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Effects of drugs on miniature end-plate currents at the mouse neuromuscular junction Pennefather, Peter 1982

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EFFECTS OF DRUGS ON MINIATURE END-PLATE CURRENTS AT THE MOUSE NEUROMUSCULAR JUNCTION by PETER PENNEFATHER B.Sc., McGill U n i v e r s i t y , 1974 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department of Pharmacology, F a c u l t y of Medicine We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1982 O Peter Pennefather, 1982 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of \ f)o.\Tfy\ 16 c o f o<j y The University of B r i t i s h Columbia 1956 Main M a l l Vancouver, Canada V6T 1Y3 D a t e Opn I 3 0 19 E X DE-6 (3/81) Supervisor: ( i i ) ABSTRACT D i g i t a l averaging and a n a l y s i s of miniature endplate currents (MEPCs) from mouse diaphragm was used to c h a r a c t e r i z e the normal MEPC and i t s modi-f i c a t i o n by a v a r i e t y of drugs. Under normal c o n d i t i o n s the decay of MEPCs showed c o n s i s t e n t d e v i a t i o n s from a simple exponential c o n s i s t i n g i n a progressive increase of r a t e constant, followed by a slow t a i l . Receptor blockade by d-tubocurarine (dTC), a-bungarotoxin, and other agents thought to occupy ACh-binding s i t e s reduced MEPC amplitude, a c c e l e r a t e d MEPC decay by about 30 7o(making i t about equal to decay r a t e of channels opened by exogenous a c e t y l c h o l i n e ) , and eliminated the e a r l y d e v i a t i o n s from an expo-n e n t i a l decay; dTC a l s o abolished the l a t e t a i l . Examination of the i n t e r -a c t i o n of a c e t y l c h o l i n e s t e r a s e (AChE) poisoning and receptor blockade on MEPC height and time course i n d i c a t e d that normally most quantal ACh i s captured by receptors and, as predicted by t h e o r e t i c a l c o n s i d e r a t i o n , a rath e r large degree of receptor blockade i s necessary to reduce MEPC height. MEPC t a i l s were exaggerated by AChE poisoning and exogenous ACh or carba-c h o l . The l a t t e r agents reduced MEPC height i n a fas h i o n i n c o n s i s t e n t with blockade of ACh binding and concurrent modulation of the t a i l suggested an important r o l e of d e s e n s i t i z e d receptors i n t a i l generation. A number of other drug actions are also described q u a n t i t a t i v e l y : (a) channel prolonga-t i o n , t y p i c a l of al c o h o l s but also found with ketones and some amines; (b) 'channel plugging', t y p i c a l of l o c a l anaesthetics but a l s o found with many other agents, i n c l u d i n g long chain a l c o h o l s , and (c) an a c t i o n to reduce MEPC s i z e without reducing net response to exogenous agonist t y p i c a l of v o l a t i l e a n a e s t h e t i c s , associated with increase r a t h e r than decrease of ACh binding to receptor. C r i t e r i a f o r d i s t i n g u i s h i n g d i f f e r e n t modes of m o d i f i -c a t i o n of receptor f u n c t i o n are discussed. ( i i i ) TABLE OF CONTENTS Abstract i i Table of contents i i i L i s t of Figures x L i s t of Tables x v i i L i s t of Abbreviations x i x Acknowledgements x x Dedication x x i PART I . GENERAL INTRODUCTION 1) The neuromuscular j u n c t i o n and neuromuscular tran s m i s s i o n 2 2) H i s t o r i c a l development of ideas concerning drugs and r e c e p t o r s . 9 a) O r i g i n of the drug receptor concept: Langley and E h r l i c h (c. 1910) 10 b) Mass a c t i o n : A . J . Clark (1926) 13 c) Drug e f f i c a c y : Stephenson (1957) 15 d) A pharmacological d e f i n i t i o n of re c e p t o r s : ( c . 1962) 18 e) D i r e c t evidence f o r the existence of receptors 20 f) Drug-receptor i n t e r a c t i o n s — an a c t i v e process 22 3) The ACh receptor as a molecular e n t i t y 25 a) Preface 25 b) The i s o l a t e d receptor from Torpedo e l e c t r i c organ 26 c) The subunit s t r u c t u r e of the ACh receptor i s o l a t e d from e l e c t r i c organ and s k e l e t a l muscle 28 d) Receptor enriched m i c r o v e s i c l e s derived from Torpedo e l e c t r o p l agues 32 ( i v ) e) Fun c t i o n a l r e c o n s t i t u t i o n of p u r i f i e d r eceptors i n t o l i p i d b i l a y e r s of defined composition 36 f ) Fine s t r u c t u r e of the AChR 41 g) Binding c h a r a c t e r i s t i c s of the AChR 42 ( i ) Are a l l acceptor s i t e s on the AChR the same 42 ( i i ) How many acceptor s i t e s per ACh receptor 47 4) Diverse types of drug-receptor i n t e r a c t i o n 49 a) Preface 49 b) Drug receptor i n t e r a c t i o n s at the acceptor s i t e on the n i c o t i n i c receptor 50 ( i ) N i c o t i n i c agonists 50 ( i i ) Mutual d i s p o s i t i o n of acceptor s i t e s on n i c o t i n i c receptors 53 ( i i i ) A f f i n i t y l a b e l s f o r the acceptor s i t e 56 ( i v ) Competitive antagonists that act at the acceptor s i t e of the n i c o t i n i c receptor 58 (v) Appendix 60 (1) S t r u c t u r a l formulae of drug that i n t e r a c t with the AChR acceptor s i t e 60 c) Pharmacological phenomena i n v o l v i n g the intermediary component of the receptor 69 ( i ) Preface 69 ( i i ) D e s e n s i t i z a t i o n of the ACh receptor by n i c o t i n i c agonists 69 (1) Fast and slow d e s e n s i t i z a t i o n 69 (2) Biochemical measures of d e s e n s i t i z a t i o n 75 (3) Molecular mechanisms of f a s t and slow d e s e n s i t i z a t i o n 79 ( i i i ) C o o p e r a t i v i t y i n a c t i v a t i o n of the ACh receptor 84 (v) d) A c t i o n of drugs at the e f f e c t o r component of the re c e p t o r . . . 89 ( i ) Preface 89 ( i i ) A ction of 'membrane s t a b i l i z e r s ' on the e l e c t r o s e n s i -t i v e Na + channel of nerve 90 ( i i i ) Steady s t a t e k i n e t i c s ; acceptor v_s. e f f e c t o r blockade 103 5) Methods f o r analy z i n g the k i n e t i c s of ACh receptor a c t i v a t i o n . . 