. Rabinovsky E.D., Gelir E., Gelir S., Lui H., Kattash M., DeMayo F.J., Shenaq S.M., & Schwartz R.J. (2003) FASEB Journal 17, 53-55. Takahashi T., Ishida K., Itoh K., Konishi Y., Yagyu K.I., Tominaga A., Miyazaki J.I., & Yamamoto H. (2003) Gene Therapy 10, 612-620. Supported by the Muscular Dystrophy Association (USA) and the National Health and Medical Research Council. JDS was supported by Melbourne International Research and Fee Remission Scholarships.

Proceedings of the Australian Physiological Society

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AuPS/ASB Meeting - Canberra 2005 Free Communications 8: Biophysics Thursday 29th September 2005 Chair: Paul Smith

Bicarbonate is not a physiological substrate of Photosystem II I.L. McConnell, W. Hillier and T. Wydrzynski, Photobioenergetics Group, The Research School of Biological Sciences, The Australian National University, Canberra, ACT 0200, Australia. Photosystem II (PSII) produces essentially all the molecular oxygen (O2) in the atmosphere. The photochemistry of this large transmembrane protein complex has radically changed the planet since its evolutionary origin over 2.5 billion years ago. PSII catalyses a light dependent charge separation to drive electron transfer from water to plastoquinone and, as a by product of the reaction, oxidizes water, which releases O2. The oxidation chemistry proceeds via a catalytic component called the oxygen evolving complex (OEC). The OEC consists of a bio-inorganic core containing four manganese ions joined by oxo bridges, and a calcium ion (Mn4OxCa1). Additionally, it is coordinated to a number of surrounding amino acids from the supporting protein matrix. The mechanism of oxygen formation is subject to considerable speculation. During the last several decades there has been simmering debate over the precise nature of the immediate substrate of the OEC. It is widely accepted that water is the ultimate substrate based on labeling studies with H218O. However bicarbonate has been considered as an alternative intermediate and this has never been fully discounted. The interest in HCO3- has recently been rekindled by the inclusion of a (bi)carbonate ligand in the OEC in a crystal structure of PSII at 3.5 Å resolution (Ferreira et al., 2004). Using 18O labeled bicarbonate in conjunction with membrane inlet mass spectroscopy the substrate flux into O2 has been measured in three species of photosynthetic organisms, including a cyanobacterial species requiring high bicarbonate (400 mM) to grow. Evidence is presented that bicarbonate is not the physiological substrate of the OEC. Bicarbonate can only be oxidized by one in a few thousand PSII. Ferreira, K.N., Iverson, T.M., Maghlaoui, K., Barber, J. & Iwata, S. (2004) Science 303, 1831–1838.in 0

Proceedings of the Australian Physiological Society

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Spectra of reef fish – a physics approach to colourful patterns Misha Vorobyev and Justin Marshall, Vision Touch and Hearing Research Centre, School of Biomedical Sciences, University of Queensland, QLD 4072, Australia. Coral reef fishes use brightly coloured body patterns for advertisement and camouflage. How are these conflicting demands combined? Simple inspection of colourful patterns of reef fish provides little insight into this problem, because fish vision differs substantially from ours. To investigate the utility of fish colours, we calculated the number of quanta absorbed by cone photoreceptors (cone quantum catches) in fish eyes viewing fish. These calculations were based on measurements of reflectance spectra of fish skin, spectra of light under water and spectral sensitivity of fish cone photoreceptors (Losey et al., 2003; Marshall et al., 2003). Cone quantum catches provide full description of colour, because surfaces that differ in their reflectance spectra, but yield similar quantum catch in photoreceptors, cannot be discriminated. To reconstruct views of a fish as seen through fish eyes, we encoded each point of a fish image with the set of quantum catches that were calculated for the reflectance spectrum in a corresponding point (Vorobyev et al., 2001; Marshall & Vorobyev, 2003). This method allowed us to visualise the information available to fish brain, but it does not take into account the neural processing of photoreceptor signals.

The Figure shows a royal dottyback, Pseudochromys paccagnellae, as seen by an achromatic colour channel of a barracuda. We placed this fish against the background of water (upper panel) and of coral (lower panel). The head of this fish provides little contrast with coral, while the highly contrasting tail may serve as a signal. This fish usually hides its tail in the burrow and exposes its head. Many reef fishes are sensitive in the UV part of the spectrum, but none of them have photoreceptors that are sensitive to orange and red. Therefore, bright for our eyes red and orange colours of many reef fishes may look dull for fish. Often patches of skin that look red for us reflect in the UV-blue part of the spectrum; these reflectances yield signals in UV or blue sensitive photoreceptors in fish eye, and may strongly contrast with yellow patches that usually absorb in the UV-blue part of the spectrum. Since the illumination spectrum varies significantly under water, fish colours also change. Another important physical factor that affects colour appearance is spectrally selective absorption and scatter of light by water. One of the consequences of light scatter is a veiling effect, which reduces contrast (the Figure, right column). Since scatter is most prominent in the UV part of the spectrum, UV reflectance cannot be transmitted at long distance. Many small fishes probably use UV as a ‘secret communication channel’ that conveys signals visible at close distance, but is invisible for predators from long distance. Another trick, used by colourful fish to avoid being seen from far distance, is to combine strongly contrasting blue and yellow colours whose optical mixture is similar to the spectrum of background (Marshall & Vorobyev, 2003). Due to the scatter of light in water and the poor optical resolution of fish eyes, the body pattern of such fish cannot be resolved when viewed from a distance. Therefore a colourful fish appears to be well camouflaged. Losey, G.W., McFarland, W.N., Loew E.R., Zamzow J. & Marshall, N.J (2003) Copeia 3, 433-454. Marshall N.J., Jennings K.J., Losey, G.W. & McFarland, W.N. (2003) Copeia 3, 455-466. Marshall, N.J. & Vorobyev, M. (2003) In: Sensory Processing in Aquatic Environments. Ed. S.P. Collin & N.J.Marshall, Springer, pp 194-223. Vorobyev, M., Marshall, J., Osorio, D., Hempel de Ibarra, N. & Menzel, R. (2001) Color Research and Application 26, S214-216.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/121P

Resonances of the human vocal tract and some of their uses John Smith and Joe Wolfe , School of Physics, University of New South Wales, NSW 2052, Australia. The human vocal tract behaves approximately as an acoustical waveguide with a series of resonances whose frequencies may be varied by adjusting the position of tongue, lips and teeth. In voiced speech, these resonances interact with the harmonics of the lower frequency signal from the vibrating vocal folds to produce associated peaks, or formants, in the output spectrum. Such formants are characteristic of vowels in speech. Singers sometimes use these resonances in musical rather than linguistic ways. For sopranos, the vibration frequency of their vocal folds may be much higher than the normal values for the lowest resonance, and consequently a reduced interaction would cause a loss of power. Direct measurements of the resonance frequencies of the vocal tract of classically-trained sopranos during singing show that they consistently increase them to match the frequency of their singing. This significantly increases the loudness and the uniformity of tone, at the expense of comprehensibility. The fundamental frequency of other singers is usually less than the value of the lowest resonance and so they would experience no advantage in tuning this resonance. However the power could be increased if the resonance frequency were tuned to a harmonic of the fundamental frequency. Our measurements indeed show that some altos, tenors and baritones use this strategy when appropriate. The role of the vocal tract resonances is quite different when playing a wind instrument. The sound is then generated by the vibrating lip or reed valve rather than by the vibrating vocal folds. The frequency of vibration is then primarily determined by one of the strong resonances of the wind instrument itself. Our measurements show that varying the resonances of the vocal tract can then still slightly alter the vibration frequency and change the harmonic structure or timbre of the produced sound. The research described has involved several members and associates of our Acoustics Laboratory. http://www.phys.unsw.edu.au/speech http://www.phys.unsw.edu.au/music

Proceedings of the Australian Physiological Society

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AuPS/ASB Meeting - Canberra 2005 Free communications 9: Education Friday 30 September 2005 Chair: Ann Sefton

Enhancing the first-year experiences of undergraduate students enrolled in large classes Roger W. Moni, Karen B. Moni, Lesley Lluka and Philip Poronnik, School of Biomedical Sciences The University of Queensland, St. Lucia, 4072, Brisbane, Australia. BIOL105 (Human Biology) is a 2-unit, integrated, interdisciplinary course offered by the School of Biomedical Sciences (The University of Queensland) to first-year students. Physiology forms a major component of the content. Student enrolments are high (n =315 in semester one, combined Pharmacy and Human Movement Studies cohort) to very high (n = 860 in the semester two, Science cohort). BIOL1015 provides a foundation of disciplinary knowledge, conceptual frameworks, practical skills and socio-affective orientations. As a keystone course, student experiences can strongly influence engagement in learning, consequent course selection and career paths linked to global knowledge economies. This paper describes the substantial re-culturing of BIOL1015 in 2005, framed around an explicit learning model and the building of a learning community. It presents findings from a comprehensive evaluation of the effectiveness of the course in enhancing their first-year experience. The course has been renewed with substantive, benchmarked improvements to learning and assessment experiences for students. Two innovative assessment tasks were designed and implemented. The first task was to write a Personal Response (weighted as 10%) around contemporary biological issues. The aim was to develop students’ abilities to communicate to various audiences. This task comprised a two-page written assignment of their response to recent audio interviews from Radio National’s “Science Show” presented on a CD. As a complementary assessment task (weighted as 5%) students wrote a review of one other students’ Personal response, guided by explicit criteria. The second task was an e-Conference which enabled students to actively and collaboratively contribute to learning about contemporary issues of biology. Students worked in collaborative pairs which formed larger clusters of 16 pairs. Each cluster addressed one broad interdisciplinary topic related to the course content. Students worked on-line within their cluster to develop: (a) a brief paper, weighted 9% (b) a related PowerPoint presentation, 4% and (c) presented one question and one answer to members of their cluster, 2%. Submissions were assessed by on-line tutors using pre-specified criteria. Students were supported in these tasks by explicit teaching and use of exemplars during lectorials (combined lecturetutorial format). Participants in the evaluation included 246 students (80% of the total combined cohort), four academic staff and one research assistant. The course was evaluated in terms of the: impact of teaching, effectiveness of course design, levels of students support, course delivery and student perceptions. Data were gathered from the following sources: print-based individual questionnaires; field notes; focus group interviews; analysis of student work using detailed criteria sheets, and analysis of assessment results. Interview and open-ended questionnaire items were analysed using qualitative methodologies (open, axial and selective coding). Survey data were analysed using non-parametric quantitative methodologies. Key themes emerging from the data were grouped into three categories. These were: teaching and management; learning pathways; and learning collaboratively. As expected, students expressed both positive and negative views about the e-Conference as a teaching and learning activity. Some students had difficulties articulating their learning pathways explicitly. An unexpected finding was the importance of social dynamics in affecting students’ learning. Findings indicate that the adopted model of learning could be extended to include a social dimension. The findings also support the importance of collaboration, dialogue and reflection as key learning constructs from previous research.

Proceedings of the Australian Physiological Society

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Creating an effective learning community in a large-class service teaching physiology course H. Ernst and K. Colthorpe, School of Biomedical Sciences, The University of Queensland, St Lucia, QLD 4072, Australia. Ideally students should enjoy a community of practice that includes students, academics and industry representatives, and that facilitates learning based on inquiry, discovery and practice. In large-class service teaching courses this is seldom feasible. However, we believe that by creating effective learning communities in these courses, we are able to engender creative interest from students and enhance their learning experiences. In such learning communities students and teaching staff all engage with each other to acquire knowledge and share understanding, counteracting the trend towards isolation students feel in such large cohorts and their disillusionment with the field of study and its relevance to their chosen profession. In this study we set out to facilitate the creation of an effective learning community in a 3rd year Physiology course for Pharmacy students by (1) focusing on clear learning objectives, (2) delivering interactive lectures that concentrate on major physiological concepts, (3) providing discovery based practicals, (4) convening an on-line discussion board, (4) providing practice questions to reassure students about the validity of the learning objectives and to stimulate indepth, out-of-class learning activities, and by (5) convening a final voluntary tutorial before the end-of-the semester exam for students in need of further nurturing. We received very positive student feedback and student performance in the end-of-the semester assessment was markedly increased compared to the previous year, from an average of 66% in 2003 to an average of 89% in 2004.

Proceedings of the Australian Physiological Society

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The place of physiology in an integrated medical curriculum T.O. Neild, Department of Human Physiology, Flinders University, GPO Box 2100, Adelaide SA, 5001. Australia. In 1996 Flinders University took its initial cohort of students into Australia’s first Graduate Entry Medical Programme (GEMP) (Finucane et al., 2001). The teaching of basic sciences such as physiology in the first two years was by Problem-based Learning (PBL) tutorials with a small number of supporting lectures and practical sessions. The tutorials involved analysis of PBL cases which were clinical patient scenarios written to highlight particular areas of body function in a relevant context. There were no groups of lectures under the heading of “Physiology”, or under the names of any of the other preclinical departments that had previously taught their own disciplines. This of course generated concerns that basic science would no longer be learned in any depth. The extent to which physiology appeared in the preclinical years of the course was measured by analysing the “Learning Objectives” – a group of brief statements that define the course designers’ intentions for student learning, week by week. Learning Objectives define the course content and are decided at the earliest stage of course design before PBL cases are written or lectures planned. The first 59 teaching weeks of the 4 year GEMP involved teaching basic human biology integrated with clinical science, using PBL cases. Over this period there were a total of 494 Learning Objectives in the area of basic biomedical science, on average 8 or 9 per week. The number of Learning Objectives that could be considered as relating to physiology, eg: “Describe the mechanisms (muscles, pressures and volumes) underlying spontaneous ventilation” “Know the pattern of blood flow through the kidney and how this is regulated under different circumstances” “Understand how gastric acid is produced” were counted. There were 119 physiology Learning Objectives, constituting 24% of the basic biomedical science learning expected in the first 2 years of the GEMP. These Learning Objectives were spread across 39 of the 59 weeks, showing that physiology learning was explicitly expected in 66% of the weeks. This is probably an underestimate of the physiology content. There were Learning Objectives such as: “The physiological basis and significance of added heart sounds” and many others relating to pathophysiology which were not included in the count, even though such issues would traditionally have been taught by physiologists in past courses. It is therefore not surprising that, despite the absence of sessions called “Physiology”, the staff of the Department of Human Physiology were highly in demand to write PBL cases and participate in supporting sessions. The lack of an explicitly labelled physiology section in the course has not lessened the amount of physiology that the students are required to learn. On the contrary, the high proportion of physiology material in the explicit Learning Objectives emphasises the fundamental role of the discipline in the early stages of medical training. Finucane, P., Nicholas, T.E., & Prideaux, D. (2001) Medical Teacher 23, 76-79.

