Electrophysiological Effects of Fractions Isolated from the Venom of Parabuthus Granulatus on Calcium Channels in Cardiac Myocytes
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Electrophysiological effects of fractions isolated from the venom of Parabuthus granulatus on calcium channels in cardiac myocytes agister Scientiae in Physiology at the sity, Potchefstroom Supervisor: Prof. K. Dyason Co-supervisor: Mr. J.L. du Plessis 2004 Potchefstroom I would like to thank The Almighty Father for the blessings, opportunities and talent He provided me to make my studies successful. I would like to thank my supervisor, Prof. Karin Dyason. Without her sacrifices, expert knowledge, time and guidance, the completion of my dissertation would not have been possible. To my family, who supported me in many ways. Their guidance and encouragement were the most appreciated. To my husband, a special word of thanks for supporting me through the difficult times during the study. A word of thanks to the following persons: Mrs. Carla Fourie, not only for the isolation of the ventricular myocytes, but also for the knowledge and support during the two years of this study. Dr. Francois van der Westhuizen (subject group Biochemistry), for his assistance in determining the fiaction protein concentrations. Prof. Lourival Possani for the separation of the fraction and subfiactions. Prof. J.J. van der Walt, for his continuous interest and valuable input. Contents Abbreviations and symbols .................................................................................. i List of figures and tables .................................................................................... iv OPSOMMING ....................................................................................................... v SUMMARY ......................................................................................................... vi CHAPTER 1 .General introduction. problem statement and aims .................1 1.1 Problem statement and aims .....................................................................................4 CHAPTER 2 .Literature study ................................................................................. 5 1 . Distribution of medically important scorpions .................................................. 5 2 . Scorpion venom ................................................................................................. 6 2.1 Composition of scorpion venom .......................................................................6 2.2 Peptide toxins in scorpion venom ...................................................................... 7 2.3 Scorpion envenomation ....................................................................................8 3 . Ion channels ....................................................................................................... 9 3.1 Ca2+ channels ................................................................................................... 10 3.1.1 Classification ................................................................................................ 11 3.1.2 Distribution and properties ..........................................................................13 3.2 Cardiac Ca2+ channels .................................................................................... 16 3.2.1 Structure ....................................................................................................... 16 3.2.1 .1 Structure-function relationship of the a, subunit ...................................... 17 . 3.2.1.2 Auxlhary subunits ......................................................................................20 . 3.2.2 Distr~but~on.................................................................................................. 21 . 3.2.3 Electrophysiological character~st~cs............................................................. 22 3.2.4 Phosphorylation ........................................................................................... 23 3.2.5 Pharmacology .............................................................................................. 24 3.2.5.1 Organic antagonists ................................................................................... 25 3.2.5.2 Inorganic antagonists ................................................................................ 27 3.2.5.3 Organic agonists ........................................................................................ 27 3.2.5.4 Interactions of natural toxins with Ca2+channels .....................................29 3.2.6 Physiological role of Ca2+ channels ....................................................... 31 4 . Pathology of Ca2+ channels .............................................................................. 33 5 . Scientific and therapeutic value of natural peptides ........................................ 34 Guidelines for the Author .Toxicon .................................................................. 37 CHAPTER 3 .Article .......................................................................................... 46 The electrophysiological effects of fractions isolated from the venom of Parabuthus granulatus on calcium channels in rat cardiac rnyocytes ...................................... 46 CHAPTER 4 .Conclusions and recommendations .......................................... 77 Addendum A ......................................................................................................... 81 Addendum B ......................................................................................................... 87 Addendum C ......................................................................................................... 96 Addendum D .......................................................................................................101 References................................................................................................................. 106 Abbreviations and svmbols a alpha A the amplitude of the maximum ca2+current that inactivates AP action potential ATP adenosine triphosphate AV node atrioventricular node B beta ~a~' barium BHK baby hamster kidney cells BmK AS Buthus martensi Karsch peptides AS C the time dependent ca2+current that inactivates slowly or not at all ca2+ calcium CAMP cyclic adenosine monophosphate cd2+ cadmium cDNA complementary deoxyribonucleic acid CHO chinese hamster ovary cells CNS central nervous system c1- chloride co2+ cobalt C-terminal carboxy-terminal 6 delta DHPIs dihydropyridinels DRG dorsal root ganglia E the membrane potential Abbreviations and svmbols E-C excitation-contraction Eta the equilibrium potential for ca2+ EGTA ethylene Glycol-bis (P-aminoethyl Ether) N, N, N', N', - Tetraacetic Acid. Eh membrane potential for 50 % activation ER endoplasmic reticulum E-S excitation-secretion FTX funnel web spider toxin Y gamma g gr- gmax maximum conductance through the ca2+channel GR giga ohm GTP guanosien triphosphate HEPES N- (2-hydroxyethyl) piperazine-N'-(2-ethanesulphonic acid) HIV human immunodeficiency virus HO~+ holmium HP holding potential HVA high-voltage activated I the maximum current through the ca2+channel IB~ the ~a~+current through the ca2+channel Ih inositoltriphosphate IpTx, Imperatoxin A IpTxi Imperatoxin I K+ potassium kD kilo Dalton Abbreviations and svmbols KLI kurtoxin like peptide I KLII kurtoxin like peptide I1 ~a~' lanthanum L-type long-lasting type ca2+channel LVA low-voltage activated M~*+ magnesium MR mega ohm Pg microgram PM micromolar mM millimolar ml millilitre mg milligram ms milliseconds mV millivolt Na' sodium ~i~+ nickel NMDA N-methyl-D-aspartate NT neurotransmitter N-terminal amino-terminal N-type neuronal type ca2+channel w-Aga w-Agatoxin w-CTx w-conotoxin pb2+ lead PBTxl parabutoxin 1 PBITx3 parabutoxin 3 iii Abbreviations and svrnbols PEG polyethelene glycol PgIII fraction I11 isolated from P. granulatus venom PICA protein-kinase A PKC protein-kinase C PKG protein-kinase G P-loop pore loop PS picosiemens P/Q-type purkunje type ca2+channel RP-HPLC reverse phase high performance liquid chromatography R-type resistant type ca2+channel S slope of voltage dependence SA node sinoatrial node SEM standard error of the mean SF1 subfraction I isolated from PgIII SF11 subfraction I1 isolated from PgIII SFIII subfraction I11 isolated from PgIII SR sarcoplasmic reticulum T the time constant of inactivation TEA-CI tetraethylammonium chloride TEA-OH tetraethylammonium hydroxide TFA trifluoroacetic acid T-type transient type ca2+channel TTX tetrodotoxin uv ultra violet V% membrane potential where 50 % of the ca2' channels are activated Abbreviations and svmbols voltage dependent calcium channeWs test potential zinc List of figures and tables CHAPTER 2 Table 1 -The properties of the different types of VDCCs ..................................................... 15 Figure 1 .A diagrammatic representation of the VDCC ........................................................ 17 CHAPTER 3 Figure 1 . Chromatographic profile of subfractions from PgIII separated by RP.HPLC ...... 72 Figure 2 .Effect of PgIII on ~a"currents in ventricular myocytes ..................................... 73 Figure 3 . Effects of subfractions on ca2+channels in ventricular myocytes....................... 74 Figure 4 .Comparison of the voltage dependence of activation in the presence of the subfractions (A) SFI. (B) SFII. (C) SFIII..................................................... 75 Figure 5 . Representative superimposed current traces recorded from a HP of -80mV and depolarizing to -10 mV ................................................................ 76 Table 1 .Effects of subfractions on the time to