108 a) Preface 108 b) Noise a n a l y s i s 109 c) Relaxation a n a l y s i s 118 d) Singl e channel recording 120 e) The observations of Linder and Quastel, and the use of MEPCs f o r studying the k i n e t i c s of drug i n t e r a c t i o n with the ACh receptor 122 ( i ) The normal MEPC 122 ( i i ) E f f e c t s of l o c a l and general anaesthetics on the MEPC 124 6) Statement of the problem considered i n t h i s t h e s i s 127 PART I I . METHODS 7) General methods 131 ( i ) Standard superperfusion s o l u t i o n s 131 (i i ) Voltage clamp 131 ( i i i ) Recording 132 ( i v ) Recording of MEPCs 133 (v) Use of e x t r a c e l l u l a r 'MEPCs' 133 ( v i ) The use of paraoxon to i r r e v e r s i b l y poison AChE 134 ( v i i ) The use of ethanol 134 ( v i i i )Response of end-plates to carbachol 135 ( v i ) 8) D i g i t a l a n a l y s i s of MEPCs 136 ( i ) Averaging of MEPCs 136 ( i i ) C a l c u l a t i o n of MEPC parameters 137 ( i i i ) Q u a n t i t a t i o n of the devi a t i o n s of the MEPC decay from an exponential to obtain graphs of d i f f e r e n c e s from an exponential decay 139 ( i v ) D r i v i n g f u n c t i o n s and spreading f u n c t i o n s 139 9) Computer sim u l a t i o n s of MEPCs 142 PART I I I . RESULTS AND DISCUSSION A) C u r a r e - l i k e agents 10) E f f e c t of c u r a r e - l i k e agents on MEPC height 146 a) I n t r o d u c t i o n 146 ( i ) Theory 148 (1) A simple model of i t s p r e d i c t i o n 149 (2) More complex models 153 (3) S t i l l more complex models 158 b) Results 159 ( i ) P r e l i m i n a r y observations 159 ( i i ) M o d i f i c a t i o n of MEPCs by receptor blockade and AChE poisoning 162 ( i i i ) Increase of MEPC amplitude by AChE poisoning 164 ( i v ) M o d i f i c a t i o n of MEPC amplitude by dTC and hexamethonium 167 (v) Blockade of postsynaptic response to carbachol by ( v i i ) 11) E f f e c t of c u r a r e - l i k e agents on the time course of MEPCs 190 a) I n t r o d u c t i o n 190 b) Results and Discussion 194 ( i ) E f f e c t of dTC on decay r a t e and voltage s e n s i t i v i t y . . 194 ( i i ) E f f e c t of c u r a r e - l i k e agents on 'curvature' 201 ( i i i ) E f f e c t of quantal s i z e on r e v e r b e r a t i o n 215 ( i v ) M o d i f i c a t i o n of rev e r b e r a t i o n a f t e r AChE poisoning I: By c u r a r e - l i k e agents 219 (v) M o d i f i c a t i o n of rev e r b e r a t i o n a f t e r AChE poisoning I I : By changes i n quantal s i z e 230 12) E f f e c t of dTC, ethanol and membrane p o t e n t i a l on the MEPC d r i v i n g f u n c t i o n 238 a) I n t r o d u c t i o n 238 b) Results 239 ( i ) P r e l i m i n a r y observations on the a c t i o n of ethanol 239 ( i i ) MEPC d r i v i n g f u n c t i o n 244 c) Discussion 255 B) D e s e n s i t i z a t i o n 13) E f f e c t of exogenous agonist on MEPC height 264 a) I n t r o d u c t i o n 264 b) Results 265 ( i ) E f f e c t s of carbachol on MEPC height 265 ( i i ) Dose-response r e l a t i o n s h i p s 271 ( i i i ) Time course of the depression of MEPC height 274 ( i v ) E f f e c t of drugs on depression of MEPC height by carbachol 279 ( v i i i ) c) Discussion 282 d) Appendix 286 ( i ) C o r r e c t i o n of response to agonist f o r d e s e n s i t i z a t i o n 286 ( i i ) C a l c u l a t i o n of H i l l c o e f f i c i e n t f o r d e s e n s i t i z a t i o n . . 287 14) E f f e c t of exogenous agonist on the time course of MEPCs 288 a) I n t r o d u c t i o n 288 b) Results 292 ( i ) T a i l s on the normal MEPC 292 ( i i ) Main part of the MEPC 297 ( i i i ) R e l a t i o n between the e f f e c t of carbachol on time course and d e s e n s i t i z a t i o n 300 (v) Normal t a i l s a f t e r poisoning AChE 303 c) Discussion 306 C) Membrane S t a b i l i z e r s Foreword 315 15) The e t h a n o l - l i k e a c t i o n of drugs on ACh receptors 318 a) I n t r o d u c t i o n 318 b) Results 319 ( i ) Ethanol reduces the MEPC decay r a t e 319 ( i i ) Role of membrane d i e l e c t r i c constants i n generation of the e t h a n o l - l i k e a c t i o n 322 c) Discussion 326 d) Appendix 329 16) Dysergic agents 332 a) I n t r o d u c t i o n 333 b) Results 333 ( i ) E f f e c t s on MEPC height and on the response to carbachol 333 ( i i ) E f f e c t s on MEPC time course 338 ( i x ) ( i i i ) Concentration response r e l a t i o n s h i p 344 c) Discussion 346 17) Channel pluggers 352 a) I n t r o d u c t i o n 352 b) Results 354 ( i ) E f f e c t s on the i n i t i a l decay r a t e of MEPCs 354 ( i i ) Voltage s e n s i t i v i t y 358 ( i i i ) K i n e t i c parameters d e s c r i b i n g the b i p h a s i c MEPC 364 c) Discussion 379 18) General d i s c u s s i o n 390 a) Preface 391 b) The time course of the MEPC and the k i n e t i c s of binding of ACh to subsynaptic receptors 391 c) S i g n i f i c a n c e of c o o p e r a t i v i t y i n a c t i v a t i o n of subsynaptic receptors by ACh 393 d) Actions of neuromuscular blockers on subsynaptic ACh receptors 395 (x) L i s t of Figures Chapter 10 Figure 1. The r e l a t i o n between the inverse of computed MEPC peak height and (R Q/R)-1 i s p l o t t e d f o r three d i f f e r e n t models of the j u n c t i o n 154-5 Figure 2. I n d i v i d u a l MEPCs recorded from normal and myasthenic mouse diaphrams at -80 mV (10 KHz sampling r a t e ) before and a f t e r poisoning AChE 161 Figure 3. A: Bar diagram showing heights of MEPCs (at -80 mV) recorded before and a f t e r poisoning of AChE by para-oxon, i n c o n t r o l diaphragms and i n diaphragms from mice p r e v i o u s l y i n j e c t e d with a-bungarotoxin or myas-thenic IgG, with no dTC and with 0.1 pM dTC, and also with 0.4 uM dTC i n con t r o l diaphragms o n l y . B: Enhancement of MEPC height by poisoning of AChE... 165-6 Figure 4. P l o t of [ g " 0 ( g ' Q / g ' j - 1 )-g' 0(g" 0/g-'j-1)] vs. (g^/g^-g-'o/g-'j) 168 Figure 5. Log-log p l o t of f ( I ) (see t e x t ) v_s. c o n c e n t r a t i o n of A) dTC and B) hexamethonium — data from c o n t r o l d i a -phragms before and a f t e r poisoning of AChE 170-71 Figure 6. E f f e c t of receptor blockade and poisoning of AChE on the potency of dTC 173-4 Figure 7. I n h i b i t i o n of postsynaptic d e p o l a r i z i n g responses to superperfused carbachol (10 and 20 uM) by 40 nM dTC. 176 ( x i ) Figure 8. Log response - log concentration p l o t f o r carbachol ± dTC i n c o n t r o l and myasthenic muscle 179 Figure 9. R e l a t i o n between peak height of MEPCs (at -80 mV) and f u n c t i o n a l receptor d e n s i t y , as percentage of normal. 183 Chapter 11 Figure 10. Averages of MEPCs recorded at d i f f e r e n t membrane poten-t i a l s and in the presence and absence of etha n o l . . . . 193 Figure 11. Graphs of p l o t t e d s e m i l o g a r i t h m i c a l l y vs. holding p o t e n t i a l under various c o n d i t i o n s 195 Figure 12. I n t e r a c t i o n of ethanol, and AChE poisoning by paraoxon, and dTC on MEPC height and time constant 197 Figure 13. Reduction of MEPC time constant (T^ ) by dTC and hexamethonium a f t e r poisoning of AChE 199 Figure 14. Deviation of the MEPC decay phase from an e x p o n e n t i a l . 202 Figure 15. E f f e c t of dTC and other c u r a r e - l i k e agents on the dev i a -t i o n of the MEPC decay phase from an exponential 203-4 Figure 16. E f f e c t of ethanol and membrane p o t e n t i a l on the de v i a -t i o n of the MEPC decay phase form an exponential 206-7 Figure 17. Graphs s i m i l a r to those i n F i g . 