Proceedings of the Australian Physiological Society

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Using a student-centred approach to enhance understanding of the physiology of metabolism and energy balance K.L. Colthorpe and H.G.G. Ernst, School of Biomedical Sciences, University of Queensland, St Lucia, Queensland 4072, Australia. This study examined the effectiveness of modifications to a physiology laboratory class on metabolism for 2nd year occupational therapy and speech pathology students. The class was designed to create an environment in which the students could identify and address their misconceptions through interactive discussion and have an opportunity to explore and integrate information available to improve their critical thinking skills. By this process it was intended that the students would deepen their understanding of the physiological principles that underlie metabolism, integrating the knowledge they had gained in this and other elements of their course. Further, the class aimed to develop the students’ skills in appraising the value of nutritional information they are exposed to and to identify the important factors which influence an individuals’ metabolic needs. The design incorporated some initial questions, answered individually, a tutorial, a group-based workshop and a final discussion, which specifically included revisiting the initial questions. Care was taken to create a learning environment in which the students were comfortable to discuss their knowledge and ideas openly and confidently. Evaluation of the project, in the form of a questionnaire, showed that the students agreed that the class allowed them to recognise misconceptions, improved their understanding and increased their ability to evaluate information. The results in the end of semester summative examination were improved, with an increase of 37.7% in average marks for this topic compared to the previous cohort.

Proceedings of the Australian Physiological Society

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Student perceptions and use of pre-specified criteria in constructing complex concept maps in physiology Roger W. Moni, Eileen Beswick, Alex Forrest and Karen B. Moni, School of Biomedical Sciences The University of Queensland, St. Lucia, QLD 4072, Australia. Constructing quality assessment criteria can be challenging, especially when used for integrated, groupcentred, applied learning. In this paper, we describe a project investigating the development and use of criteria in a collaborative assessment task (weighted as 6%). The task involved groups (four students in each) from Second-Year Dentistry working to construct a complex concept map. The students were given a written, simulated, medical history of a patient and were required to construct a concept map illustrating relevant pathophysiological concepts and pharmacological interventions. This paper describes the second phase of this research project aimed at making educational goals of the task more explicit through investigating student and staff understandings of the criteria. The findings from the first phase (Moni, Beswick & Moni, 2005) were used to revise the assessment criteria for the task with the aim of making them more accessible to learners. The new criteria used adopted the language of both staff and students to more clearly represent expectations of each criterion and standard. The new criteria were used for the first time in 2005. This paper presents data from a cohort survey undertaken to determine students’ perceptions about the concept map and the criteria. In addition, two groups of student volunteers were videotaped during a regular, three-hour workshop as they worked to draw their concept map. The aim of this was to capture student interactions as they constructed their concept maps with the support of the new criteria sheet. Survey data were analysed using non-parametric methodologies. Differences in students’ opinions about the assessment task were identified and mapped against their final mark awarded for the task. Evidence was found that for some groups, attitudes towards the assessment task played a role in influencing their final mark. Transcripts were developed from the videotapes and these were analysed using two complementary approaches. First, an inductive strategy was adopted to define emergent categories of behaviour. Second, a deductive strategy was used to explore student behaviours according to how they aligned with principles of effective learning. Most students reported that they found the criteria sheet useful in completing the group concept map. This was more the case for students who had not used a criteria sheet before. However, there was little other evidence (e.g. from video recordings) that most students actually used the criteria sheets to guide the construction of concept maps. Our conclusion was that students perceived the criteria sheet as being more useful for lecturers to mark their work, rather than as a tool to enhance their own learning. Moni, R.W., Beswick, E. & Moni, K.B. (2005) Advances in Physiology Education, in press.

Proceedings of the Australian Physiological Society

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The opinion editorial – a novel assessment task in final year physiology Deanne Hryciw, Philip Poronnik and Roger W. Moni, School of Biomedical Sciences The University of Queensland, St. Lucia, QLD 4072, Australia. Improving the public understanding of science is likely to remain an important challenge to future professional scientists who are our current undergraduates. In this paper, we present the findings from two phases of a study investigating teaching, learning and assessment strategies aimed to improve undergraduates’ communication of science to non-professional audiences. As the first phase in 2004, we developed a “Media Role” model to identify the function of mass media as community gatekeepers of new scientific findings. This conceptual model predicts the potential benefits for all undergraduate science students in adopting styles of writing used by journalists. We then detail a writing task with a novel application for third-year Physiology students – the Opinion Editorial (weighted as 10%) and accompanying Peer Review (weighted as 5%). Survey data from final year students (n = 230) enrolled in the course - Human Physiology and Pharmacology in Disease - were collected before and after the implementation of the Opinion Editorial / Peer review. The task requirements were explicitly taught to the students by a professional journalist. In the assessment task, students adopted the role of journalists to re-write a recent, technical paper (Mattick, 2004), as an Opinion Editorial. This was assessed both by staff and peers using a detailed criteria sheet. After minimal editing, the top-ranked student Opinion Editorial was published in the UQ News. Pre-writing Task and Post-writing Task surveys (5-point Likert scale) were administered to students. Research questions included: (i) How far did writing the Op-Ed give the students a deeper understanding of the role of media and the difficulty in communicating science to the public? (ii) Was writing the Op-Ed challenging and valuable? and (iii) Did the students perceived any changes in their own communication skills? Student surveys were analysed by non-parametric methodologies. Samples of student work (n = 177) were analysed using algorithms to describe surface and conceptual features. As the second phase in 2005, we describe an intervention to determine the effectiveness of explicitly teaching students how to write an Opinion Editorial. As the pre-instructional assessment task (weighted as 8%), students read a technical article from the course and completed a written assignment intended for a nonprofessional audience. Work was marked using a criteria sheet. Subsequently, a professional journalist explicitly taught both the construct and features of an Opinion Editorial to the students. As the post-instructional assessment task, students read a different technical article from the course and then re-wrote this as an Opinion Editorial (weighted as 12%). The same criteria sheet was used. On a random basis, students in the course were presented with the first and second submissions of these two tasks. They were required to mark both against prespecified criteria (weighted as 5%). In a similar manner, the two tasks from volunteering students were presented to members of the public who were asked to complete an associated survey to capture opinions about, and understanding of, the two texts. Analyses of surveys, student results and student work were undertaken. Major findings indicated that the students valued writing to non-professional audiences, and that their final submissions corresponded well to the surface and conceptual features of published Opinion Editorial pieces. However, difficulties in constructing writing because of issues around linguistic competence, were identified. It is evident that more explicit teaching of this type of writing is needed at undergraduate level. Mattick, J. (2004) Nature Genetics 5, 316.

Proceedings of the Australian Physiological Society

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AuPS/ASB Meeting - Canberra 2005 Free communications 10: Skeletal Muscle 1 Friday 30 September 2005 Chair: Robyn Murphy

Low dose formoterol treatment reverses sarcopenia and improves muscle function in fast- but not slow-twitch skeletal muscles of aged rats G.S. Lynch and J.G. Ryall, Basic and Clinical Myology Laboratory, Department of Physiology, The University of Melbourne, Victoria 3010, Australia. Ageing is associated with progressive muscle wasting (sarcopenia) and weakness and in the frail elderly, the loss of muscle mass can be so severe it impacts on the ability to perform the tasks of everyday living (Lynch, 2004). There is a profound need for strategies to ameliorate sarcopenia and improve quality of life. One strategy is treatment with b 2-adrenoceptor agonists (b 2-agonists). Although traditionally administered at low doses for treating asthma, at higher doses, b 2-agonists have potent muscle anabolic effects. We have shown previously that treatment with the b 2-agonist fenoterol can reverse muscle wasting and weakness in old rats (Ryall et al., 2004a). However, fenoterol was also associated with impaired cardiac function, likely mediated through actions at the b 1-adrenoceptor (Gregorevic et al., 2005). The b 2-agonist formoterol has a greater duration of action than the most widely used asthma drugs, and has an increased selectivity for the b 2-adrenoceptor (Anderson, 1993). Having found formoterol to be more potent and efficacious than fenoterol, with respect to its effects on skeletal muscle mass, (Ryall et al., 2004b), we tested the hypothesis that low dose formoterol treatment would reverse the atrophy and weakness in skeletal muscles of old Fischer 344 rats, whilst having minimal unwanted effects on the heart (due to reduced actions at the 1-adrenoceptor). Young (3 months/age, n = 8), adult (16 months/age, n = 8) and old (28 months/age, n = 6) rats were treated daily with either formoterol (25 m g/kg/day, i.p ∼0.5 mL total volume) or saline vehicle for 4 weeks. Following treatment, rats were anaesthetised with sodium pentobarbitone and the fast-twitch extensor digitorum longus (EDL) and predominantly slow-twitch soleus muscles were surgically excised from the hindlimb for determination of isometric contractile properties in vitro. Following completion of the functional measurements the deeply anaesthetised rats were killed by surgical excision of the heart. Muscle mass was greater in adult than young rats, indicative of normal growth, whilst old rats exhibited a significant reduction in muscle mass compared to both young and adult rats (EDL (in mg): young 125 ± 3 vs adult 142 ± 2 vs old 80 ± 7, P < 0.05; soleus (in mg): young 113 ± 4 vs adult 129 ± 2 vs old 94 ± 9, P < 0.05). Similarly, maximum force of EDL and soleus muscles was greatest in adult rats, and significantly reduced in old rats (EDL (in mN): young 2737 ± 80 vs adult 3019 ± 40 vs old 1902 ± 210, P < 0.05; soleus (in mN): young 1373 ± 95 vs adult 1576 ± 35 vs old 1009 ± 159, P < 0.05). Treatment increased EDL muscle mass in young, adult and old rats by 23%, 23% and 40% respectively, with a concomitant increase in maximum force producing capacity. Treatment increased soleus muscle mass and maximum force producing capacity in young, but not adult or old rats.Treatment was associated with a significant increase in heart mass in young rats (743 ± 31 vs 868 ± 50 mg, P < 0.05), but not in adult or old rats. Our findings indicate a muscle specific decrease in b -adrenergic responsiveness with age, with fast- but not slow-twitch skeletal muscle responding to low-dose administration of formoterol. We conclude that formoterol can restore muscle mass and strength of fast-twitch skeletal muscles in old rats without cardiac hypertrophy. Anderson, G.P. (1993) Life Sciences 52, 2145-2160. Gregorevic, P., Ryall, J.G., Plant, D.R., Sillence, M.N. & Lynch, G.S. (2005) American Journal of Physiology 289, H344-H349. Lynch, G.S. (2004). Internal Medicine Journal 34, 294-296. Ryall, J.G., Plant, D.R., Gregorevic, P., Sillence, M.N. & Lynch, G.S. (2004a) Journal of Physiology 555, 175-188. Ryall, J.G., Plant, D.R. & Lynch, G.S. (2004b) Proceedings of the Australian Physiological Society 35, 44P. This work was supported by grants from the Muscular Dystrophy Association (USA), the National Health & Medical Research Council and the Rebecca L. Cooper Medical Research Foundation. JGR was supported by a Postgraduate Scholarship from the National Heart Foundation of Australia.

Proceedings of the Australian Physiological Society

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b -adrenergic signalling in skeletal muscle regeneration after myotoxic injury F. Beitzel1, M.N. Sillence2 and G.S. Lynch1, 1Basic and Clinical Myology Laboratory, Department of Physiology, The University of Melbourne, VIC 3010, Australia and 2School of Agriculture, Charles Sturt University, Wagga Wagga, NSW 2678, Australia. b -adrenoceptor agonists (b -agonists) have therapeutic potential for skeletal muscle wasting disorders due to their potent muscle anabolic effects. b -agonist administration promotes skeletal muscle hypertrophy via cAMP-mediated increases in protein accretion (Navegantes et al., 2001; Ryall et al., 2002). We have shown previously that b -agonist administration can enhance functional repair of rat skeletal muscle after injury (Beitzel et al., 2004). It has been suggested that adrenoceptor desensitisation may limit the therapeutic efficacy of b -agonists in skeletal muscle (Claing et al., 2002), but little is known about b -adrenergic signalling during muscle regeneration. The aim of this study was to examine aspects of b -adrenergic signalling in skeletal muscle and test the hypothesis that during regeneration, b -agonist administration does not cause b -adrenoceptor desensitisation. Male rats (275-300g) were deeply anaesthetised (ketamine 100 mg/kg and xylazine 10 mg/kg, i.p.), and the extensor digitorum longus (EDL) and soleus muscles of the right hindlimb were surgically exposed and injected with a maximal volume of the myotoxic agent, bupivacaine hydrochloride, to cause complete destruction of all muscle fibres (Beitzel et al., 2004). The EDL and soleus muscles of the contralateral hindlimb served as uninjured controls. Rats then received either the b -agonist, fenoterol (1.4 mg/kg/day, i.p.), or an equivalent volume of saline for 7 days post-injury. Following treatment, rats were anaesthetised deeply and the EDL and soleus muscles were excised for analysis. All rats were killed by cardiac excision whilst anaesthetised. b -adrenoceptor density was measured using radioligand binding assays on isolated muscle membranes (Beitzel et al., 2004). In regenerating EDL muscles there was a ∼2-fold increase in b -adrenoceptor density compared to control values. b -adrenoceptor density in regenerating EDL muscles from fenoterol treated rats was only 57% that for saline treated rats. In regenerating soleus muscles, b -adrenoceptor density was restored to control levels. Fenoterol treatment reduced b -adrenoceptor density during regeneration to 58% that for saline treated rats. Adenylate cyclase (AC) activity assays were performed on fresh isolated muscle membranes. Despite the marked reduction in b -adrenoceptor density in both regenerating EDL and soleus muscles with fenoterol treatment, receptor desensitisation did not occur, since AC activity was maintained during isoproterenol stimulation. Various AC stimulants (NaF, forskolin and Mn2+) which act at different points in the AC signalling pathway were used to examine the underlying mechanisms responsible for these observations. The findings indicated compensation for homologous downregulation of the b -adrenoceptors by the heterologous sensitisation at the level of AC. These results highlight the unique b -adrenergic signalling responses of injured/regenerating muscles compared with uninjured muscles, so as to maximise functional recovery. Beitzel, F., Gregorevic, P., Ryall, J.G., Plant, D.R., Sillence, M.N. & Lynch, G.S. Journal of Applied Physiology 96, 1385-1392, 2004. Claing, A., Laporte, S.A., Caron, M.G. & Lefkowitz, R.J. Progress in Neurobiology 66, 61-79, 2002. Navegantes, L.C.C., Resano, N.M.Z., Migliorini, R.H. & Kettelhut, I.C. American Journal of Physiology 280, E663-E668, 2001. Ryall, J., Gregorevic, P., Plant, D.R., Sillence, M.N. & Lynch, G.S. American Journal of Physiology 283, R1386-R1394, 2002.

