MECHANISM OF a-LATROTOXIN ACTION AT THE FROG NEUROMUSCULAR JUNCTION Chris Tsang A thesis in confomiity with the requirements for the degree of Master of Science Graduate Department of Physiology University of Toronto " Copyright by Christopher Wilson Tsang 2000 Acquisitions and Acquisitions et Biblbgnphii Services aiwices bibliographiques The author has granted a non- L'auteur a accordé une licence non exclusive Licence allowing the exclusive permettant à la National Li- of Canada to Bibliothèque nationale du Canada de reproduce, loan, distriiute or seii reproduire, prêter, distniiuer ou copies of this thesis in microforni, vendre des copies de cette thèse sous paper or electronic formats. la forme de microficheffilm, de reproduction sur papier ou sur format electronique. The author retains ownership of the L'auteur conserve la proprieté du copyright in this thesis. Neither the droit d'auteur qui protege cette thèse. thesis nor substmtial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. Canada MECHANISM OF a-LATROTOXIN ACTION AT THE FROG NEUROMUSCLULAR JUNCTION Chris Tsang, M. Sc. Thesis, 2000. University of Toronto, Department of Physiology. ABSTRACT a-Latrotoxin (a-LTX) is a neurotoxin purified from black widow spider venom that accelerates spontaneous transmitter release independently of extracellular ca2+.Since transmitter release is ca2+-dependent,! hypothesized that a-LTX mobilized intracellular ca2+.To address this question, standard electrophysiology and confocal imaging techniques were used to measure the rate of transmitter release and ion concentration dynarnics respectively in frog motor nerve terminais bathed in CFS. Here I report that transmitter release by a-LTX follows an increase in intracellular ca2+.The ca2+pool targeted by a-LTX is likely to be mitochondrial in origin and not endoplasmic reticulum. An increase in intracellular Na' precedes the release of ~a"suggesting that the mitochondrial ~a+/~a~+exchanger may be activated. There is also evidence to suggest that the mitochondrial peneability transition pore rnay be involved. Although the release of ca2' may accompany the increase in transmitter release produced by a-LTX, it is not necessary. .. AB STRACT ....................................................................................................II ... TABLE OF CONTENTS ..................................................................................III LIST OF TABLES ...................... ... ..................................................................vi .. LIST OF FIGURES .......................................................................................... VII LIST OF ABBREVIATIONS ...............................................................................ix ACKNOWLEDGEMENTS ................................................................................. x 1 INTRODUCTION .......................................................................................................... 1 1. 1 THE BLACK WlDOW SPIDERS .......................................................................... 1 1.1 -1 Habitat .............................................................................................*............. 1 1.1.2 Envenornation ................................................................................................ 1 1.2 a-LATROTOXIN ................... ... ............................................................................ 3 1.2.1 Purification.......................................... .. ............................... ...... 3 1.2.2 Structure ..................................................................................... .... ............... 4 1.3 ACTION OF a-LATROTOXIN.............................................................................. 6 13. Transmitter release .............................................+.......................................... 6 1.3.2 Fine Structure ................................................................................................ 7 1.4 MECHANISM OF a-LATROTOXI N ACTION ....................................................... 8 1 A.1 Pore-Forming Hypothesis .............................................................................. 8 1.4.2 Receptor Hypothesis ................................................................................... 10 7.4.2.7 Neurexins .........................................................................+............. .......... 10 . 7.4.2.2 Latrophrlrn / CIRL ............................................................. ... ................ 1 2 1.4.2.2.1Sequence .................... .. ...................................................................... 12 1.4+2.2.2Biochemica/Propetties .................... ... ................................................... 14 ~.4.2.2.3~omoîoguesand Distribution ......................... .. ..*................**........*........1 5 1.5 PURPOSEOFTHISSTUDY .....................................,.... 15 1.5.1 Hypothesis ........................................ .. ....... 15 2 MATERIALS AND METHOOS ............................................................. ,. 16 2.1 ANIMALS AND EXPERIMENTAL TREATMENT .......... .... .........*......*............16 2.2 EXPERIMENTAL SOLUTIONS ......................................................................... 16 iii 2.3 IMMUNOCYTOCHEMISTRY..................... ........... ..,, ......................................... 16 2.4 DYE LOADiiUG .......................................................................................... 19 2.5 BLOCKADE OF SPONTANEOUS ACTION POTENTIALS ......................... ,., ..,20 2.6 BLOCKADE OF POSTJU NCTIONAL RECEPTORS ...... .... ......... ..................... -20 2.7 RECORDING TRANSMITTER RELEASE......................................................... 21 2.7.1 Analyzing Spontaneous Transrnitter Release ..............................................21 2.8 FLUORESCENCE MICROSCOPY................... .. ............................................ 23 2.8.1 Perisynaptic Schwann Cell Calcium ............................................................ 23 2.8.2 Image analysis............................................................................................. 23 2.8.3 Chemicals .................................................................................................... 24 2.9 CHEMICALS.................. ... ............................... ......... ,., .....*..., -2.1 0 STATISTICAL ANALYSI S ................................................................................ -24 -2.1 1 FIGURES .................................,., ...................................................................... 24 3 RESULTS .......................,, ....., ................................................................................... 25 3.1 IS CL? LOCATED AT THE FROG NMJ? .................................... ,..,. ................-25 3.2 DOES a-LTX INCREASE INTRACELLULAR ca2+WlTH TRANSMITTER RELEASE IN CFS? .................................................................................................... 27 3.2.1 1s the Tissue Bath Norninally Free of ca2'? ............................................... 27 3.2.2 a-LTX and Presynaptic caZ+........................................................................ 27 3.2.3 a-LTX and Transmitter Release ................................................................. 29 3.2.4 Dose-dependence of a-Ln< Action ............................................................. 29 3.3 DOES a-LTX RELEASE ca2' FROM ER? ................................................... -31 3.3.1 The Effect of U-73122 ..................... ,.,, .......... ,., .......................... 31 3.3.2 The Effect of Thapsigargin ................................................................. -31 3.4 DOES a-LTX RELEASE ca2+FROM MITOCHONDRIA? ................................ 36 3.4.1 The Effect of CCCP ................................................................................... 36 3.5 DOES a-LTX INCREASE INTRACELLULAR ca2+BY INCREASING INTRACELLULAR Na'? ................... ....... ...........................................................41 3.6 CYCLOSPORIN-A ..................................................................................~..........46 3.7 1s ca2+NECESSARY FOR a-LTX-INDUCED EXOCYTOSIS? ......................... 48 3.7.1 TheEffectofBAPTA-AM ............................................................................. 48 4 DISCUSSION ............................................................................................................. 52 ................. 4.1 SUMMARY ..................... .. ................. 52 4.2 a-LTX-STIMULATED TRANSMITTER RELEASE............................................. 52 4.2.1 Vesicle Depletion ........................................................................................ 53 4.2.2 Independence of Extracellular ca2+............................................................. 53 4.2.3 Dependence on Extracellular Divalent Cations............................................ 54 4.3 a-LTX STlMULATED ca2*MOBlLlZATlON ...................................................... 55 4.3.1 Ca2+ Signais In Other Ceif Systems ............................................................. 56 2+ 4.4 NEURONAL Ca STORES ................................................... ,, ............. ...., ........ 56 4.4.1 The Endoplasmic Reticulum ........................................................................57