16 obtained a f t e r deconvoluting each average of MEPCs by the r a t e constant ofthe r i s i n g phase of the MEPC (s) f o r c o n t r o l s and 0.4 M ethanol 211 ( x i i ) Figure 18. Increase of decay r a t e as MEPC height decays 212 Figure 19. E f f e c t of quantal s i z e on the d e v i a t i o n of the MEPC decay r a t e from an exponential 217 Figure 20. P l o t of the r e l a t i o n ( f 0 - f j ) v s ^ [T"o - ' f j C g j / T ^ ) ] , a f t e r poisoning of AChE 221 Figure 21. P l o t of the reduction of time constants vs. the apparent reduction of subsynaptic receptor d e n s i t y a f t e r AChE has been poisoned 224-225 Figure 22. Averages of MEPCs a f t e r poisoning AChE, grouped accord-ing to Tj from one j u n c t i o n at -60 mV, p l o t t e d semi-log vs. time 231 Figure 23. Scatter graphs of log Yj vs. height f o r two j u n c t i o n s at -60 mV a f t e r paraoxon and at -80 mV i n 0.8 M ethan o l , with AChE i n t a c t at -80 mV 232 Figure 24. Re l a t i o n between time constant (Tj) and peak MEPC height 233 Chapter 12 Figure 25. Increase of MEPC height produced by ethanol under c o n t r o l c o n d i t i o n s and i n the presence of dTC — 0.64 or 0.4 yM, or a f t e r poisoning AChE 240 Figure 26. P l o t of r e l a t i v e MEPC height ( c o n t r o l / e t h a n o l ) vs. con t r o l MEPC height at various l e v e l s of receptor blockade 241 (x i i i ) F igure 27. E f f e c t of ethanol on the decay phase of the MEPC a f t e r poisoning AChE 242 Figure 28. The r i s i n g phase of MEPCs 245-6 Figure 29. Semi-log p l o t s of ( t o t a l area of d r i v i n g f u n c t i o n minus accumulated area) vs. time f o r c o n t r o l at -120, -100,... -40 mV 247 Chapter 13 Figure 30. E f f e c t on endplate conductance and MEPC amplitude of superperfusion of endplate region with carbachol 266 Figure 31. Averages of MEPCs during short exposure to carbachol.. 267 Figure 32. Time course of i n h i b i t i o n of MEPC height by carbachol. 269 Figure 33. Response-concentration r e l a t i o n s h i p f o r the increase i n holding current produced by carbachol corrected f o r f a s t d e s e n s i t i z a t i o n 272-3 Figure 34. H i l l p l o t of the r e l a t i o n between f a s t d e s e n t i z a t i o n and carbachol concentration 272-3 Figure 35. The lag between a c t i v a t i o n of postsynaptic receptors and reduction of MEPC height by carbachol 275-6 Chapter 14 Figure 36. Semilog p l o t s of MEPC grand averages recorded under normal co n d i t i o n s showing the ex i s t e n c e of a l a t e low t a i l i n the MEPC decay phase 291 ( x i v ) Figure 37. Semilog p l o t s of the decay phases of MEPC; grand averages showing the e f f e c t s of exogenous a g o n i s t s , ethanol, dTC and poisoning of AChE on the MEPC 293-4 Figure 38. R e l a t i o n s h i p between the time constant of the MEPC t a i l and the reduction of MEPC height produced by carbachol 302 Figure 39. Grand averages grouped accordingly to time constants of 1713 MEPCs recorded from 30 j u n c t i o n s at -60 mV, a f t e r poisoning AChE 304 Figure 40. R e l a t i v e height of the t a i l ( i . e . t a i l amplitude/MEPC height) of grand average MEPCs shown i n F i g . 38 p l o t -ted against the areas of the main part of these MEPCs. 305 Chapter 15 Figure 41. MEPC time constant p l o t t e d against ethanol concentra-t i o n 320 Chapter 16 Figure 42. E f f e c t of c e n t r a l depressant agents on MEPP amplitude and the d e p o l a r i z a t i o n produced by superperfused c a r -bachol (I) 333 Figure 43. E f f e c t of c e n t r a l depressant agents on MEPP amplitude and the d e p o l a r i z a t i o n produced by superperfused car-bachol ( I I ) 334-5 (XV) Figure 4 4 . Scattergrams of MEPC time constant (T^) vs. MEPC height, p l o t t e d l o g - l o g , f o r MEPCs recorded i n con-t r o l s o l u t i o n and i n the presence of 10 mM paralde-hyde a f t e r poisoning AChE 337 Figure 4 5 . Change i n MEPC time constant with i n c r e a s i n g concentra-t i o n s of paraldehyde, paraldehyde i n the presence of 0.4 M ethanol, pentane, and pentane i n the presence of 0 .4 M ethanol 341 Figure 4 6 . Concentration response r e l a t i o n s h i p f o r the reduction of MEPC height by butanol and paraldehyde 343 Chapter 17 Figure 4 7 . Biphasic MEPCs produced by octanol and p e n t o b a r b i t a l . . 355 Figure 4 8 . Biphasic MEPC decay phase produced by a v a r i e t y of agents: E f f e c t of membrane p o t e n t i a l 359-60 Figure 4 9 . T r i p h a s i c MEPC decay phase produced by p o s i t i v e l y charged agents 361 Figure 50 . A c t i o n of procaine (25 and 100yM) on the i n i t i a l MEPC decay r a t e i n the presence and absence of ethanol (0.4 M), and at d i f f e r e n t membrane p o t e n t i a l s 362 Figure 5 1 . E f f e c t of membrane p o t e n t i a l and drug concentration on the estimated r a t e of a s s o c i a t i o n (k-^[Dj) and d i s s o -c i a t i o n (k_^) of procaine with the open channel s t a t e . 366 Figure 5 2 . E f f e c t of membrane p o t e n t i a l , AChE a c t i v i t y and ethanol on the estimated r a t e of a s s o c i a t i o n and d i s s o c i a t i o n of morphine (50uM) with the open channel 367 ( x v i ) F i g u r e 5 3 . A p p a r e n t a d e r i v e d f r o m t h e t i m e c o u r s e o f t h e MEPC d e c a y p h a s e a s s u m i n g a 1 i s n e g l i g i b l e 368 F i g u r e 5 4 . V a l u e s o f a' c a l c u l a t e d u s i n g e q u a t i o n s d e r i v e d f r o m m o d e l e q u a t i o n 1 7 . 5 and a s s u m i n g v a l u e s o f a ( s o l i d l i n e ) e s t i m a t e d as d e s c r i b e d i n t e x t 373 (xvi i ) LIST OF TABLES Table 1. D i s s o c i a t i o n constant f o r two s i t e model d e s c r i b i n g the r e l a t i o n between s p e c i f i c binding and li g a n d con-c e n t r a t i o n 44 Table 2. Thermodynamic parameters d e s c r i b i n g the r a t e l i m i t -ing s t a t e s i n ACh i n t e r a c t i o n with normal and desen-s i t i z e d receptors 82 Table 3. R e l a t i o n s h i p between r a t e constants, determining the channel opening and c l o s i n g r a t e , that can give give r i s e to s i n g l e components i n a u t o c o r r e l a t i o n f u n c t i o n s of current f l u c t u a t i o n s produced by agonist at the neuromuscular j u n c t i o n 114 Table 4. MEPC amplitude, 'time constant' of decay, and s e n s i t i v i -ty to (+)-tubocurarine; m o d i f i c a t i o n by poisoning of AChE, myasthenic IgG and a-BuTX 163 Table 5. I n h i b i t i o n of responses to superperfused carbachol and of MEPC height, and c a l c u l a t e d r e d u c t i o n i n f r e e receptors by dTC 177 Table 6. Parameters d e s c r i b i n g the d e v i a t i o n of the MEPC decay from an exponential; e f f e c t of ethanol and membrane p o t e n t i a l 216 Table 7. E f f e c t of ethanol and membrane p o t e n t i a l on the r a t e constant and area of the MEPC d r i v i n g f u n c t i o n and on MEPC height 252 Table 8. E f f e c t of b r i e f (30-120 second) superperfusion with a c e t y l c h o l i n e and carbachol on MEPC height 270 ( x i i i ) Table 9. E f f e c t s of drugs on the change of holding current and the reduction of MEPC height produced by carbachol.. 