Proceedings of the Australian Physiological Society

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Streptomycin reduces stretch-induced membrane permeability in isolated muscles from mdx (dystrophic) mice N.P. Whitehead, M. Streamer and D.G. Allen, School of Medical Sciences, University of Sydney (F13), NSW 2006, Australia. Duchenne muscular dystrophy (DMD) is a genetic disease, which causes severe muscle degeneration, leading to profound muscle weakness and early death. DMD is caused by the absence of a protein, dystrophin, which is attached to the surface membrane of muscle fibres. The recent focus of our laboratory has been to investigate whether a component of the damage is caused by entry of Ca2+ through stretch-activated channels (SACs) in the surface membrane. We have recently shown in single muscle fibres from mdx (dystrophic) mice that following stretched (eccentric) contractions, Ca2+ influx can be prevented and isometric force improved by the addition of three known SAC blockers, one being the antibiotic streptomycin (Yeung et al., 2005). It is known that stretched contractions cause greater membrane permeability of mdx muscles compared to wild-type (Petrof et al., 1993). In the present study we investigated whether this stretch-induced membrane permeability was due to Ca2+-dependant membrane damage as a result of Ca2+ entry through SACs. Extensor digitorum longus (EDL) muscles were dissected from 8-10 week old wild-type and mdx mice. Muscles with clips attached to the tendons were mounted in a chamber between a force transducer and the lever of a motor. In some experiments, streptomycin (200m M) was added to the perfusate 60 min before the stretched contractions. Muscles were set to the length that produced maximal isometric force (optimum length, Lo). Procion orange, a membrane impermeable fluorescent dye, was added to the perfusate in order to detect fibres with increased membrane permeability. Muscle damage was induced by 10 stretched contractions, where the muscle was stretched by 30% of its length from L0, during a 400 ms tetanus. Following the stretched contractions, isometric force was measured at 30 and 60 min and the muscle was frozen in isopentane cooled in liquid nitrogen at either 0, 30 or 60 min. Muscle cross-sections (10m m) were viewed with a fluorescent microscope and the area of procion orange positive muscle fibres was calculated as a percentage of the entire muscle cross-sectional area. Following the stretched contractions, isometric force measured from control mdx muscles (n=5) fell to 33.4% ± 3.3. In experiments on mdx muscles where streptomycin was added to the perfusate before the stretched contractions (n=8), the reduction in force was significantly less, reaching 44.6% ± 1.6 (p<0.05, t-test). Wild-type muscles had a smaller decrease in force than mdx muscles following the stretched contractions (60.3% ± 1.6, n=3) and there was no effect of streptomycin (60.3% ± 2.2, n=3). Procion orange uptake for control mdx muscles was 5.0% ± 0.9 (n=3) immediately after the stretched contractions and then increased to 10.3% ± 0.8 (n=4) at 30 min and 15.1% ± 2.5 (n=4) at 60 min. At all times, streptomycin significantly reduced procion orange uptake (p<0.05, t-test), with values of 1.6% ± 0.7 (n=3), 5.3% ± 1.4 (n=3), and 4.9% ± 1.4 (n=5) at 0, 30, and 60 min, respectively. Wild-type muscles had very little procion orange uptake, with mean values of 0.7% (without streptomycin, n=2) and 1.1% (with streptomycin, n=2). This study showed that following stretched contractions, membrane permeability of mdx muscles increased progressively over 60 min, and importantly, most of this permeability could be prevented by the SAC blocker, streptomycin. Taken together, these results suggest that the increased membrane permeability is mainly due to Ca2+ entry through SACs and not the result of transient mechanical tears of the membrane during the stretched contractions (Petrof et al., 1993). The mechanism by which increased intracellular Ca2+ causes muscle damage to dystrophic muscle is unclear but might be attributable to an increased production of reactive oxygen species and/or the activation of calcium-dependent proteases or phospholipase A2. These damage pathways are now being explored in our current series of experiments. Petrof, B.J., Shrager, J.B., Stedman, H.H., Kelly, A.M. & Sweeney, H.L. (1993) Proceedings of the National Academy of Sciences 90, 3710-3714. Yeung, E.W., Whitehead, N.P., Suchyna, T.M., Gottleib, P.A., Sachs, F. & Allen, D.G. (2005) Journal of Physiology 562, 367-380. Supported by ARC & NHMRC. NPW is supported by a Rolf Edgar Lake Fellowship, Faculty of Medicine, University of Sydney.

Proceedings of the Australian Physiological Society

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Muscle weakness in a mouse model of nemaline myopathy can be reversed with exercise and reveals a novel myofibre repair mechanism A.J. Kee, J.E. Joya, V. Nair-Shalliker, M.-A. Nguyen, M. Ghoddusi and E.C. Hardeman, Muscle Development Unit, Children’s Medical Research Institute, Westmead, NSW 2145, Australia. Nemaline myopathy (NM) is an inherited muscular disorder characterised by muscle weakness and the presence of distinct rod-shaped accumulations of sarcomeric proteins (nemaline rods) in muscle fibres. We previously generated a transgenic mouse model for nemaline myopathy that expresses in all skeletal muscles a mutant α-tropomyosin-slow (Met9Arg) protein that causes NM in humans (Laing et al., 1995). This mouse shows all of the features of the human disease including late-onset muscle weakness (4-6 mo of age) and nemaline rods, the defining feature of the disease (Corbett et al., 2001). A debilitating feature of NM in humans is prolonged muscle weakness after periods of inactivity. In the present study, we have examined endurance exercise as means of improving recovery following muscle inactivity in the transgenic NM mouse model. Physical inactivity was induced by bilateral hind limb immobilisation (using surgical tape) in a maximal dorsoflexed position that stretches the ventral muscles (e.g. soleus) and shortens the dorsal muscles (e.g. extensor digitorum longus, EDL). Mice were fully anaesthetised during the immobilisation procedure with ketamine/xylazine (100 and 10 mg/kg body weight, respectively). The mice were then subjected to one of three, 4 week recovery regimens: 1) minimal physical activity (cage rest), 2) low intensity voluntary free-wheel exercise, or 3) high intensity treadmill exercise (1.5h/day; 5 days/week; 20 m/min; 5% incline). Fours weeks of immobilisation resulted in muscle fibre atrophy and severe muscle weakness in both wild-type (WT) and NM mice. However, NM mice were weaker than the WT mice after immobilisation, and exercise, not cage-rest, was required to regain whole body strength. Immobilisation of the EDL in the shortened position, led to an increase in the number of nemaline rods in the NM mice and surprisingly these rods that were formed with immobilisation appeared to be resolved with endurance exercise. Together these results suggest that nemaline rods may have a role in muscle weakness in NM. Chronic stretch-immobilisation of the soleus muscle for 10 days resulted in myonecrosis and continued stretch-immobilisation for a further 18 days resulted in complete regeneration of the damaged fibres. Although muscle regeneration did occur in NM mice during immobilisation it occurred without the classical features of regeneration (centrally located myonuclei) indicating an alteration in the normal repair process of muscle in NM. In conclusion, exercise is effective at attenuating disuse-induced muscle weakness in the NM mouse model. The novel muscle repair process in the NM maybe a response to primary myofibrillar damage that occurs in NM and maybe distinct from the classical repair observed in muscular dystrophies. Corbett, M.A., Robinson, C.S., Dunglison, G.F., Yang, N., Joya, J.E., Stewart, A.W., Schnell, C., Gunning, P.W., North, K.N. & Hardeman, E.C. (2001) Human Molecular Genetics, 10, 317-328. Laing, N.G., Wilton, S.D., Akkari, P.A., Dorosz, S., Boundy, K., Kneebone, C., Blumbergs, P., White, S., Watkins, H., Love, D.R. & Haan, E. (1995) Nature Genetics, 9, 75-79.

Proceedings of the Australian Physiological Society

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Contraction-mediated damage in mdx dystrophic mouse tibialis anterior muscles is not affected by the membrane sealant poloxamer D.R. Plant, J.G. Ryall and G.S. Lynch, Basic and Clinical Myology Laboratory, Department of Physiology, The University of Melbourne, VIC 3010, Australia. Dystrophin deficiency causes Duchenne muscular dystrophy (DMD), a severe inherited and progressive disease of striated muscle in humans. Dystrophin is a subsarcolemmal protein responsible for linking the cytoskeleton to the extracellular matrix, and it is postulated to play a mechanical role in stabilising the muscle fibre membrane (sarcolemma) during contraction. The muscles of the mdx dystrophic mouse, an animal model for DMD, also lack dystrophin, which makes them more susceptible to contraction-induced injury (Dellorusso et al., 2002). The increased susceptibility to stretch-mediated Ca2+ overload, leading to cell contracture and death, is prevented by treatment with the membrane sealant poloxamer 188 (P-188; Yasuda et al., 2005). P-188 can incorporate into damaged membranes and effectively ‘plug’ holes caused by lengthening contractions. We tested the hypothesis that treatment with P-188 would reduce damage and promote membrane integrity in muscles from mdx mice following contraction-induced injury. On the day prior to experimentation, 4-6 month old mdx and wild type (C57BL/10 ScSn) mice were injected with Evans blue dye (EBD; 100 mg/kg). Mice were anaesthetised by intraperitoneal injection with pentobarbitone sodium (60 mg/kg), the right external jugular vein exposed, and a bolus dose of P-188 (460 mg/kg body mass; dissolved in 200 µL sterile saline), or vehicle only, infused intravenously. The right tibialis anterior (TA) muscle was surgically exposed and the distal tendon firmly attached to the lever arm of a servomotor/transducer with the knee immobilised by a secure clamp. The right sciatic nerve was also exposed to deliver supramaximal square wave pulses via a needle electrode. The TA muscle was immersed in warmed paraffin oil to maintain temperature at 37°C and maximum isometric tetanic tension (Po) recorded at the muscles optimum length in situ (intact nerve and blood supply). The muscle was subjected to two stretches of 40% strain (relative to muscle fibre length; initiated from the plateau of isometric contractions, Consolino & Brooks 2004). The magnitude of damage was assessed 5, 10 and 15 minutes later by the deficit in Po (force deficit = (Po (initial) - Po (post strain))/Po (initial)%). The TA muscle was then carefully dissected free and rapidly frozen for later cryosectioning. At the conclusion of experimentation mice were killed by cervical dislocation whilst deeply anaesthetised. Muscle cross sections (8µm) were analysed using a fluorescence microscope for quantification of intracellular infiltration of EBD. Preliminary findings indicate that force deficit was greater in mdx than wild type mice (43 ± 9% vs 25 ± 9%, P < 0.05), but was unaffected by P-188 treatment. The proportion of EBD positive fibres was greater in mdx than wild type mice (15 ± 7% vs. 2 ± 1%, P < 0.05), and was reduced in mdx mice treated with P-188 (4 ± 2%, P < 0.05), irrespective of injury. The proportion of EBD positive fibres was not affected by the injury protocol in either wild type or mdx mice. The results indicate that P-188 does not affect the force deficit following contraction- induced injury but may play a role in maintaining sarcolemmal integrity in muscles from mdx mice, which might prevent Ca2+ overload and promote cell survival. Consolino C.M. & Brooks, S.V. (2004) Journal of Applied Physiology, 96, 633-638. Dellorusso C., Crawford R.W., Chamberlain, J.S., Brooks, S.V. (2002) Journal of Muscle and Cell Motility 22, 467-475. Lee R.C., River, L.P., Pan, F.S., Ji, L., Wollmann, R.L. (1992) Proceedings of the National Academy of Sciences 89, 4524-4528. Yasuda S., Townsend, D., Michele, D.E., Favre, E.G., Day, S.M. & Metzger, J.M. (2005) Nature (in press) DOI: 10.1038/nature03844. Supported by the Muscular Dystrophy Association (USA) and the National Health & Medical Research Council

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Aberrant splicing of ryanodine receptor reduces Ca2+ release via an inter-domain interaction in myotonic dystrophy type 1 T. Kimura1,2, M. Nakamori2, J.D. Lueck3, P. Pouliquin1, R.T. Dirksen3, M.P. Takahashi2, S. Sakoda2 and A.F. Dulhunty1, 1Muscle Research, John Curtin School of Medical Research, Australian National University, Canberra ACT, Australia, 2Clinical Neuroscience (Neurology), Graduate School of Medicine, Osaka University, Suita, Osaka, Japan and 3Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY, USA. Myotonic dystrophy type 1 (DM1) is a multisystem disorder with autosomal dominant inheritance. Expansion of CTG repeats in the 3’ untranslated region of a putative protein kinase gene occurs in DM1. Downstream, it was reported that several mRNAs were aberrantly spliced in muscles from DM1 patients, but the cause of muscle weakness is unknown. We investigated splicing of two major proteins of the sarcoplasmic reticulum, the ryanodine receptor 1 (RyR1) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase. The fetal variants, ASI(-) of RyR1 which lacks residue 3481-3485, and SERCA1b which differs at the C-terminal were significantly increased in skeletal muscles from DM1 patients and the transgenic mouse model of DM1 (HSALR). To examine the functional difference between the ASI(+) and the ASI(-) RyR1 isoforms, we characterized [3H]ryanodine binding to microsomal vesicles of HEK293T cells transfected with ASI(+) and ASI(-) RyR constructs. [3H]ryanodine binding is a standard technique for assessing the open probability of RyR channels, because ryanodine binds solely to open channels and the binding is proportional to open probability. Channel open probability was also measured form RyRs incorporated into using artificial lipid bilayers. Finally Ca2+ release was examined using Ca2+ imaging techniques in dypedic myotubes (lacking RyR1) transfected with ASI(+) and ASI(-) RyR1 cDNA. The affinity of [3H]ryanodine binding to ASI(+) was higher than that to ASI(-). Channel open probability was significantly decreased and mean open time was significantly shorter in ASI(-) than in ASI(+). Consistent with the lower activity of ASI(-) channels, the RyR1-knockout myotubes expressing ASI(-) exhibited a decreased incidence of Ca2+ oscillations during caffeine exposure compared with that observed form myotubes expressing ASI(+) RyR (Kimura et al., 2005). To determine how this aberrant splicing affects the activity of RyR channels, we tested whether the splicing region is involved in inter-domain interaction using synthetic peptides (Yamamoto et al., 2000). Both peptides corresponding to the Thr(3471)-Gly(3500) around the ASI region in the presence (ASI(+)) and the absence (ASI(-)) of exon ASI activated native RyRs. However, peptide ASI(-) activated the channels more than peptide ASI(+). The results suggest that ASI(-) peptide interrupts an inhibitory interdomain interaction in the native RyR more strongly than the ASI(+) peptide. We therefore suggest that ASI(-) region may interact more tightly with other domains and produce stronger inhibition of ASI(-) RyR, resulting in reduced activity of the ASI(-) RyR. Kimura, T., Nakamori, M., Lueck, J.D., Pouliquin, P., Aoike, F., Fujimura, H., Dirksen, R.T., Takahashi, M.P., Dulhunty, A.F. & Sakoda, S. (2005) Human Molecular Genetics, 14, 2189-200. Yamamoto, T., El-Hayek, R. & Ikemoto, N. (2000) Journal of Biological Chemistry, 275, 11618-25.