280 Table 10. Summary of the e f f e c t of carbachol and ACh on MEPC time course 295 Table 11. The r e l a t i v e potency of agents that act to prolong MEPCs and end-plate channels 323 Table 12. Comparison of the e f f e c t s of ethanol and butanol on the conductance change induced by 5 yM ca r b a c h o l , and on the time constant fTj ) of MEPC decay 325 Table 13. E f f e c t s of tubocurarine and methoxyflurane , alone and in combination on MEPC height and time constant ( T j ) , before and a f t e r poisoning AChE and on the change i n conductance produced by carbachol (10 yM) 339 Table 14. R e l a t i v e potency of 'dysergic' agents 345 Table 15. A. E f f e c t of channel pluggers on i n i t i a l decay r a t e of MEPCs B. E f f e c t s of channel pluggers on height of MEPC averages when expressed as a percentage of the height i n the absence of drug 356 Table 16. K i n e t i c parameters, d e s c r i b i n g the channel plugging a c t i o n , derived form time course of the bi p h a s i c MEPC using equations 17.13 - 17.15 374 Table 17. E f f e c t s of pH on and k ^ determined from decay phase of MEPC grand averages 378 ( x i x ) LIST OF ABBREVIATIONS Term Ab b r e v i a t i o n A c e t y l c h o l i n e ACh A c e t y l c h o l i n e receptor AChR A c e t y l c h o l i nesterase AChE Ampere A a-Bungarotoxin a-BuTX Carbachol carb Dalton D Decamethonium CIO D i e l e c t r i c constant e D i r e c t current DC D i t h i o t h r e i t o l DTT End-plate current EPC End-plate p o t e n t i a l s EPP Figure F i g . Herz Hz Hexodecamethonium C16 H i s t r i o n i c o t o x i n HTX Immunoglobulin G IgG Meter m Miniature end-plate currents MEPCs Miniature end-plate p o t e n t i a l s MEPPs Molar M u l t i p l i c a t i o n of r a t e constant by A temperature = 10°C Second Semen Svedburg Time constant of MEPC between exp(-0.5) and exp(-1.5) of peak t!, (+)-Tubocurarine V o l t V ^10 s S (conductance) S ( s i z e ) 1 dTC (xx) ACKNOWLEDGEMENTS Acknowledgements I would l i k e to thank the H. R. MacMillan F e l l o w s h i p Committee f o r t h e i r f i n a n c i a l support, by way of a Frank Wesbrook pre-doctoral f e l l o w s h i p ; Dr. Ernest P u i l f o r h i s advice and guidance; the support s t a f f of the Department of Pharmacology at the U n v i e r s i t y of B r i t i s h Columbia f o r t h e i r a s s i s t a n c e during the various stages of t h i s work, i n p a r t i c u l a r the help of Ms. Tracy Slocombe in the f i n a l preparation of t h i s t h e s i s was indispensable and much appreciated. ( x x i ) DEDICATION To Dr. David Quastel f o r h i s support and the freedom he gave me to c a r r y out t h i s work, but most of a l l , f o r h i s opinions and i n s i g h t s . - 1 -PART I GENERAL INTRODUCTION - 2 -CHAPTER 1 The neuromuscular junction and neuromuscular transmission This thesis deals with the action of drugs that interfere with neuro-muscular transmission by altering subsynaptic receptor function. This was investigated by measuring the effects of these drugs on the height and time course of miniature end-plate currents. Neuromuscular transmission takes place at the end-plate region of the muscle. The term 'end-plate' was f i r s t coined by Bowmann (1840) and refers to the dist inct ive morphology of nerve endings on mammalian muscle f ibers . At the point where the motor nerve inserts i t s e l f into the muscle, i t divides into a series of branches, = 1 yM in diameter. Together with sate l -ite cel ls and Schwann cel ls these branches form a disk or p late - l ike appo-sit ion with a diameter of about 40 yM (Salpeter and Eldefrawi, 1973). It is notable that in the frog the nerve terminal arborization is not clumpea and can extend for up to 300 yM (Letinsky, Fichbeck and McMahan, 1976). Neuromuscular transmission involves the release of a brief pulse of the neurotransmitter acetylcholine (ACh) into the synaptic c le f t and i ts subse-quent action on subsynaptic ACh receptors. This release is triggerec by a presynaptic nerve action potential . The released ACh interacts with subsyn-aptic receptors and causes the opening of ion channels that allow Na + and K+ to cross the semipermeable sarcolemma. As a resul t , the ab i l i t y of the - 3 -s u b s y n a p t i c membrane t o m a i n t a i n a r e s t i n g membrane p o t e n t i a l i s r e d u c e d and t h e r e i s a d e p o l a r i z a t i o n c a l l e d t h e ' e n d - p l a t e p o t e n t i a l ' ( E P P ) . T h i s d e p o l a r i z a t i o n a c t i v a t e s e l e c t r o s e n s i t i v e i o n c h a n n e l s i n t h e e x t r a s y n a p t i c m u s c l e m e m b r a n e , w h i c h g i v e s r i s e t o a m u s c l e a c t i o n p o t e n t i a l and m u s c l e c o n t r a c t i o n . The s y n a p t i c c l e f t i s a n a r r o w ( 5 0 nm) c l e f t b e t w e e n t h e n e r v e t e r m i n a l and t h e s u b s y n a p t i c m e m b r a n e . The s u b s y n a p t i c membrane i s e x t e n s i v e l y f o l d e d . I t d i p s i n t o t h e m a i n b o d y o f t h e m u s c l e t o f o r m a s e r i e s o f t r e n -c h e s t h a t a r e 5 0 - 1 0 0 nm i n w i d t h , 2 0 0 - 4 0 0 nm i n d e p t h , and g e n e r a l l y r u n a c r o s s t h e p a t h o f t h e n e r v e t e r m i n a l b r a n c h e s ( s e e C o u t e a u x , 1 9 8 2 ; F e r t u c k and S a l p e t e r , 1 9 7 6 ) . B e c a u s e o f t h e p r e s e n c e o f t h e s e t r e n c h e s , t h e s y n a p -t i c c l e f t c a n be d i v i d e d i n t o p r i m a r y and s e c o n d a r y p o r t i o n s , a c c o r d i n g t o w h e t h e r t h e s u b s y n a p t i c membrane f a c e s e i t h e r t h e n e r v e t e r m i n a l membrane o r t h e s u b s y n a p t i c membrane on t h e o t h e r s i d e o f a f o l d . I n t h e f i r s t c a s e , t h e c l e f t i s s a i d t o be p r i m a r y , i n t h e s e c o n d c a s e i t i s s a i d t o be s e c o n -d a r y . O n l y a b o u t 2 0 p e r c e n t o f t h e p o s t s y n a p t i c membrane ( a t t h e mouse NMJ) f a c e s t h e p r i m a r y s y n a p t i c c l e f t ( M a t t h e w s - B e l l i n g e r and S a l p e t e r , 1 9 7 8 ) . The d i s t r i b u t i o n o f ACh r e c e p t o r s c a n be s t u d i e d b y m a k i n g u s e o f a p r o t e i n c a l l e d a b u n g a r o t o x i n (a - B u T X ) t h a t i s d e r i v e d f r o m a s n a k e v e n o m . I t b i n d s s e l e c t i v e l y and a l m o s t i r r e v e r s i b l y t o t h e ACh r e c e p t o r a t t h e n e u r o m u s c u l a r j u n c t i o n . I t c a n be r a d i o l a b e l e d and u s e d t o s t u d y t h e d i s -t r i b u t i o n o f ACh r e c e p t o r i n t h e s u b s y n a p t i c c l e f t b y means o f a u t o r a d i o -g r a p h y . a - B u T X b i n d i n g s i t e s a r e c o n c e n t r a t e d on t h e s u b s y n a p t i c membrane f a c i n g t h e p r i m a r y c l e f t and on t h e ' l i p s ' o f t h e s u b s y n a p t i c t r e n c h e s . T h e s e r e g i o n s a p p e a r t h i c k e r t h a n a d j a c e n t r e g i o n s when e x a m i n e d w i t h e l e c -t r o n m i c r o s c o p y . Q u a n t i t a t i v e a u t o r a d i o g r a p h y i n d i c a t e s t h a t t h e d e n s i t y o f - 4 -a-BuTX s i t e s on the region of the thickened p o s t j u n c t i o n a l membrane i s about 20,000/uin. The value f a l l s to about 2000/um at the bottom of the trenches. This d i s t r i b u t i o n i s e s s e n t i a l l y the same at mouse, f r o g , and Torpedo neuromuscular j u n c t i o n s and i s probably true of a l l ver t e b r a t e neuromuscular j u n c t i o n s (Matthews-Bellinger and S a l p e t e r , 1978; Heuser and Sal p e t e r , 1979; Robbins et a l . , 1980). Thus r e l e a s e d ACh has only a short distance to t r a v e l before f i n d i n g receptors. The mean l i f e t i m e of ACh released i n t o the c l e f t i s normally very b r i e f (>lms) because of the presence of a c e t y l c h o l i n e s t e r a s e (AChE), an enzyme that breaks down ACh i n t o the r e l a t i v e l y i n a c t i v e products, c h o l i n e ano acetate. AChE i s l o c a l i z e d over the e n t i r e p o s t j u n c t i o n a l membrane at an average den- s i t y of about 2400/ym ( S a l p e t e r , P l a t t n e r and Rodgers, 1972), and appears to be attached to the amorphous basement membrane that f i l l s the synaptic c l e f t (see H a l l and K e l l y , 1971; Betz and Sakmann, 1973; McMahan, Sanes and M a r s h a l l , 1978). What escapes h y d r o l y s i s r a p i d l y d i f f u s e s away from receptors (see Eccles and Jeager 1958). The ACh w i l l be d i l u t e d as i t leaves the primary c l e f t , and only has a short distance to t r a v e l to reach the outer l i m i t s of the synaptic c l e f t . ACh i s released i n d i s c r e t e packages or quanta ( F a t t and Katz, 1952) of about 10,000 molecules (see K u f f l e r and Yoshikami, 1975). When the nerve terminal membrane p o t e n t i a l i s at the r e s t i n g l e v e l (-90 mV), quanta are released randomly, at a mean r a t e of about 1/s; each quantum produces a miniature end-plate p o t e n t i a l (MEPP). D e p o l a r i z a t i o n of the presynaptic nerve terminal e i t h e r by a nerve terminal a c t i o n p o t e n t i a l (del C a s t i l l o and Katz, 1954a), r a i s e d K + ( L i l e y , 1956), or f o c a l p o l a r i z a t i o n (del C a s t i l l o - 5 -and Katz, 1954c; Katz and M i l e d i , 1967) increases the frequency of quantal r e l e a s e . L i l e y (1956) noted that there i s an exponential r e l a t i o n s h i p be-tween r e l e a s e r a t e and membrane p o t e n t i a l and that t h i s r e l a t i o n s h i p p r e d i c t s a r e l e a s e r a t e during a presynaptic a c t i o n p o t e n t i a l such that about one hundred quanta are r e l e a s e d . Such an a c c e l e r a t i o n of the r e l e a s e r a t e can account e n t i r e l y f o r the EPP. The nerve terminal i s f i l l e d with small (= 50 nm) v e s i c l e s . These were f i r s t described at about the same time as MEPPs (Robertson, 1954; Palade, 1954), so i t was natural to propose that these v e s i c l e s contained ACh, and that MEPPs were produced when these v e s i c l e s r e l e a s e d t h e i r contents i n t o the synaptic c l e f t as the r e s u l t of exocytosis (Palade, 1954; del C a s t i l l o and Katz, 1954b). Synaptic v e s i c l e s appear to congregate i n the nerve terminal above the postsynaptic trenches along 100 nm wide th i c k e n i n g s of the axolemma. Hubbard and Kwanbunbumpen (1968) found l o c a l i z e d thickenings of the axolemmal membrane and congregation of nerve terminal v e s i c l e s oppo-s i t e postsynaptic trenches i n 60 percent of the cases examined. They sug-gested that the t h i c k e n i n g represented the component in the nerve terminal membrane which mediates t r a n s m i t t e r r e l e a s e . These th i c k e n i n g s have subsequently come to be c a l l e d 'active zones' and there are g e n e r a l l y about 1000 per end-plate at i n t e r v a l s of 0.5-1 um along the nerve terminal branches (see Peper, Dreyer, Sandi, Akert and Moore, 1974; Heuser and Reese, 1981). The hypothesis that these zones are inv o l v e d with the t r a n s m i t t e r r e l e a s e mechanism has been studied e x t e n s i v e l y by Reese and h i s colle a g u e s , who have found f a i r l y convincing supporting evidence (see Heuser, Reese and Landis, 1974; Heuser and Reese, 1981). - 6 -The above d e s c r i p t i o n of neuromuscular s t r u c t u r e i m p l i e s that quanta are released at a large number of release s i t e s spread out along the nerve terminal (see a l s o del C a s t i l l o and Katz, 1954b). Since the number of re l e a s e s i t e s seems to be several times greater than the number of quanta r e l e a s e d i n a normal EPP, and rel e a s e at any one of these s i t e s i s a random process (see Hubbard, L l i n a s and Quastel, 1969), i t seems l i k e l y that quanta, re l e a s e d during a presynaptic a c t i o n p o t e n t i a l , can act on the sub-synaptic membrane independently of one another. Indeed H a r t z e l l , K u f f l e r and Yoshikami (1975) have presented d i r e c t evidence that when AChE i s a c t i v e , there i s no i n t e r a c t i o n between quanta re l e a s e d during an EPC. Le s t e r , K o b l i n and Sheridan (1978) have suggested the term 'synaptic u n i t 1 to describe the area opposite the presynaptic a c t i v e zone centered on the postsynaptic f o l d and extending halfway to the next a c t i v e zone on each s i d e . Neuromuscular transmission can be thought of as many of these synap-t i c u n i t s being simultaneously, but independently, a c t i v a t e d . Thus, i t i s not necessary to have neuromuscular transmission i n order to study the e f f e c t of drugs