Proceedings of the Australian Physiological Society

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AuPS/ASB Meeting - Canberra 2005 Free communications 11: Skeletal muscle 2 Friday 30 September 2005 Chair: Gordon Lynch

The effect of altering the rest period during interval training on adaptations to muscle metabolism, ion regulation and exercise performance J. Edge, D. Bishop and C. Goodman, School of Human Movement and Exercise Science, The University of Western Australia, 35 Stirling Highway, Crawley WA 6009, Australia. Exercise training has been shown to reduce the ionic and metabolic disturbance within skeletal muscle during exercise. This may be beneficial to athletes involved in various sporting pursuits, as a greater ionic/metabolic disturbance during a given exercise task is linked with early muscle fatigue. Interval training is a popular training method used by athletes to improve both power and endurance performance. While the adaptations to various interval-training programs have been documented, little research has compared the affects of manipulating the rest period between intervals, on muscle and performance adaptations. The purpose of the present study was to determine the effects of altering the rest period between intense, exercise intervals (during 5 weeks interval training), on adaptations to repeated sprint performance, aerobic fitness and muscle metabolism and ion regulation. Methods. Twelve, recreationally trained females (mean ± SD: age 20 ± 3 y, mass 62.3 ± 10 kg), participated in this study. Tests consisted of a graded exercise test (GXT) to determine VO2peak and the lactate threshold (LT), followed 48 and 96 h later, by two, high-intensity exercise tests. On these days, subjects performed a constant intensity cycle test (CIT: 45 s at 200% VO2peak). On test day one, 60 s after the CIT, subjects performed a repeated-sprint test (5 ´ 6 s sprints, 24 s rest between sprints). On day two, subjects had muscle biopsies before and after the CIT and did not perform the repeated-sprint test. Capillary blood was sampled at the end of each stage of the GXT and before and after the CIT and repeated-sprint test to determine blood lactate and hydrogen ion (H+) concentration. Muscle biopsies (vastus lateralis) were taken to determine muscle ATP, PCr, lactate and H+ accumulation and muscle buffer capacity. Subjects were randomly assigned to one of two training groups. Group one performed high-intensity interval training, with 1 min rest periods between intervals (HIT-1), while group two performed high-intensity interval training with 3 min rest periods between intervals (HIT-3). Each subject had a matched partner (matched on the LT) in the opposing group, with whom they were required to complete an equal amount of work during each training interval and session (10 ´ 2 min at 150% LT, 3 d.wk-1 ´ 5 weeks). Results. There were significant increases in VO2peak (11% HIT-1 vs 9% HIT-3; p<0.05) and the LT (8% HIT-1 vs 15% HIT-3; p<0.05) for both groups, with no differences between groups. There were also significant improvements in mean peak power (W.kg-1; 9% HIT-1 vs 10% HIT-3; p<0.05) and total work (J.kg-1, 13% HIT-1 vs 11% HIT-3; p<0.05) completed during the repeated sprint-test for both groups with no differences between groups. There were no significant changes in muscle buffer capacity or immediate post-CIT blood lactate or H+ following the training period (p>0.05). There were significant reductions in the changes to muscle ATP (∼30%), PCr (∼30%), lactate (∼80%) and H+ (∼60%) following training for both groups (p>0.05), however, no differences between groups. Discussion/Conclusions. Similar to others using endurance activity (1-2 h), we have shown that interval training (3 days per week ´ 5 weeks) can significantly reduce the metabolic disturbance during short-term, highintensity exercise (45 s). Our results also show that, when intense interval training (∼100% of VO2peak intensity), is interspersed with either short (1 min) or longer (3 min) rest periods between each interval, there is little difference in the metabolic and ionic adaptations within the muscle or changes to VO2peak, the LT or repeatedsprint performance. It is likely that the reduced metabolic disturbance during the 45 s of high-intensity exercise, contributed to the improved repeated-sprint performance, as the concentration of PCr and H+ within muscle has previously been shown to affect repeated-sprint performance. It appears that the workload during each interval may be more important to training adaptations following very high-intensity exercise, than the length of the rest period between efforts. This may have important implications for those wanting to improve exercise performance or health, as a greater training stress during intense intervals (shorter rest periods), did not result in a greater training adaptation.

Proceedings of the Australian Physiological Society

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Long lasting muscle fatigue: partial disruption of EC-coupling by the elevated cytosolic calcium during contractions E. Verburg, T.L. Dutka and G.D. Lamb, Department of Zoology, La Trobe University Bundoora Campus, Melbourne, VIC 3086, Australia. We have previously shown that a 10s period of very high cytosolic [Ca2+] (20m M) can disrupt excitationcontraction (EC)-coupling at the signal transduction between the t-tubuli and the Sarcoplasmic Reticulum (SR) Ca2+-release channels in the triad junction (Lamb et al., 1995). It has also been shown that the repeated periods of elevated cytosolic [Ca2+] during repeated tetani are associated with reduced Ca2+-release and long-lasting fatigue (Chin & Allen, 1996). It is however unclear how low levels of [Ca2+] can disrupt EC-coupling in mammalian muscle, and what aspects of the increased levels of cytosolic [Ca2+] during contractions are causing the disruption of EC-coupling. In this study we have used a mechanically-skinned fibre preparation, in which the normal EC-coupling system remains intact. Extensor Digitorum Longus muscles were excised from 4-11 month old Long-Evans Hooded rats that had been killed by an overdose of halothane. Single fibres were dissected from the muscle and skinned. The fibre was then transferred to a solution mimicking the cytosol. Twitch and tetanic force responses were triggered by depolarising the T-system with electrical field stimulation. Periods of elevated cytosolic [Ca2+] were induced by transferring the fibre to a ‘Ca2+-rigor’-solution containing a set [Ca2+]. In this solution no ATP or CrP was present, preventing Ca2+-uptake by the SR, and thus applying a homogenous [Ca2+] throughout the fibre. Alternatively, elevated [Ca2+] was achieved by eliciting four or five 50Hz-tetani in the presence of 5 mM Caffeine.

The figure shows that even a concentration as low as 2m M free Ca2+ throughout the fibre can disrupt ECcoupling, with a bigger effect at concentrations that elicit > 90% of maximal force (n ³ 4 in each case). The time of elevated [Ca2+] required for the effect was however quite long, 1 min. Fifteen normal 0.2 s long tetani, or 4-5 tetani with caffeine and 0.2 mM BAPTA present, did not result in a significant decrease in peak tetanic force. However, the total time at high [Ca2+] eliciting > 90% force would have been £ 2s in the normal tetani. Only in the presence of caffeine, when tetani were at least twice as long as the normal ones and peak [Ca2+] in the triad junction probably a lot higher, was EC-coupling partially disrupted. This shows that the [Ca2+] has to be raised to a very high level and/or be applied for a relatively long period in order to have a deleterious effect. It also suggests that the relatively high [Ca2+] attained locally in the triad junction is more important than the concentration attained in the bulk of the cytoplasm. During normal contractile activity, it can be expected that calcium-induced disruption of EC-coupling would have a significant impact after a very large number of contractions, and hence it may be one of the mechanisms causing the long-lasting muscle fatigue observed after prolonged hard exercise. Chin E.R. & Allen D.G. (1996) Journal of Physiology 491, 813-824. Lamb G.D., Junankar P.R. & Stephenson D.G. (1995) Journal of Physiology 489, 349-362.

Proceedings of the Australian Physiological Society

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The role of reactive oxygen species on stretch-induced muscle damage in dystrophic mice D.G. Allen1 and E.Y. Yeung2, 1School of Medical Sciences, University of Sydney F13, NSW 2006, Australia and 2Department of Rehhabilitation Sciences, Hong Kong Polytechnic University, Hong Kong. Recently we showed that mdx (animal model of Duchenne muscular dystrophy) muscle fibres are more susceptible to stretch-induced muscle damage and there is an associated rise in resting [Ca2+]i (Yeung et al., 2005). We propose that elevated [Ca2+]i causes reactive oxygen species (ROS) production, leading to muscle damage. Thus treatment with ROS scavenger may exert a protective effect against stretch-induced muscle damage. To test this hypothesis, single fibres isolated from the flexor digitorum brevis of the mdx mice were subjected to 10 stretched contractions (eccentric contractions), stretched by 30 % of optimal length (Lo) during each tetanus. Measurements of intracellular calcium with fluo-4 were obtained using confocal microscopy. Calibration of fluo-4 intensities were performed using the procedure described by Kao et al. (1989). The resting [Ca2+]i in the mdx fibres was 227 ± 44 nM (n = 5), significantly higher than that in the wildtype fibres (100 ± 6 nM, n=3, P < 0.05). Under control conditions in the mdx muscle, [Ca2+]i increased slowly following stretched contractions to 690 ± 64 nM (n= 9) after 20 min. The ROS scavenger 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron, 5 mM) was applied during and for 30 min following the stretched contractions in 6 mdx fibres. Not only did Tiron prevent the rise in [Ca2+]i (145 ± 21 nM, P<0.0001) at 20 min, it also improved the force following stretched contractions from 35 ± 4% to 59 ± 7 % (P<0.05). These results indicate that production of ROS play a role in stretch-induced muscle damage in mdx fibres and, further, suggest that ROS may have a role in the activation of stretch-activated channels which produce the Ca2+ entry.

Kao, J.P., Harootunian, A.T. & Tsien, R.Y. (1989) Journal of Biological Chemistry, 264, 8179-84. Yeung, E.W., Whitehead, N.P., Suchyna, T.M., Gottlieb, P.A., Sachs, F. & Allen, D.G. (2005) Journal of Physiology 562, 367-80.

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Effects of raising the temperature from 25°C to 37°C on twitch responses in fast-twitch mechanically skinned muscle fibres of the rat C. van der Poel, J. Edwards and D.G. Stephenson, Department of Zoology, La Trobe University, VIC 3086, Australia. It has been known for many years that intact mammalian muscle fibre preparations brought to the normal body core temperature (37°C) rapidly and irreversibly deteriorate in their ability to produce force (Lännergren & Westerblad, 1987). Consequently, most experiments on isolated mammalian skeletal muscle are conducted at sub-physiological temperatures. In a previous study (van der Poel & Stephenson, 2004) we showed that as the temperature is brought to 37°C, the rate of mitochondrial production of superoxide (O2·−), the parent molecule in the reactive oxygen species (ROS) cascade, rises and that a relatively large fraction of the superoxide produced in the mitochondria can be measured extracellularly. In this study we investigated the effect of raising the temperature to 37°C on the twitch responses induced by triggering action potentials in the sealed transverse tubular (t-) system of single fast-twitch mechanically skinned fibres of the rat by electric stimulation (Posterino et al., 2000). Long-Evans hooded rats were killed by an overdose of halothane in accordance with the procedure approved by La Trobe University Animal Ethics Committee. Extensor digitorum longus (EDL) muscles were dissected out at room temperature, attached to a force transducer and placed in physiological solution at room temperature containing (mM): HEPES, 90; Mg2+, 1; HDTA, 49.95; ATP, 10; CP, 8; Na+, 36; K+, 126; Ca2+, <10-6; pH 7.1. Fibres were initially equilibrated at 25°C and then transferred to equivalent solutions at 37°C. Twitch force responses at 37°C were obtained by electrically stimulating the fibres every 2 mins with supramaximal square pulses until the fibre failed to produce any force. After 7 min at 37°C, the amplitude of single twitches dropped to only 17.33 ± 10.22% (n = 5) of initial response. To test if this decrease was associated with O2·− production, an uncharged, membrane permeable SOD mimetic Tempol (1 mM), which effectively removes O2·− without being used as a substrate, was applied. In its presence, Tempol prevented to a large extent this decrease to only 61.31 ± 8.72% (n = 3). The ability of the contractile apparatus to produce maximum Ca2+ activated force was not different between treatments as shown by the similar force responses per cross sectional area obtained at the end of each experiment in maximally Ca2+-activating solutions at 22ºC (ANOVA, P = 0.82). Also the sarcoplasmic reticulum (SR) Ca2+ content was not different between treatments as indicated by the similarity of force responses elicited following direct activation of the SR Ca2+-release channels in the presence of low [Mg2+] (0.015 mM) ((ANOVA, P = 0.61) (low [Mg2+] responses at 22°C as % of maximum Ca2+-activated force at 22°C: 91 ± 14% max force for control fibres that were kept only at 22°C (n = 6) vs 96 ± 15% max force for fibres that became unresponsive to electrical stimulation at 37°C (n = 6)). Separate experiments indicated that neither the SR Ca2+ handling properties nor the sensitivity to Ca2+ of the contractile apparatus were affected by exposure to 40°C for up to 10 min. Thus, the results imply that O2·− production inside mitochondria at 37ºC is associated with the depression in Excitation-Contraction coupling at a step preceding the SR involvement. The most likely interpretation of our results is that the intracellular O2·− production in the mitochondria decreases the excitability of the t-system in muscle fibres, thus explaining the deterioration in ability of the intact muscle fibre preparations to produce force at 37°C. The results also show that the use of Tempol, a membrane permeant O2·− dismutase (SOD) mimetic can markedly prevent muscle function deterioration at 37°C. Lännergren, J, & Westerblad, H. (1987) Journal of Physiology 390, 285-293. Posterino, G.S., Lamb, G.D. & Stephenson, D.G. (2000) Journal of Physiology 527, 131-137. van der Poel, C., & Stephenson, D.G. (2004) Proceedings of the Australian Physiological Society 35, 75P. Supported by ARC and NH&MRC