on t h i s process. Drugs that act p r e s y n a p t i c a l l y w i l l a f f e c t the frequency of quanta while drugs that act p o s t s y n a p t i c a l l y w i l l a f f e c t the amplitude and time course of the pe r m e a b i l i t y change produced by an ACh quantum. The MEPP i s not a s u i t a b l e measure of the change i n i o n i c p e r m e a b i l i t y produced by an ACh quantum because i t i s d i s t o r t e d by the membrane c a p a c i -tance. This problem only a r i s e s when the membrane p o t e n t i a l changes, and can be avoided by using the voltage clamp technique. A voltage clamp i s a feedback c i r c u i t t h a t maintains membrane p o t e n t i a l at a constant l e v e l des-p i t e changes i n membrane p e r m e a b i l i t y . The point voltage clamp of Takeuchi - 7 -and Takeuchi (1959) was used i n the present experiments. This technique involves penetrating the muscle f i b e r with two glass m i c r o e l e c t r o d e s , i n the v i c i n i t y of the end-plate. One electrode i s used to monitor membrane poten-t i a l , the other i s used to supply current s u f f i c i e n t to maintain a constant membrane p o t e n t i a l i n the end-plate r e g i o n . Changes i n p e r m e a b i l i t y are measured in terms of changes in the current necessary to hold the membrane p o t e n t i a l constant. Thus, the EPP becomes the end-plate current (EPC) and the MEPP becomes the miniature end-plate current (MEPC). With t h i s t e c h-nique i t i s c r i t i c a l t hat both electrodes be c l o s e to one another and c l o s e to the current source produced by the p e r m e a b i l i t y change being measured. Thus, an MEPC recorded at a mouse end-plate i s l i k e l y to be le s s d i s t o r t e d than one generated at a f r o g end-plate where the nerve terminal branches are not t i g h t l y clumped and quanta are released over a wide area. MEPCs occur spontaneously and are r e l a t i v e l y easy to record ana q u a n t i f y using w e l l e s t a b l i s h e d e l e c t r o p h y s i o l o g i c a l techniques. The aim of t h i s t h e s i s i s to demonstrate how q u a n t i t a t i v e measurement and a n a l y s i s of the MEPC time course or shape, under a v a r i e t y of pharmacological c o n d i t i o n s , allows inferences to be drawn regarding the normal k i n e t i c processes govern-ing ACh-receptor i n t e r a c t i o n and how these processes are modified by drugs. The approach taken was i n d i r e c t to the extent t h a t some k i n e t i c proces-ses could not be measured d i r e c t l y . Rather, mathematical models were con-s t r u c t e d to account f o r the shape of the MEPC and changes i n t h i s shape brought about by drugs. But the approach of measuring the e f f e c t s of drugs on MEPCs has two major advantages over the more common steady-state ap-- 8 -proaches to the problem of measuring drug receptor i n t e r a c t i o n s . F i r s t , one i s sure that those receptors that are being i n v e s t i g a t e d are i n f a c t those involved in synaptic transmission and MEPC generation. Secondly, e f f e c t s of drugs on the p h y s i o l o g i c a l , nonsteady s t a t e system can be s t u d i e d ; the k i n e t i c processes involved i n t r a n s m i t t e r - r e c e p t o r i n t e r a c t i o n may be mani-f e s t e d q u i t e d i f f e r e n t l y i n nonsteady s t a t e s i t u a t i o n s then when a steady s t a t e has been achieved. - 9 -CHAPTER 2 H i s t o r i c a l development of ideas concerning drugs ana receptors This t h e s i s i s concerned with how drugs i n t e r a c t w i t h r e c e p t o r s . Thus i t i s important to discuss the concepts of drugs and receptors in order to put the work i n t o p e r s p e c t i v e . There are many d e f i n i t i o n s of a drug, but i t i s g e n e r a l l y agreed t h a t the term can be a p p l i e d to any substance which, when added to a l i v i n g system ( i n vivo or i n v i t r o ) , w i l l cause a change in the f u n c t i o n of that system. Thus a pharmacological a c t i o n i s an i n t e g r a l part of the d e f i n i t i o n of a drug. When methylene blue i s used f o r the h i s -t o l o g i c a l i d e n t i f i c a t i o n of a stucture in a f i x e d t i s s u e i t i s not a c t i n g as a drug. However, i f i t binds to the same s i t e s i n a f u n c t i o n i n g t i s s u e and changes that f u n c t i o n then i t i s a c t i n g as a drug. Some drugs need not associ a t e with the t i s s u e to produce an a c t i o n ; e.g., a n t a c i d s , absorbents, a s t r i n g e n t s , d e m u l c i f i e r s , l u b r i c a n t s , bulk pur-g a t i v e s , osmotic d i u r e t i c s , plasma expanders, chemical antidotes ( F a s t i e r , 1964), and these may be c a l l e d n o n s p e c i f i c drugs. This type makes up only a small m i n o r i t y of drugs. With most drugs i t i s necessary to post u l a t e that there i s a c e l l u l a r c o n s t i t u e n t in the target t i s s u e which somehow re c o g n i z -es the drug and mediates the drug e f f e c t . These c o n s t i t u e n t s are g e n e r a l l y r e f e r r e d to as drug r e c e p t o r s . In order to get a c l e a r e r idea of what i s meant by the term receptor, i t i s useful to f o l l o w the h i s t o r i c a l develop-ment of the drug receptor concept. - 10 -a) O r i g i n of the receptor concept: Langley and E h r l i c h (c. 1910) By the end of the 19th century, chemists began to i s o l a t e the a c t i v e i n g r e d i e n t s r e s p o n s i b l e f o r the therapeutic actions of medicines. For exam-pl e , i t was found that in the case of several medicines, the a c t i v e i n g r e -dient was water s o l u b l e and could be p r e c i p i t a t e d i n a pure form with ammo-ni a . Such substances became known as the a l k a l o i d s and in c l u d e d , morphine, s t r y c h n i n e , n i c o t i n e , and qu i n i n e . By 1857, Claude Bernard had shown that i n d i v i d u a l drugs had s p e c i f i c actions on d i s t i n c t parts of the body. For example, doses of strychnine that caused convulsions d i d not cause neuromus-cula r p a r a l y s i s while curare caused p a r a l y s i s without causing convulsions, moreover, the ac t i o n of curare could be l o c a l i z e d to the end-plate r e g i o n . I t a f f e c t e d n e i t h e r muscle c o n t r a c t i o n nor neuronal conductance (Bernard, 1857). Crumm-Brown and F r a z i e r (1868) found that a quaternary ammonium d e r i v a t i v e of st r y c h n i n e l o s t the convulsive property of the parent com-pound, but gained a c u r a r i f o r m a c t i o n . Indeed they showed th a t quaternary ammonium bases in general possessed