Proceedings of the Australian Physiological Society

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Exposure of mammalian skeletal muscle to sub-physiological temperatures reduces its ability to function at physiological temperatures J. Edwards, C. van der Poel and D.G. Stephenson, Department of Zoology, La Trobe University, VIC 3086, Australia. Most studies using isolated mammalian skeletal muscle preparations are conducted at temperatures well below physiological temperatures (17-25°C) because the performance of isolated mammalian skeletal muscle preparations dramatically and irreversibly drops when preparations are re-exposed to normal body core temperatures around 37°C (Lännergren & Westerblad 1987; Ranatunga 1998; Coupland & Ranatunga 2003). This loss in force may be the result of re-heating the preparation during experimental procedures. In order to test the hypothesis that re-heating isolated skeletal muscle fibre preparations to physiological temperature causes damage to the muscle, rat EDL fibre bundles were excised (30-50 fibres) either at 22°C or in a temperature-controlled room at 37°C from Long Evans (Hooded) rats killed by halothane overdose in accordance with the LTU Animal Ethics Committee. The muscles were then attached to a force transducer, stretched to optimum length and tetanically stimulated every 10 min until force could not be measured whilst immersed in a Krebs-Ringer solution (KRS) maintained at 37°C. KRS contained (mM); NaCl 122, KCl 2.8, CaCl2 1.3, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25 and D-glucose 5, (constantly bubbled with carbogen: 95% oxygen, 5% carbon dioxide). The results show that after 30 min of exposure to solution maintained at 37°C, tetanic force dropped dramatically to 3.4 ± 0.1% of initial tetanic force in muscle preparations that were dissected at 22°C and then reheated, whereas after the same length of time, tetanic force dropped to only 68.0 ± 7.8% of initial tetanic force in muscle preparations dissected and kept throughout at 37°C. This marked decrease in tetanic force appears to be associated with an increase in free radical O2•− production when preparations are re-heated. These results show that preventing isolated mammalian skeletal muscle from dropping below core body temperature during dissection helps maintain its function when working at 37°C. Coupland, M.E. & Ranatunga, K.W. (2003) Journal of Physiology 548(Pt 2), 439-49..in 0 Lännergren, J. & Westerblad, H. (1987) Journal of Physiology 390, 285-93. Ranatunga, K.W. (1998) Experimental Physiology 83(3), 371-6..br Supported by ARC and NH&MRC.;

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AuPS/ASB Meeting - Canberra 2005 Free communications 12: Systems physiology Friday 30 September 2005 Chair: Rick Lang

Novel nifedipine-insensitive high voltage activated calcium channels play a role in vascular tone of cerebral arteries M.F. Navarro-Gonzalez and C.E. Hill, Division of Neuroscience, John Curtin School of Medical Research, Australian National University, Canberra, ACT 2601, Australia. Calcium channels are common therapeutic targets for the treatment of cardiovascular disorders such as hypertension (Ishikawa et al., 1997), however they have not always been as successful as might be expected against vasospasm and stroke. Recently a novel high voltage activated, nifedipine-insensitive, mibefradilsensitive calcium channel was described in small mesenteric arteries of guinea pigs and rats (Morita et al., 1999, 2002), and called the “M-type” voltage dependent calcium channel (mVDCC). This channel has also been found in rabbit mesenteric arteries, where it is suggested to play a role in diameter regulation (Itonaga et al., 2002). The aim of the present study was to determine if cerebral arteries possess similar nifedipine-insensitive VDCCs which could be used as targets for cerebrovascular disorders. Juvenile (14-17 day old) male Wistar rats were anaesthetized with ether and decapitated. The basilar artery was removed from the brain and superfused with physiological Krebs solution at 33-37°C. Diameter was monitored as a measure of vascular tone using an edge-tracking computer program. Membrane potential was measured with sharp intracellular microelectrodes (100-180 MW ), and current pulses (1-2 min) were applied to short segments of artery (less than 800µm) using discontinuous current clamp mode (Axoclamp 2B). Change in intracellular calcium concentration ([Ca]i) was measured with the ratiometric calcium indicator Fura-2 AM and a photometry system. After 30 minutes the arteries developed spontaneous rhythmical oscillations in diameter (vasomotion) and membrane potential with the most negative potential around -45mV. Application of the L-type VDCC blocker, nifedipine, abolished vasomotion but did not alter tone, membrane potential or [Ca]i in basilar arteries, while inhibition of the IP3 pathway with U73122 also abolished vasomotion but caused hyperpolarization, relaxation and a decrease in [Ca]i. Small hyperpolarizing current steps which took the membrane potential to -50mV caused immediate abolition of vasomotion and relaxation. Relaxation occurred in the presence or absence of nifedipine. Application of the T- and M-type VDCC blocker, mibefradil, hyperpolarized and relaxed the artery, decreasing [Ca]i, while the T-type VDCC blocker, nickel chloride, only relaxed the artery at a high non-specific concentration (1mM). After the artery was hyperpolarized and relaxed with U73122, application of 40mM KCl caused depolarization and constriction in the presence of nifedipine. A similar result was obtained when 2-APB, an IP3 inhibitor was present together with nifedipine, suggesting that the effect of voltage was not on calcium release from intracellular stores. Taken together the results suggest that nifedipine-insensitive, mibefradil-sensitive VDCCs play a role in vascular tone in the rat basilar artery. These channels are activated at depolarized potentials and rapidly closed by small hyperpolarizations. They are thus unlikely to be T-type VDCCs, which are activated at more negative potentials and rapidly inactivated during prolonged depolarization. We suggest that these nifedipine-insensitive high voltage-activated calcium channels may provide a novel therapeutic target for cerebrovascular disorders. Ishikawa, K., Nakai, S., Takenaka, T., Kanamasa, K., Hama, J., Ogawa, I., Yamamoto, T., Oyaizu, M., Kimura, A., Yamamoto, K., Yabushita, H. & Katori, R. (1997) Circulation, 95, 2368-2373. Itonaga, Y., Nakajima, T., Morita, H., Hanano, T., Miyauchi, Y., Ito, Y. & Inoue, R. (2002) Life Sciences, 72, 487-500. Morita, H., Cousins, H., Onoue, H., Ito, Y. & Inoue, R. (1999) Circulation Research, 85, 596-605. Morita, H., Shi, J., Ito, Y. & Inoue, R. (2002) British Journal of Pharmacology, 137, 467-476.

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Accentuation during diabetes of differential connexin expression between the preglomerular and postglomerular renal vasculature J.H. Zhang and C.E. Hill, Division of Neuroscience, JCSMR, ANU, Acton, ACT 2602, Australia. Gap junctions may play an important role in regulating renal blood flow and glomerular responses. Our previous studies have demonstrated extensive expression of connexins (Cxs) 37, 40 and 43 in endothelial cells and Cx37 in smooth muscle cells of the preglomerular renal vasculature and of Cxs37 and 40 in the reninsecreting cells and intraglomerular mesangial cells. In contrast, there was limited cell coupling in the efferent arterioles with only Cx43 found in the endothelium (Zhang & Hill, 2004). Since elevated glucose has been reported to down-regulate Cx43 in vascular cells in vitro (Kuroki et al., 1998; Sato et al., 2002), our aim was to determine the impact of diabetes on Cx expression in the renal vasculature. Diabetes was induced with sequential daily doses of streptozotocin in citrate buffer (120/80 mg/kg, pH 4.4, intraperitoneally) in male C57BL/6 mice (8-10 weeks) while vehicle injected mice were used as controls. Diabetes was defined as a nonfasting blood glucose level ³ 18 mmol/L on two consecutive days. Mice were deeply anaesthetized (rompun/ketamine 5/25mg/kg body wt. i.p.), the kidneys removed, fixed in ice-cold acetone and 30 m m coronal cryosections cut. Cx distribution was determined at 2, 4, 6, 8 and 10 weeks after the onset of diabetes, using immunohistochemistry and Cx subtype-specific and celltype-specific antibodies. Quantification of Cx changes associated with diabetes was made using the software program Analytic Imaging Station 3. At 2 weeks of diabetes, Cx43 expression in the endothelium of the efferent arterioles was reduced and in many glomeruli was absent by 8 weeks. By 4 weeks, the glomeruli had increased in size and the expression of Cx37 in mesangial cells within the glomerulus had expanded from the vascular pole, while there was no change in Cx37 in the preglomerular vasculature. Cx40 expression in the glomerulus was also increased but not when considered in relation to the enlarged size of the glomerulus. Cx40 was now found in the smooth muscle cells of the afferent arterioles. These changes in Cx expression were maximal by 8 weeks. At 10 weeks of diabetes, Cx43 was detected in the renin secreting cells and in the adjacent smooth muscle cells of the afferent arterioles. We conclude that, during diabetes, cellular coupling within the preglomerular vasculature and intraglomerular mesangial cells is increased while the restricted coupling on the postglomerular side is further reduced. We propose that these changes may accentuate the independence of responses in afferent and efferent arterioles and contribute to the hyperfiltration and pathophysiological damage seen in the diabetic kidney. Kuroki, T., Inoguchi, T., Umeda, F., Ueda, F & Nawata, H. (1998) Diabetes 47, 931-936. Sato, T., Haimovici, R., Kao, R., Li, A.F. & Roy, S. (2002) Diabetes 51, 1565-1571. Zhang, J.H. & Hill, C.E. (2004) Proceedings of the Australian Physiological Society, 35, 87P.

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High-amplitude oscillations in human skin blood flow are distinct from known cardiac or respiratory influences C.D. Haley, C.J. Gordon, N.A. S. Taylor and A.B. Jenkins , Department of Biomedical Science, University of Wollongong, Wollongong, NSW 2522, Australia. ˙ ) oscillations in humans We have recently described the presence of high-amplitude skin blood flow (Q sk (Haley et al., 2004a) and animals (Haley et al., 2004b). These oscillations had a characteristic frequency of ∼0.4 Hz, spanning ∼700-800 ms, and were comprised of high-amplitude peaks that are up to 7-fold greater than basal ˙ . We hypothesised that the high-amplitude oscillations may be related to previously identified lowerQ sk ˙ caused by respiration (0.15-0.4 Hz) and cardiac frequency (0.4-1.6 Hz) amplitude (±30 %) oscillations in Q sk (Stefanovska et al., 1999). Hence, we sought to compare the spectra of respiratory and cardiac activities, with ˙ time-frequency spectrum. the Q sk ˙ (ventral aspect), cardiac frequency and respiration were measured simultaneously (20 Hz) Forearm Q sk from eight males (27.9 yr (SD 6.4), 181.1 cm (SD 4.8), 75.8 kg (SD 7.5), during semi-recumbent rest at 25°C (50% R.H.), on two separate days, for a 5-6 min period. Skin blood flow, was estimated using laser-Doppler flowmetry (TSI Laserflo BPM2, Vasamedics Inc., U.S.A.), cardiac frequency was collected using a three-lead electrocardiogram (ECG: model 100, Humtec, Australia) and respiration was measured from rib cage movement ˙ data were analysed (mercury-in-silastic strain gauge: Hokansen EC-4SB, U.S.A.). Respiratory, ECG and Q sk using a wavelet transform in the frequency domain of 0.05-2 Hz; the dominant frequency band for each variable was calculated as the central portion accounting for 95% of the total integrated power of the time-averaged frequency spectrum. Data are means ± standard errors. ˙ spectrum (0.72 ±0.03) was significantly different (P<0.05, The dominant frequency band for the Q sk paired t-test) from both the respiratory (0.22 ±0.02) and ECG spectra (1.31 ±0.01). Variations between studies ˙ dominant frequencies were not significantly correlated with between study variations in either (n=16) in the Q sk cardiac (r=0.02, P =0.94) or respiration (r=-0.15, P=0.60) dominant frequencies. These results indicate that the ˙ oscillations are not directly related to either cardiac frequency or respiratory function. high-amplitude Q sk Instead, we propose that these oscillations are related to local factors, such as changes in transmural pressure, or the release of substances that alter vascular endothelial and smooth muscle function. Haley, C.D., Zeyl, A., Taylor, N.A.S. & Jenkins, A.B. (2004a) Journal Thermal Biology, 29, 717-723. Haley, C.D., Gordon, C.J., Taylor, N.A.S. & Jenkins, A.B. (2004b) Journal Thermal Biology. 29, 779-783. Stefanovska, A., Bracic, M & Kvernmo, H.D. (1999) IEEE Transactions on Biomedical Engineering, 46(10), 1230-1239.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/142P

Activation of at least three classes of ion channels by b -adrenoceptor activation in pregnant uterine smooth muscle H.C. Parkington, M.A. Tonta, S. Simon, S.A. Cohen, A. Satragno, R.J. Lang and H.A. Coleman, Department of Physiology, Monash University, Vic 3800, Australia. Preterm labour complicates 5-10% of births, has significant repercussions for neonatal morbidity and mortality and may have consequences for lifelong health. Agents that stimulate b -adrenoceptors are commonly used to suppress preterm uterine contractions, yet despite considerable effort, a complete understanding of the mechanisms involved is lacking. We have previously shown that activation of b -adrenoceptors in sheep myometrium markedly reduces the sensitivity of the contractile apparatus to Ca2+ and induces large hyperpolarization that is inhibited by blockade of ATP-sensitive K+ (KATP) channels (Parkington et al., 2000). Stimulation of b -adrenoceptors shifts the activation curve for large-conductance, Ca2+-activated K+ (BKCa) channels to the left in human myometrium (Zhou et al., 2000). In the present study we probed the effects of b -adrenoceptor activation in late pregnant sheep myometrium using a variety of approaches: (1) simultaneous recording of membrane potential and tension in myometrial strips; (2) patch clamp recordings of single channel and whole cell currents in freshly isolated cells from these same ewes; and (3) simultaneous recording of extracellular electrical (EMG) and contractile activity in the uterus of conscious ewes at days 130-140 of pregnancy (term ∼145 days). Under general halothane anaesthesia and using full sterile techniques, EMG electrodes and transducers were attached to the uterus and catheters implanted into a branch of the uterine artery and the jugular vein for local and general drug infusion, respectively. A fetal jugular catheter was implanted to monitor fetal well being. Isolated tissues were obtained during surgery and again at post mortem. Labour was induced preterm in 5 ewes by infusion of RU486 (0.5 mg/kg) (Hirst et al., 2005). Salbutamol was used to stimulate b -adrenoceptors. The hyperpolarization (15 mV in tissues from 20 ewes) evoked in cells in myometrial strips by salbutamol (10-300 nM) was blocked by glibenclamide (1 m M, n=16) and PNU-37883A (10 m M, n=4) but not by iberiotoxin (60 nM, n=3) or charybdotoxin (60 nM, n=6), indicating activation of KATP but not BKCa channels. However, salbutamol induced a prominent activation of BKCa channels in isolated cells and caused a leftward shift of the activation curve that was similar to raising the intracellular Ca2+ concentration (n=12). Blockade of BKCa channels with iberiotoxin revealed that salbutamol also activated two small channels, a KATP channel of 62pS and a channel (conductance 14 pS) that reversed near −20 mV. In strip preparations continuously superfused with glibenclamide-containing solution, the amplitude of the action potential was reduced by salbutamol, and this was blocked by iberiotoxin. In addition, in the presence of PNU-37883A, salbutamol induced a small depolarization. Infusion of salbutamol (100 m g/kg/h) into conscious ewes, 7 days after surgery, caused immediate cessation of the bursts of EMG activity and associated contraction that occurs 3-4 times per hour. Following 30 min infusion of glibenclamide (1mg/kg/h), salbutamol failed to suppress uterine activity before labour (n=7), during normal spontaneous labour (n=3), and following induction of labour preterm (n=5). These results demonstrate a significant activation of KATP by salbutamol in pregnant sheep myometrium at the single channel and tissue levels and in the intact ewe. BKCa channels are also activated, and their main effect is to reduce the amplitude of the action potential and not to cause membrane hyperpolarization. An intriguing and unexpected action of salbutamol was the activation of an inward conductance, but its role in the excessive “rebound” uterine activity observed following salbutamol withdrawal in vivo awaits further investigation. Hirst, J.J., Parkington, H.C., Young, I.R., Palliser, H.K., Peri, K.G. & Olson, D. (2005) American Journal of Obstetrics and Gynecology (in press) Parkington, H.C., Tonta, M.A. & Coleman, H.A. (2000) Proceeding of the Australian Physiological and Pharmacological Society 31, 15P. Zhou, X.B., Wang, G.X., Ruth, P., Huneke, B. & Korth, M. (2000) American Journal of Physiology 279, C1751-1759.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/143P