the common property of e x e r t i n g a cura-r i f o r m a c t i o n . Thus, i t seemed l i k e l y that drugs had s p e c i f i c a c t i o n s because they had a s p e c i f i c chemical s t r u c t u r e , but i t proved d i f f i c u l t to e x p l a i n the ob-served s t r u c t u r e a c t i v i t y r e l a t i o n s h i p s . Bernard (1857) suggested that cu-rar e acted to 'poison' the nerve terminal in the end-plate r e g i o n . This concept was extended to other drugs that i n t e r f e r e d with the nervous sys-tem. For example, Brodie and Dixon (1904) suggested that apocodeine blocked the response to sympathetic nerve s t i m u l a t i o n by poisoning the nerve t e r m i -n a l . Since the a c t i o n of adrenaline was a l s o a b o l i s h e d , they suggested that adrenaline acted to 'stimulate' nerve t e r m i n a l s . Even when i t was found - 1 1 -that adrenaline could s t i l l a f f e c t the i r i s 10 months a f t e r removal of the super i o r c e r v i c a l ganglion i t was simply suggested that nerve terminals could survive i n the absence of the main c e l l body ( E l l i o t , 1905). I t i s i n t e r e s t i n g to note that i n an e a r l i e r study on the a c t i o n of adrenaline on the bladder a f t e r denervation, E l l i o t (1904) suggested that adrenaline might be a c t i n g d i r e c t l y on something i n the muscle. However, he did not develop t h i s hypothesis f u r t h e r . The Bernardian view that curare acted on nerve terminals was d i s c r e d i t e d by the experiments of Langley (1906, 1909, 1914). I t was known that n i c o t i n e acts to block t w i t c h muscle. However, in slow muscle such as i s found i n the trunk musculature of frogs i t causes a slow c o n t r a c t i o n and hence a r i g i d p a r a l y s i s . Langley observed that curare opposed t h i s a c t i o n and caused a f l a c c i d p a r a l y s i s to develop. Furthermore, the opposing a c t i o n of these two drugs could s t i l l occur i n denervated muscles, i n which no h i s t o l o g i c a l evidence f o r the presence of nerve terminals could be found. Low doses of n i c o t i n e caused a l o c a l i z e d contracture when ap p l i e d d i r e c t l y to the regions in denervated muscle where the nerve terminals had been, and t h i s c o n t r a c t u r e could be blocked by curare (Langley, 1906; 1909; 1914). Langley performed a large number of c a r e f u l experiments t e s t i n g various i n t e r p r e t a t i o n s of these phenomena and concluded that n i c o t i n e and curare r e v e r s i b l y i n t e r a c t with the same post-j u n c t i o n a l 'receptive substance' and that t h i s r e c e p t i v e substance was most prevalent i n the end-plate region of the muscle. Not only was t h i s one of the f i r s t c l e a r formulations of the idea that drugs act by r e v e r s i b l y binding to receptors but i t a l s o implied that neural s t i m u l a t i o n of the muscle, which was also blocked by cu r a r e , might be - 12 -mediated by the r e l e a s e of a substance that acted l i k e n i c o t i n e (Langley, 1906). Though the p o s s i b i l i t y of chemical neurotransmission had already been suggested many years e a r l i e r (1877) by DuBois-Reymond (see Dale, 1937), Langley's experiments provided some of the e a r l i e s t experimental support f o r t h i s hypothesis. Chemical neurotransmission i s now recognized to be the most common form of synaptic transmission in v e r t e b r a t e s . The term 'receptor' was o r i g i n a l l y introduced by Paul E h r l i c h at the turn of the century f o r h i s studies of the n e u t r a l i z a t i o n of tetanus toxins by a n t i b o d i e s . Parascandola (1980) has pointed out that i n i t i a l l y E h r l i c h was h e s i t a n t to extend h i s ideas about receptors to the a c t i o n s of drugs. At the time l i t t l e was known about chemical bonding and concepts such as hydrogen and van der Waal bonds had not been developed. Chemical i n t e r -actions were g e n e r a l l y thought of i n terms of i o n i c and covalent bonding. These types of i n t e r a c t i o n s were not c o n s i s t e n t with the r e v e r s i b l e nature of drug a c t i o n and the f a c t that drugs could e a s i l y be e x t r a c t e d from t i s s u e by s o l v e n t s . However, i n the 1900s E h r l i c h turned h i s a t t e n t i o n to chemo-therapy and was struck by the observation that when trypanosomes became r e s i s t a n t to a p a r t i c u l a r trypanocidal dye they became r e s i s t a n t to chemi-c a l l y - r e l a t e d dyes as w e l l . The simplest explanation f o r t h i s was that the microorganism had l o s t the s i t e which bound the dye. This o b s e r v a t i o n , together with h i s f a m i l i a r i t y with the work of Langley, l e d E h r l i c h to change h i s mind and become a great proponent of the drug receptor concept ( E h r l i c h , 1913). He borrowed Fi s c h e r ' s 'lock and key' analogy f o r the spe-c i f i c i t y of enzymes and suggested that a s i m i l a r analogy could be applied to the i n t e r a c t i o n between drugs and t h e i r r e c e p t o r s . Perhaps, because he was p r i m a r i l y i n t e r e s t e d i n chemotherapy, h i s concept of a 'receptor' was - 13 -s l i g h t l y d i f f e r e n t from that of Langley f o r a 'receptive substance'. He f e l t that receptors were simply side chains of c e l l s p e c i f i c molecules that could i n t e r a c t with poisons and provide a convenient t a r g e t f o r h i s 'magic b u l l e t s ' . Langley, on the other hand, had a more p h y s i o l o g i c a l b i a s . He f e l t that drugs, by i n t e r a c t i n g with t h e i r r e c e p t o r s , i m i t a t e d or antagoniz-ed the ac t i o n of substances normally used i n the l i v i n g system i n which the drug produced t h e i r e f f e c t s (see S c h i l d , 1962). b) Mass a c t i o n : A. J . Clark (c. 1926) In the '20s and '30s, when i n v e s t i g a t o r s began i n earnest to attempt to q u a n t i f y the e f f e c t s of drugs on c e l l s , i t was noted that in many cases the response to a drug was l i n e a r l y r e l a t e d to the log of the drug concentra-t i o n . At f i r s t t h i s was i n t e r p r e t e d i n terms of d i f f e r e n t thresholds f o r drug a c t i o n i n the c e l l s that made up the responding t i s s u e s , and the thresholds of these c e l l s being log-normally d i s t r i b u t e d due to natural v a r i a b i l i t y ( S h a c k e l l , 1924; Gaddum, 1926). However, i n 1926, A. J . Clark pointed out t h a t , i f i t i s assumed that the drug response i s p r o p o r t i o n a l to the amount of drug combining