Expression of a constitutively active K+ channel prevents cell division in the mouse preimplantation embryo M.L. Day1, C.G. Bailey2, J.E. Rasko2, M.H. Johnson3 and D.I. Cook1, 1Physiology, School of Medical Sciences, University of Sydney, NSW 2006, Australia, 2Gene Therapy, Centenary Institute of Cancer Medicine & Cell Biology, University of Sydney, NSW 2042, Australia and 3Department of Anatomy, Downing St., University of Cambridge, CB2 3DY, UK. The activity of a large-conductance, voltage-gated K+ channel changes during the cell cycle in all stages of mouse preimplantation development. This channel is active during M and G1 phases and inactive during S and G2. In parallel with the oscillations in K+ channel activity are changes in the cell membrane potential, being hyperpolarized when the channel is active. The channel appears to be regulated by a cytoplasmic cell cycle that can function independent of the activation of the Cdk1/Cyclin B complex, but does also interact with the nuclear cell cycle. The objective of this study was to determine whether the cycling of K+ channel activity in the mouse early embryo is required for progression of the cell cycle. In these studies we used an adenoviral construct containing a constitutively active mutant of the K+ channel IRK1 (D172N-IRK1) and GFP under separate promoters. A control adenoviral construct that only contained GFP was used to determine non-specific effects of adenovirus transduction. Quackenbush strain mice were superovulated by intraperitoneal injections of pregnant mares’ serum gonadotrophin and human chorionic gonadotrophin (hCG) 48 hours apart. Mice were killed by cervical dislocation approximately 48 hours after hCG injection and 2-cell embryos isolated. Embryos were then transduced with the adenoviruses by incubation in medium M16 containing 1´ 105 pfu/ml adenovirus. Successful transduction of embryos was determined after 16 hours by the expression of GFP. Whole-cell patchclamping confirmed the expression of an inwardly-rectifying K+ current in the GFP positive 4-cell embryos. Expression of D172N-IRK1 caused the membrane potential to be hyperpolarized (-59.1 mV) compared with the membrane potential in non-transduced embryos (-34.7 mV). Development of embryos to the 8-cell stage was reduced from 76.9% in non-transduced embryos to 16.3% in embryos expressing D172N-IRK. These results suggest that cyclic changes in K+ channel activity are important for cell cycle progression in the early embryo. Whether the inhibitory affect is due to hyperpolarization of the membrane potential or loss of cytosolic K+ requires further study.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/144P

AuPS/ASB Meeting - Canberra 2005 Symposium 7: Function and Regulation of Ion Transport Membrane Proteins Friday 30 September 2005 Chair: Ron Clarke

The transition between gating states in inward rectifier K+ channels J. Gulbis, Structural Biology Division, The Walter and Eliza Hall Institute, 1G Royal Parade, Parkville, VIC 3052, Australia. Potassium channels are integral membrane proteins that facilitate a controlled flow of charge across cell membranes. Electrical activity depends the capacity of the channel to stably adopt alternate physiological conformers – ‘closed’ and ‘open’. Although previous crystal structures of K+ channels reveal significant plasticity of the pore, it is unclear whether the conformational differences between individual structures correlate solely with gating state, or if they are representative of familial connections. Two subtly different Xray structures of a prokaryotic inward rectifier K+ channel (KirBac3.1) from Magnetospirillum magnetotacticum are presented here. The assembly with the more constricted ion conduction pathway is markedly asymmetric in the intracellular domains, whereas the other channel is sufficiently open to allow insertion of a large polyamine into the conduction pathway. The KirBac3.1 structures complement that of a close homologue, KirBac1.1, crystallised in an unequivocally non-conducting ‘closed’ conformation. By eliminating family-specific differences, the structures define key molecular indicators of gating state. Incremental re-arrangements occurring in the pore and intracellular domains are likely to reflect distinct stages in the closed-to-open transition.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/145P

Channelrhodopsin 1,2, a new class of ion channels: functional description and cellular applications Georg Nagel1, Peter Hegemann2, Suneel Kateriya2 and Ernst Bamberg 1, 1Max-Planck-Institut für Biophysik, Frankfurt, Germany and 2Institute of Biology, Humboldt University, Berlin, Germany. Microbial-type rhodopsins are found in archaea, prokaryotes and eukaryotes. Some of them represent light activated ion pumps like the well known proton pump bacteriorhodopsin or they act as photosensors for phototactic behaviour in archaea. These proteins have in common the usual rhodopsin-like seventransmembrane helices motif. By expressing microbial-type rhodopsins from the green alga Chlamydomonas reinhardtii in oocytes from Xenopus laevis or in HEK 293 cells we identified two light gated channels. Both channels open rapidly after light excitation and generate a large permeability for protons (ChR1) and for monovalent and divalent cations (ChR2), respectively. The action spectra give strong evidence for the participation of these light gated ion channels on the phototactic behaviour of the alga. The predicted seven transmembrane α helices structure of ChR1,2 is characteristic for G protein-coupled receptors but reflect a completely new motif for a cation-selective ion channel. Because of its unique properties as a light gated ion channel, which depolarizes cells directly without any delay, ChR2 offers the possibility to use it as a tool for manipulating the electrical properties excitable cells or Ca-dependent processes simply by light in a non-invasive manner. Nagel, G., Ollig, D., Fuhrmann, M., Kateriya, S., Mustl, A.M., Bamberg, E. & Hegemann, P. (2002) Science, 296, 2395-2398. Nagel, G., Szellas, T., Huhn, W., Kateriya, S., Adeishvili, N., Berthold, P., Ollig, D., Hegemann, P. & Bamberg, E. (2003) Proceedings of the National Academy of Sciences of the United States of America, 100, 13940-13945. Boyden, E.S., Zhang, F., Bamberg, E., Nagel, G., & Deisseroth, V. (2005) Nature Neurosciences in press. -->

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/146P

Membrane lipid composition and its effect on Na+,K+-ATPase molecular activity: insights from mammals, birds and ectotherms N. Turner1,2,4, P.L. Else1,2, B.J. Wu1,2 and A.J. Hulbert1,3, 1Metabolic Research Centre, 2Department of Biomedical Science and 3School of Biological Sciences, University of Wollongong, Wollongong NSW 2522, Australia (4Present address: Diabetes and Obesity Program, Garvan Institute of Medical Research, 384 Victoria St, Darlinghurst, NSW 2010, Australia.) The basal metabolic rate (BMR) of animals varies dramatically, being several-fold higher in endotherms compared to ectotherms, and much greater, on a mass-specific basis, in smaller vertebrates compared to larger vertebrates. Despite this large variation in metabolic rate between species, a significant and relatively constant proportion of metabolism is associated with membrane-linked energy consuming processes (e.g. Na+ cycling), regardless of the absolute level of BMR. The majority of these membrane-associated processes are mediated by membrane-bound proteins, and here we have measured the molecular activity (turnover rate) of the Na+,K+-ATPase enzyme, a ubiquitous membrane protein that is a significant contributor to BMR, in a range of tissues* from species (five mammals, eight birds and three ectotherms) that vary greatly in their metabolic intensity. Additionally, we have analysed membrane acyl composition in the same tissues to determine the role of the lipid milieu surrounding membrane proteins, in regulating their activity. Na+,K+-ATPase molecular activity varied approximately 20-fold across the different species (1,600 29,000 ATP.min-1), and was generally greater in animals with a higher BMR (i.e. small vertebrates > large vertebrates and endotherms > ectotherms). These variations in molecular activity were associated with differences in membrane lipid composition, with membranes from more metabolically active species having a higher unsaturation index (i.e. number of double bonds per 100 fatty acid chains). The trends in membrane unsaturation were primarily due to significant and substantial variations in the concentration of the highly polyunsaturated omega-3 fatty acid, docosohexaenoic acid (22:6(n-3)), which ranged between 0.5% and 40% of the total fatty acids across the different species. When linear correlations were calculated between Na+,K+-ATPase molecular activity and the relative percentage of individual fatty acids in the membrane, 22:6(n-3) displayed the strongest correlation for any fatty acid in the combined data sets for both the endothermic species (R=0.69, N=39,P<0.0001) and the ectothermic species (R=0.78, N=12, P=0.003). Our results suggest that membrane lipid composition, and particularly 22:6(n-3) content, may play a role in determining the pace (or rate) of metabolism, via an effect on the molecular activity of membrane-bound proteins. * Tissues were obtained from euthanased animals.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/147P

Testing the membrane pacemaker model of metabolism Paul L. Else1, Nigel Turner1, Todd W. Mitchell1, Ben J. Wu1 and Anthony J. Hulbert2, 1Metabolic Research Centre, Department of Biomedical Science University of Wollongong, Wollongong, NSW 2522, Australia and 2Metabolic Research Centre, School of Biological Sciences, University of Wollongong, Wollongong, NSW 2522, Australia. (Introduced by R. Clarke) Much of the metabolic chemistry of life occurs in the lipid rich environment of membranes. Although membrane lipid composition is often seen as relatively constant this belies both processes that continuously remodel these structures as well as the differences between species. In a series of comparisons that differ greatly in metabolic rate (namely ectotherms vs endotherms, newborn vs adult rats, large mammals vs small mammals, large birds vs small birds) we have observed a correlation with the fatty acid composition of cellular membranes and metabolic rate. Low metabolic rates are associated with monounsaturated (i.e. lipids with fatty acids with only one C=C) and high metabolic rates are associated with polyunsaturated (i.e. lipids with fatty acids with two or more C=C) membranes. In essence there is a link between membrane lipid composition and metabolism; this link forms the basis for the membrane pacemaker theory of metabolism. Much of our work on the relationship between membrane lipid composition and metabolism has been derived from examining the sodium pump (Na+K+-ATPase). Constituting up to 20% of the resting metabolism, the sodium pump in different species has vastly different rates of molecular activity (i.e. rate of substrate turnover) with higher rates of molecular activity associated with polyunsaturated and lower rates associated with monounsaturated membranes. In order to test these correlations, species membrane lipid cross-over experiments were performed. Basically, sodium pumps from tissues (kidney or brain) of species with high and low sodium pump molecular activities were crossed-over with membrane lipid from the same tissue of each species (namely; rat against toad, cow against crocodile and adult rat against neonate rat). In all cases, the results showed molecular activities shifted in the direction of the added membrane lipid source. Namely, original membrane lipid restored original molecular activity, sodium pumps with high molecular activity when added with lipid from membrane with low sodium pump molecular activities resulted in decreased activity and conversely sodium pumps with low molecular activity when added with lipid from membrane with high sodium pump molecular activities resulted in increased activity. The order of change in some cases was as much as a 2-3 fold increase or decrease in molecular activity. These results clearly suggest that membrane lipid composition may play a significant role in determining the molecular activity of membrane bound proteins such as the sodium pump, and in so doing set the pace of metabolism. One consequence of this possibility is that membrane lipid composition can be influenced by dietary fat intake and that may have significant implications for metabolic based processes.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/148P

Regulation of the Na,K-ATPase Helge Rasmussen, Department of Cardiology, Royal North Shore Hospital, and Department of Medicine, University of Sydney, NSW, Australia. Na+,K+-ATPase, the enzymatic equivalent of the membrane Na-K pump, maintains transmembrane electrochemical gradients for Na+ and K+. These gradients serve in secondary active transport processes that regulate cellular ions as well as organic compounds. The Na+-K+ pump therefore has a central role in cell function, and its activity is tightly regulated by a variety of hormones, acting on the pump via cell surface receptors coupled to intracellular messenger pathways. Regulation of the Na+-K+ pump is of particular interest in the heart because of the role intracellular Na+ plays in excitation-contraction coupling and in the "electro-mechanical phenotype" of heart failure. Effects of hormones are controversial. Most controversies arise from inappropriate experimental methods, not taking into account the pump’s dependence on both transported ligands at intracellular and extracellular sites and on membrane voltage. We use the whole-cell patch clamp technique to measure electrogenic Na+-K+ pump current (Ip, arising from the 3:2 Na+:K+ exchange ratio) in ventricular myocytes. Provided wide-tipped patch pipettes are used, the technique allows accurate control of pump ligands on both sides of the cell membrane and control of membrane voltage. We have examined effects of hormones coupled to protein kinases A, C and G (PKA, PKC, PKG). Direct phosphorylation of the Na+-K+ pump by protein kinases have been implicated in its regulation for many years. However, such phosphorylation is difficult to demonstrate in vitro unless the pump molecule is denatured. We examined the effect of the catecholamine noradrenaline (NA), typically believed coupled to PKA activation via b 1 and b 2 adrenergic receptors. NA induced an increase in Ip. However, the increase persisted after blockade of b 1/b 2 or inhibition of PKA. In contrast, NA-induced pump stimulation was abolished by ODQinduced inhibition of nitric oxide-activated guanylyl cyclase, an enzyme coupled to the b 3 receptor. Stimulation was reproduced by the selective b 3 agonist BRL 37344. Angiotensin II induced a decrease in Ip that was abolished by inhibition of PKC, a kinase often implicated in pump phosphorylation/regulation. However, PKC-mediated phosphorylation of the pump molecule itself seemed unlikely because additional experiments indicated that the effect of PKC was was dependent on NAD(P)H oxidase activation; the Na+-K+ pump may be regulated by a direct effect of reactive oxygen species on the pump molecule itself. Atrial natriuretic peptide (ANP) induced an increase in Ip. Most effects of ANP are mediated by the NPRA receptor, a ‘membrane guanylyl cyclase’ that is insensitive to ODQ. However, ODQ abolished ANPinduced pump stimulation implicating nitric oxide-activated guanylyl cyclase. In agreement with this inhibition of cGMP-activated protein kinase (PKG) also abolished stimulation. It is concluded that hormone, receptor and protein kinase-mediated Na+-K+ pump regulation is intricately related to nitric oxide and reactive oxygen species metabolism and that direct phosphorylation of the pump molecule is probably not involved.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/149P

AuPS/ASB Meeting - Canberra 2005 Symposium 8: Epithelial Transport of Ions and Metabolites Friday 30 September 2005 Chair: Stefan Bröer, David Cook

A systems biology approach to understanding the role of peptide transporters in biology H. Daniel, Meissner, B. Spanier, D. Weitz and I. Frey, Molecular Nutrition Unit, Technical University of Munich, Am forum 5, D-85350 Freising-Weihenstephan, Germany. Cell membrane transporters for di- and tripeptides are found in bacteria, yeast, plants, invertebrates and vertebrates including mammals. They mediate the cellular uptake of essentially all possible di- and tripeptides and numerous pharmacologically active peptidomimetics by a proton-dependent electrogenic symport mechanism. In mammals, the two di-tripeptide transporters that have been characterized in detail are PEPT1 and PEPT2. PEPT1 mediates as a low affinity but high capacity system the influx of peptides from dietary protein digestion in the gut into intestinal epithelial cells whereas PEPT2 as the high affinity subtype transporter is found in a variety of epithelial cells (i.e. lung, mammary gland, choroid plexus) and prominent expression in renal cells with a role in the reabsorption of filtered peptides. For understanding the biological importance of peptide transporters we follow two lines of research; a gene guided approach by comparing the structure and functions of the same proteins in various organisms (E. coli, C. elegans, zebrafish, mice, rabbit, humans) and a technology-driven approach by applying transcriptomics, proteomics and metabolomics for phenotype analysis in animals (C. elegans and mice) lacking either one of the peptide transporters. The cloning and functional characterization of E. coli peptide transporters with only a low sequence homology (YGDR) but high functional similarity to mammalian PEPT1 provides new insights into structurefunction relationship. Carriers from the various species when studied by electrophysiology after expression in Xenopus oocytes show very similar features despite marked sequence differences. Gene deletions followed by analysis of phenotypical consequences have been carried out in C. elegans and mice. In the nematode, a deletion of the PEPT1 homologous gene provides clues for the role of the intestinal peptide transporter in delivery of bulk qualities of amino acids for growth and development and for a critical crosstalk with the insulin/IGF receptor pathway. There is also a significant effect on stress-resistance of the animals when lacking PEPT1 (Meissner et al., 2004). A mouse line lacking a functional PEPT2 protein did not show any obvious phenotypical changes despite impaired transport of model peptides in kidney and choroids plexus (Rubio-Aliaga et al., 2003). However, when kidney tissue samples of KO and WT mice were submitted to gene expression analysis by cDNA microarray, proteome analysis by 2D-SDS-PAGE and peptide mass fingerprinting via MALDI-TOF-MS and metabolite fingerprinting via GC-MS a variety of metabolic alterations were identified. Pathways of amino acid handling showed impairments and also pathways that process keto acids and carbohydrates. Metabolism of cysteine and moreover of cysteinyl-glycine (Cys-Gly), the break-down product of GSH by g -GT was identified as altered as well. Analysis of urine samples suggests that PEPT2 in renal cells is primarily responsible for uptake of CysGly from the tubular fluids. Meissner, B., Boll, M., Daniel, H. & Baumeister, R. (2004) Journal of Biological Chemistry 279(35):36739-45. Rubio-Aliaga, I., Frey, I., Boll, M., Groneberg, DA., Eichinger, H.M., Balling, R. & Daniel, H. (2003) Molecular and Cellular Biology 23(9):3247-52.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/150P

Na+-H+ exchange regulatory factors NHERF-1 and NHERF-2: roles in albumin endocytosis in the proximal tubule P. Poronnik1, C. Ferguson2, R. Parton2, C.H. Yun3 and D.H. Hryciw1, 1School of Biomedical Science, The University of Queensland, Brisbane, QLD 4072, Australia, 2Institute of Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia and 3Department of Medicine, Division of Digestive Diseases, Emory University, Atlanta, USA. A key function of the renal proximal tubules is to constitutively reabsorb the several grams of albumin that pass across the glomerular barrier per day. This occurs via receptor-mediated endocytosis and requires the formation of a macromolecular complex that involves the scavenger receptor megalin, the Cl− channel ClC-5 and the Na+-H+ exchanger isoform 3 (NHE3). The exact composition of the complex and role of these proteins remains, however, unclear. Patients with Dent’s disease (genetic defects in ClC-5) and ClC-5 knockout mice have persistent proteinuria, demonstrating an obligate role for ClC-5 in albumin uptake. We have previously shown that the cytosolic C-terminus of ClC-5 interacts with cofilin and Nedd4-2 to regulate albumin uptake (Hryciw et al., 2003; Hryciw et al., 2004). As ClC-5 contains a potential C-terminal PDZ binding motif, we investigated if ClC-5 interacted with the NHERF-1/2 PDZ scaffolds and the role of this interaction in albumin uptake. For this study, we used the widely accepted model of renal albumin uptake, the opossum kidney (OK) proximal tubule cell line. Western blotting was used to confirm that these cells expressed NHERF1/2 and electron microscopy was used to confirm subcellular localisation. Co-immunoprecipitation was used to determine whether NHERF1/2 bound to ClC-5 in OK cell lysates. GST-fusion proteins were used to determine which PDZ domain of NHERF-2 bound to ClC-5 and maltose-binding fusion proteins used to identify the binding site for NHERF-2 on the C-terminus of ClC-5. Endogenous NHERF-2 and NHERF-1 were silenced by the use of siRNA transfection plasmids and albumin uptake was measured by standard fluorescent methods. Cell surface biotinylation was also used to monitor changes in ClC-5 under these conditions. Using electron microscopy we demonstrated that OK cells expressed both NHERF-1 and NHERF-2 with NHERF-1 primarily at the microvilli while NHERF-2 was on intracellular membranes consistent with sites of albumin endocytosis. Co-immunoprecipitation in OK cell lysates showed that NHERF-2 but not NHERF-1 bound to ClC-5 in vivo. GST-pulldowns revealed that the C-terminus of ClC-5 bound to NHERF-2 and that this interaction occurred via PDZ-2 of NHERF-2. Further, in vitro experiments with maltose-binding protein fusions confirmed that NHERF-2 bound to an internal site on the C-terminus of ClC-5 and not to the terminal PDZ binding motif of ClC-5. Functional analysis of this interaction demonstrated that silencing of NHERF-2 significantly reduced albumin uptake, accompanied by a reduction in cell surface expression of ClC-5. This suggests that NHERF-2 plays a key scaffolding role in the endocytic complex. In contrast, when NHERF-1 was silenced, there was an increase in albumin uptake paralleled by an increase in surface levels of ClC-5. Our data are consistent with a model in which the efficacy of albumin uptake is dependent on the availability of the components of the macromolecular complex. NHERF-1 is typically responsible for restricting the lateral mobility of NHE3 in the membrane and we propose that knockdown of NHERF-1 may increase the availability of NHE3 to the endocytic complex, resulting in more ClC-5 being recruited into the complex thereby increasing albumin uptake. NHERF-2, on the other hand, plays an integral role in the endocytic complex itself. Hryciw, D.H., Ekberg, J., Lee, A., Lensink, I.L., Kumar, S., Guggino, W.B., Cook, D.I., Pollock, C.A. & Poronnik, P. (2004) Journal of Biological Chemistry, 279, 54996-5007. Hryciw, D.H., Wang, Y., Devuyst, O., Pollock, C.A., Poronnik, P. & Guggino, W. (2003) Journal of Biological Chemistry 278, 40169-76.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/151P

Sulphate ions in mammalian physiology: lessons from sulphate transporter knock-out mice P.A. Dawson1, B. Gardiner2, S. Lee1, M.C. Ku1, S.M. Grimmond2 and D. Markovich1, 1School of Biomedical Sciences, and 2Institute of Molecular Bioscience, University of Queensland, Brisbane, QLD 4072, Australia. Inorganic sulphate (SO42-) is the fourth most abundant anion in mammalian plasma and is essential for numerous metabolic and cellular processes (Markovich, 2001). In humans and rodents, sulphate reabsorption is mediated by the Na+-SO42- cotransporter (NaS1) located at the brush border membrane, and Sat-1, a SO42--anion exchanger located on the basolateral membranes of proximal tubular cells. Both NaS1 null (Nas1-/-) and sat-1 null (sat-1-/-) mice exhibit hyposulphataemia, highlighting the importance of these transporters in maintaining SO42- homeostasis. Since Nas1-/- mice exhibit reduced growth and liver abnormalities, including hepatomegaly (Dawson et al. 2003), we aimed to investigate the hepatic gene expression profile of Nas1-/- mice using oligonucleotide microarrays. The mRNA levels of 130 genes with functional roles in metabolism, cell signalling, cell defence, immune response, cell structure, transcription or protein synthesis were altered (66 induced, 64 down-regulated) in Nas1-/- mice when compared to Nas1+/+ mice. The most up-regulated transcript levels in Nas1-/- mice were found for the sulphotransferase genes, Sult3a1 (∼500% increase) and Sult2a2 (100% increase), whereas the metallothionein-1 gene, Mt1, was amongst the most down-regulated genes (70% decrease). Several genes involved in lipid metabolism, including Scd1, Acly, Gpam, Elov16 and Acs15, were found to be up-regulated (³ 30% increase) in Nas1-/- mice. Increased levels of hepatic lipid (∼16% increase), serum cholesterol (∼20% increase) and LDL (∼100% increase), and reduced hepatic glycogen levels (∼50% decrease), were found in Nas1-/- mice. In addition, Nas1-/- mice have an increased hepatotoxicity to acetaminophen (250-mg/kg i.p.) associated with increased serum ALT activity (>300% increase) and reduced hepatic GSH levels (>60% decrease). Nas1-/- mice live longer (∼25% increase) than their Nas1+/+ littermates, and have a decreased incidence (0/7 affected, P<0.025) of hepatic tumours, when compared to Nas1+/+ mice (4/7 affected) at 2 years of age. In summary, the hyposulphataemic Nas1-/- mouse provides a previously uncharacterised animal model of increased lifespan and altered hepatic metabolism. Dawson, P.A., Beck, L. & Markovich, D. (2003) Proceedings of the National Academy of Science U.S.A. 100, 13704-13709. Markovich, D (2001) Physiological Reviews 81, 1499-1534.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/152P

Regulation of epithelial Na+ channels A. Dinudom, I.-H. Lee and D.I. Cook, School of Medical Sciences, University of Sydney, NSW 2006, Asutralia. Body sodium and fluid homeostasis is largely determined by the activity of Na+ transport proteins that are expressed in the kidney and the GI tract. Among these, the epithelial Na+ channels (ENaC) play an important role in Na+ transport by the distal kidney and the distal colon. This is evident from the observations that gain of function mutations of ENaC, as occur in Liddle’s syndrome, cause hyperabsorption of Na+ in the distal collecting duct of the kidney, leading to salt-sensitive hypertension, whereas loss of function mutations, as occur in pseudohypoaldosteronism type I, cause hypotension. It is well established that the activity of ENaC is tightly regulated. The most important regulators of ENaC are aldosterone and arginine vasopressin which increase activity of the channel during extracellular fluid volume depletion. ENaC activity is also regulated by the concentration of Na+ in the luminal fluid facing the apical membrane. This regulation is mediated by cytosolic Na+ concentration which inactivates the channels by a mechanism involving the G protein, Go, and an ubiquitin-protein ligase, either Nedd4 or Nedd4-2, which ubiquitinates the channels and triggers their endocytosis (Dinudom et al., 1998). Recent studies have suggested that, in addition to its genomic effects, aldosterone may activate ENaC via a mechanism that involves the serum- and glucocorticoid-stimulated kinase, Sgk. This kinase is believed to phosphorylate Nedd4-2 so as to prevent it binding the channels. Contrary to this belief, we have found in wholecell patch-clamp studies on mouse mandibular duct cells that inclusion of recombinant, constitutively-active Sgk in the pipette solution does not prevent inactivation of ENaC by increased intracellular Na+. We found instead that ENaC activity is increased by another protein kinase, the G-protein coupled receptor kinase, Grk2. Our experiments in salivary duct cells further showed that Grk2 phosphorylates the b subunit of ENaC and that this phosphorylation prevents Na+ feedback inhibition of the channel by preventing the binding of Nedd4/Nedd4-2 to channel (Dinudom et al., 2004)). We then investigated the regulation of ENaC by Grk2 in Fisher Rat Thyroid (FRT) cells, a model epithelium. We found that expression of Grk2 in FRT cells expressing ENaC caused a twofold increase in the activity of ENaC compared to FRT cells in which ENaC alone is expressed. Conversely transfection of siRNA directed against Grk2 into FRT cells expressing ENaC inhibited ENaC activity. Interestingly, the mechanism by which Grk2 regulates ENaC in FRT cells differed from the mechanism in salivary duct cells. We found in FRT cells that a kinase-dead mutant of Grk2 activated ENaC in a same manner as wild-type Grk2, and that activation of the channels by Grk2 was due to binding of α-subunits of the Gq,11 family of G proteins by the Regulatory G-protein Signalling (RGS) domain of Grk2. The exact identity of the G protein that inhibits ENaC in FRT cells, and the mechanism by which it does so, are currently being investigated. Dinudom, A., Harvey, K.F., Komwatana, P., Young, J.A. & Cook, D.I. (1998) Proceeding of the National Academy of Sciences USA, 95: 7169-7173. Dinudom, A., Fotia, A.B., Lefkowitz, R.J., Young, J.A., Kumar, S. & Cook, D.I. (2004) Proceeding of the National Academy of Sciences USA, 110: 11886-11890.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/153P

Disorders of neutral amino acid resorption in epithelial cells S. Bröer, School of Biochemistry & Molecular Biology, Australian National University, Canberra, ACT 0200, Australia. Recent successes in the molecular cloning and identification of apical neutral amino acid transporters have shed a new light on inherited neutral amino acidurias, such as Hartnup disorder and iminoglycinuria. Hartnup disorder is caused by mutations in the neutral amino acid transporter B0AT1 (SLC6A19) (Kleta et al., 2004; Seow et al., 2004). The transporter is found in kidney and intestine, where it is involved in the resorption of all neutral amino acids (Bröer et al., 2004). It belongs to the SLC6 family, comprising transporters for neurotransmitters, osmolytes and creatine. B0AT1 transports neutral amino acids together with 1 Na+-ion but in contrast to other members of the SLC6 family is chloride independent. The SLC6 family also contains a number of ‘orphan transporters’ the physiological function of which has remained elusive. Identification of SLC6A19 as a Na+-dependent amino acid transporter suggested that orphan neurotransmitter transporters might in fact be amino acid transporters. SLC6A20 turned out to be the long-sought IMINO system, a Na+ and Cl--dependent proline transporter (Kowalczuk et al., 2005). SLC6A20 is highly expressed in the kidney and intestine and may play a role in iminoglycinuria, a disorder characterised by hypersecretion of proline and glycine in the urine. Although SLC6A20 transports proline but not glycine, it is considered a candidate for iminoglycinuria because excess of proline in the proximal tubule could compete for glycine uptake by the proline/glycine transporter PAT1 (SLC36A1). Further functional analysis of SLC6 orphan transporters demonstrated that SLC6A15 is a transporter for large neutral amino acids plus proline. The transporter is highly expressed in the brain and kidney. In the kidney it may serve as a high-affinity back-up transporter for selected amino acids in the distal parts of the proximal tubule. Functionally SLC6A15 is related to B0AT1 and was hence named B0AT2. It transports neutral amino acids together with 1 Na+ and is chloride independent. In summary, a new family of Na+-dependent amino acid transporters has been identified, the members of which are involved in the transport of amino acids in epithelial cells and the nervous system. Bröer A., Klingel K., Kowalczuk S., Rasko J.E., Cavanaugh J. & Bröer S.. (2004) Journal of Biological Chemistry 279: 24467-24476. Kleta, R., Romeo, E., Ristic, Z., Ohura, T., Stuart, C., Arcos-Burgos, M., Dave, M.H., Wagner, C.A., Camargo, S.R., Inoue, S., Matsuura, N., Helip-Wooley, A., Bockenhauer, D., Warth, R., Bernardini, I., Visser, G., Eggermann, T., Lee, P., Chairoungdua, A., Jutabha, P., Babu, E., Nilwarangkoon, S., Anzai, N., Kanai, Y., Verrey, F., Gahl, W.A. & Koizumi, A. (2004). Nature Genetics 36: 999-1002. Kowalczuk, S., Bröer, A., Munzinger, M., Tietze, N., Klingel, K. & Bröer, S. (2005) Biochemcal Journal 386: 417-422. Seow, H.F., Bröer, S., Bröer, A., Bailey, C.G., Potter, S.J., Cavanaugh, J.A. & Rasko, J.E. (2004) Nature Genetics 36: 1003-1007.

Proceedings of the Australian Physiological Society

http://www.aups.org.au/Proceedings/36/154P

AuPS/ASB Canberra 2005 Meeting - Author Index Adams, D.J. 40P, 72P, 74P Agon, V.V. 51P Aguilar, M.I. 90P Allen, D.G. 22P, 33P, 65P, 104P, 105P, 131P, 137P Allen, R.J.W. 20P Amisaki, T. 54P Amoroso, S. 51P, 52P Anderson, J.L. 71P Apell, H.-J. 51P Aromataris, E. 110P Arthur, P.G. 23P, 66P Aw, J. 31P, 70P Bailey, C.G. 144P Bamberg, Ernst 146P Barclay, C.J. 35P, 114P Barritt, G. 110P Barry, P.H. 28P, 41P Bartlett, P.F. 74P Bastug, Turgut 21P Beam, Kurt 97P Beard, N.A. 68P, 69P Beilby, M.J. 56P, 60P Beitzel, F. 130P Bennetts, B. 34P Bennie, J.A. 16P Beswick, E. 127P Bezanilla, Francisco 1P, 2P Bishop, D. 135P Bisset, D. 5P Bjorksten, A.R. 17P Blazev, R. 115P, 118P Board, P.G. 99P Bröer, A. 80P Bröer, S. 79P, 80P, 154P Breit, S.N. 50P, 91P Brown, L.J. 50P, 59P, 91P Brown, M.J. 17P, 31P Bubb, W.A. 55P Buckley, P. 87P Byrne, T. 83P Cairns, S.P. 32P Caldwell, J.N. 12P Cameron-Smith, D. 70P Cannell, M.B. 65P Carey, K. 70P Casarotto, M.G. 99P Ceroni, L. 11P Chan, S. 67P Chanda, Baron 2P Chandra, M. 28P Chapman, B.E. 55P Chattopadhyay, Amitabha 96P

Chaulet, H. 22P Cheung, S. 68P Chhabra, D. 108P Chung, S.H. 5P, 18P, 19P Clare, B. 39P Clarke, C.E. 49P Clarke, R.J. 51P, 52P Cohen, A.E. 53P Cohen, S.A. 143P Cole, L. 30P Coleman, H.A. 143P Colthorpe, K.L. 124P, 126P Conroy, S-J. 76P Cook, D.I. 77P, 144P, 153P Cornford, S. 83P Corry, B. 5P, 19P, 37P Cotter, J.D. 13P Cromer, B.A. 34P Cui, Y. 99P Curl, C.L. 107P Curmi, P.M.G. 50P, 91P D'Amore, A. 107P Dallemagne, C.R 88P Dalton, R. 103P Daniel, H. 150P Davies, P. 25P Davies, W.L 76P Dawson, P.A. 81P, 152P Day, M.L. 144P Dedova, I. 108P DeLaCruz, E 108P Delbridge, L.M.D. 106P, 107P Di Maria, C.A. 66P Ding, H. 10P, 11P Dinudom, A. 153P Dirksen, R.T. 134P Dooley, D.M. 53P dos Remedios, C.G. 108P Duff, A.P. 53P Dulhunty, A.F. 68P, 69P, 84P, 99P, 134P Dutka, T.L. 30P, 136P Edge, J. 135P Edwards, J. 138P, 139P Ekberg, J. 77P Ellis, A. 11P Ellis, P.J. 53P Else, P.L. 147P, 148P Ernst, H.G.G. 124P, 126P Everitt, A.B. 47P Everitt, A.E. 27P Favaloro, J.L. 9P, 106P Ferguson, C. 151P

Fischer, L. 58P Forrest, A. 127P Fowler, C.J. 75P Fraser, S.F. 31P Freeman, H.C. 53P Frey, I. 150P Fujiwara, S. 54P Furness, J.B. 75P Gage, P.W. 27P, 45P, 46P, 47P, 48P, 102P Gardiner, B. 152P Garland, C.J. 7P Garrett, A.T. 13P Gervásio, O.L. 26P Ghoddusi, M. 132P Gong, X. 15P, 17P, 31P, 70P Goodman, C.A. 16P, 17P, 31P, 70P, 135P Goossens, N.G. 13P Gordon, C.J. 12P, 142P Graham, R.M. 22P Grimmond, S.M. 152P Guggino, W.B. 77P Gulbis, J. 145P Guss, J.M. 53P Haley, C.D. 12P, 142P Hall, K. 90P Hambly, B.D. 59P Hamill, O.P. 93P Hammer, J. 113P Hardeman, E.C. 132P Harrap, S.B. 107P Harrop, S.J. 91P Harvey, P.J. 99P Hawthorne, R.L. 6P, 43P Hayakawa, Kimihide 95P Head, S.I. 67P, 71P Hegemann, Peter 146P Henry, R. 82P Hill, C.E. 140P, 141P Hill, M.A. 10P Hillier, W. 57P, 120P Hoh, J.F.Y. 116P Holmes, G. 83P Honen, B. 103P Honore, Eric 94P Hool, L.C. 23P, 39P, 66P Horan, C.R. 48P Hosaka, K. 112P Hou, X. 90P Hoyles, M. 18P Hryciw, D.H. 77P, 79P, 128P, 151P

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Huggins, C.E. 106P Hulbert, A.J. 147P, 148P Hunne, B. 75P Hwang, Y.T. 10P Imtiaz, M.S. 112P Jenkins, A.B. 142P Johnson, M.H. 144P Joya, J.E. 132P Ju, Y.K. 22P, 65P Karunasekara, Y. 99P Kateriya, Suneel 146P Kee, A.J. 132P Keller, S.L. 89P Kelly, D.R. 61P, 63P Kemm, R.E. 86P Kemp, J.G. 118P Kemp-Harper, B.K. 9P Kent, A.B. 15P Kimura, M. 116P Kimura, T. 134P Kirk, K. 20P Klingel, K. 80P Kloda, A. 38P, 40P Klonis, Nick 92P Korn, S.J. 3P Kotecha, N. 10P Kowalczuk, S. 80P Kruger, W.A. 78P Krum, H. 31P, 70P Ku, M.C. 152P Kuchel, P.W. 55P Kumar, S. 77P Kurosky, A. 93P Lamb, G.D. 30P, 117P, 136P Lambert, J.J. 41P Lang, R.J. 113P, 143P Langley, D.B. 53P Larina, Olga 111P Larkum, A. 52P Laver, D.R. 98P, 103P Lee, A. 77P, 79P Lee, I.-H. 153P Lee, S. 81P, 152P Leikis, M.J. 15P, 16P Lensink, I.L. 77P Leppik, J.A. 15P, 31P Lewis, R.J. 72P Lewis, T.M. 28P, 41P Litjens, T. 110P Littler, D.R. 50P, 91P Liu, Z.-W. 36P Lluka, L. 123P Lueck, J.D. 134P Luu, T.L. 27P, 46P Lynch, G.S. 119P, 129P, 130P, 133P

Lynch, J.W. 6P, 42P, 43P, 44P Ma, T.A. 79P Mackenzie, L. 61P, 63P Mahomudally, E.M. 60P Markovich, D. 81P, 152P Maroto, R. 93P Marshall, J. 121P Martin, L. 90P Martinac, B. 4P, 29P, 36P, 37P, 38P, 39P, 93P Mathie, A. 49P Mazzanti, M. 91P McConnell, I.L. 120P McKenna, M.J. 15P, 16P, 17P, 31P, 70P McLennan, P.L. 64P, 82P McLeod, M.D. 51P McMahon, L.P. 15P Mechler, A. 90P Medved, I. 17P Meeker, W. 113P Meissner, B. 150P Meyer, G. 29P Mitchell, T.W. 148P Moens, P.D.J. 109P Moni, K.B. 123P, 127P Moni, R.W. 123P, 127P, 128P Monteith, G.R. 78P Moon, A.R. 60P Moopanar, T.R. 33P Moorhouse, A.J. 28P, 41P Morley, J.W. 71P Morris, N.R. 14P Morton, C.J. 34P Motin, L.M. 72P Mulders, W.H.A.M. 73P Munzinger, M. 80P Murphy, K.T. 15P, 17P, 70P Murphy, R.M. 117P Murphy, S. 25P Murphy, T.V. 10P Mynott, A.V. 50P, 91P Nagel, Georg 146P Nair-Shalliker, V. 132P Nakamori, M. 134P Navarro-Gonzalez, M.F. 140P Neild, T.O. 125P Ney, A.D. 42P Neylon, C.B. 75P Ng, H-L. 34P Ngui, K. 75P Nguyen, M.-A. 132P Nguyen, T. 39P, 110P Norris, N. 99P O'Connell, B. 115P O'Mara, M. 18P

Oakley, C.E. 59P Owen, A.J. 82P Paarker, M.W. 34P Parker, Ian 111P Parkington, H.C. 113P, 143P Parton, R. 151P Patterson, M.J. 13P Pepe, S. 64P, 106P Perozo, E. 29P Peters, J.A. 41P Petersen, A.C. 15P, 17P, 31P, 70P Petrou, S. 25P Petrov, E. 36P Phillips, W.D. 26P Philp, D.J. 55P Pierce, K.D. 28P Plakhotnik, T. 58P Plant, D.R. 133P Pollock, C.A. 77P Poole, D. 75P Poronnik, P. 77P, 78P, 79P, 123P, 128P, 151P Porrello, E.R. 107P Pouliquin, P. 134P Pow, D. 79P Premkumar, A. 48P Proietto, J. 106P Qu, W. 28P Quinnell, R. 52P Rasko, J.E. 144P Rasmussen, Helge 149P Raso, A. 93P Rayfield, A. 79P Razeghifard, R. 57P Rehrer, N.J. 13P Rigby, P. 37P Roberts, M. 110P Robertson, D. 73P Ryall, J.G. 129P, 133P Rychkov, G.Y. 34P, 100P, 110P Sabapathy, S. 14P Safer, D. 108P Saint, D.A. 61P, 62P, 63P Sakoda, S. 134P Saliba, K.J. 20P Satragno, A. 143P Schertzer, J.D. 119P Schneider, D.A. 14P Schwartz, J. 87P Sebban, P. 51P Sefton, Ann 85P Seymour, V.A.L. 45P Shepherd, V.A. 56P, 60P Sillence, M.N. 130P Simon, S. 143P

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Small, D.H. 90P Smith, John 122P Snow, R.J. 31P, 70P, 117P Sobey, C.G. 8P Sokabe, Masahiro 95P Sostaric, S. 17P, 31P, 70P Spanier, B. 150P Stafford, M.R. 74P Stapleton, D. 34P Starke-Peterkovic, T. 51P Stephenson, D.G. 118P, 138P, 139P Stephenson, G.M.M. 115P, 118P Steward, C.H. 31P Streamer, M. 131P Subasinghe, S. 90P Sugiharto, 41P Takahashi, M.P. 134P Takemori, S. 116P Tan, H. 25P Tatsumi, Hitoshi 95P Taylor, N.A.S. 12P, 142P Theiss, M.L. 64P Thomas, W.G. 107P Thorn, Peter 111P Tieleman, Peter 101P Tierney, M.L. 27P, 45P, 46P, 47P Tietze, N. 80P Tilley, Leann 92P Tongpao, L. 78P Tonta, M.A. 143P Trezise, A.E.O. 76P Triggle, C.R. 11P Turner, N. 147P, 148P Unwin, N. 24P van der Poel, C. 138P, 139P van Helden, D.F. 103P, 112P Vandenberg, J.I. 49P Varsànyi, M. 68P Veatch, S.L. 89P Verburg, E. 136P Viola, H.M. 23P, 66P Vora, T. 19P Vorobyev, M. 58P, 121P Ward, M.L. 105P Watson, M. 83P Webb, T.I. 44P Wei, L. 68P, 69P Weiss, S.M. 102P Weitz, D. 150P Wendt, I. 113P Whitehead, N.P. 131P Widén, C. 114P Wiehler, W. 11P Williams, I.A. 104P Williamson, A. 57P

Wolfe, Joe 122P Wood, T.G. 93P Wu, B.J. 147P, 148P Wydrzynski, T. 57P, 120P Yagi, N. 116P Yamaguchi, M. 116P Yang, Z. 42P, 44P Yeung, E.Y. 137P Yin, J. 18P Yuan, S.Y. 62P Yun, C.H. 79P, 151P Zhang, J.H. 141P Zhao, J. 112P Zhu, H.P. 62P Zoltkowoski, B. 113P Zvyagin, A. 58P

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Joint annual meeting of the Australian Physiological Society and

AuPS/ASB Meeting Canberra 2005 Joint annual meeting of the Australian Physiological Society and Australian Society for Biophysics Canberra 2005 1 ...

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