with a f i n i t e number of receptors i n the t i s s u e and that t h i s combination i s r e v e r s i b l e (or, at l e a s t , t r a n s i e n t ) , the shapes of the dose-response curves could be accounted f o r by the laws of mass a c t i o n . These laws had already proved useful i n d e s c r i b i n g the adsorp-t i o n of gases onto metal fila m e n t s (Langmuir, 1918) and the a s s o c i a t i o n of oxygen with haemoglobin ( H i l l , 1910). - 14 -Clark (1926) determined the dose-response r e l a t i o n s h i p of ACh under a v a r i e t y of c o n d i t i o n s i n a v a r i e t y of t i s s u e s ( i . e . , f r o g h e a r t , f r o g r e c -t u s , guinea pig ileum) and was struck by the s i m i l a r i t y of the the dose-response curves; a l l appeared to f i t a simple mass a c t i o n formula. The v a r i a b l e t h r e shold theory gave no i n s i g h t i n t o the p o s s i b l e modes of a c t i o n of the drugs and r e q u i r e d the u n l i k e l y assumption that the v a r i a b i l i t y of the threshold was s i m i l a r or even i d e n t i c a l under many c o n d i t i o n s in many t i s s u e s . The hypothesis that responses were p r o p o r t i o n a l to the r e v e r s i b l e occupancy of binding s i t e s ( i . e . , mass action) accounted f o r the dose-response curves e q u a l l y w e l l , and had the added advantage of suggesting a physical/chemical mechanism of d r u g - c e l l i n t e r a c t i o n . Clark was w e l l aware of the l i m i t a t i o n s of applying a concept developed to describe a w e l l -defined system of gas and polished metal, to processes t a k i n g place in l i v i n g t i s s u e , and although he r e a l i z e d that there was no good reason to b e l i e v e that the drug response should be p r o p o r t i o n a l to receptor occupancy, n e i t h e r could he f i n d any good reason to r e j e c t the hypothesis. Clark's receptor theory also suggested p l a u s i b l e explanations of drug antagonism and of the observation that c e r t a i n homologous s e r i e s of che-m i c a l l y r e l a t e d compounds could contain both drugs which produced a response and drugs that blocked t h i s response. Drugs which produced responses on t h e i r own were c a l l e d agonists and were thought to bind r e v e r s i b l y to the receptor in such a way as to cause a change in the receptor that i n i t i a t e d a response, while drugs which r e v e r s i b l y occupied the receptor without t r i g -gering a response were antagonists. Antagonists were thought simply to prevent access of agonist to the receptor. Experiments with homologous - 15 -s e r i e s of compounds suggested that antagonists possess enough of the chemi-c a l s t r u c t u r e of the agonist to be recognized by the r e c e p t o r , but at the same time e i t h e r not enough, or too many a d d i t i o n a l s t r u c t u r a l p r o p e r t i e s , to be able to t r i g g e r the response ( C l a r k , 1937). c) Drug E f f i c a c y : Stephenson (1957) I t took 20 years to improve upon t h i s theory. I t was only i n 1957 that Stephenson suggested some useful m o d i f i c a t i o n s of C l a r k ' s receptor theory. The most important was h i s concept of e f f i c a c y . Stephenson had examined the dose-response r e l a t i o n s h i p s of a s e r i e s of n - a l k y l trimethyl-ammonium ( n - a l k y l TMA) s a l t s using the guinea pig terminal ileum c o n t r a c t i o n as a response. Stephenson was hoping to be able to compare the potencies of c l o s e l y r e l a t e d true agonists and true antagonists s i n c e previous work had shown that short chain members of the s e r i e s mimicked the actions of ACh while the long chain members blocked the a c t i o n of ACh (Raventos and C l a r k , 1937). However, the t r a n s i t i o n between agonist and antagonist proved to be more complicated than had been expected; the heptyl to decyl TMA s a l t s had both agonist and antagonist a c t i v i t y . These agents could produce some response, but even i n high concentrations could not produce as large a maximal response as the s h o r t e r a l k y l chain s a l t s . On the other hand, these longer chain d e r i v a t i v e s antagonized the response to the s h o r t e r chain d e r i v a t i v e s i n a surmountable or competitive manner. Agents which possessed both agonist and antagonist p r o p e r t i e s were c a l l e d ' p a r t i a l a g o n i s t s ' . In s p i t e of the agonist properties of these p a r t i a l a g o n i s t s , Stephenson (1957) was able to devise a method of measuring t h e i r antagonist potency. He found the concentration required to produce a 50 percent blockade of the - 16 -response to a f u l l agonist decreased about 0.6 f o l d per methylene group; t h i s concentration was s i m i l a r to the concentration r e q u i r e d to produce 50 percent of whatever maximal response could be produced by the p a r t i a l ago-n i s t . Thus, t h i s potency seemed to be a measure of the a f f i n i t y of the p a r t i a l agonist f o r the recep t o r . F u l l a g o n i s t s , on the other hand, appear-ed to be much more potent than predicted by the change i n a f f i n i t y per methylene group seen with the p a r t i a l a g onists. However, when the ileum was kept in the organ both f o r 2 or 3 days the potency of f u l l agonists was reduced and some of the shorter chain n - a l k y l TMA s a l t s now appeared to have p a r t i a l agonist p r o p e r t i e s . These experiments suggested, t h a t , w i t h i n t h i s s e r i e s of n - a l k y l TMA s a l t s , some compounds could produce a maximal response when they occupied only a small proportion of the receptors on the muscle, w h i l e others could not produce a maximal response, even i f they occupied a l l of the a v a i l a b l e receptors. In other words, d i f f e r e n t drugs could occupy d i f f e r e n t propor-t i o n s of the a v a i l a b l e receptors i n order to produce the same response. He coined the term ' e f f i c a c y ' to describe the property of the drug that deter-mined the r e l a t i v e proportion of receptors that i t had to occupy to produce a given response. Those drugs which produced a 50 percent response when 50 percent of the receptors were occupied were a r b i t r a r i l y assigned an e f f i c a c y (e) of 2. Thus, f o r a f u l l agonist e > 2; f o r a p a r t i a l a g o n i s t , e < 2, and f o r antagonist, e = 0. Stephenson also pointed out that in many cases concentration-response curves did not s t r i c t l y f i t the curve predicted by the laws of mass a c t i o n for a r e v e r s i b l e bimolecular i n t e r a c t i o n between a drug and a recep t o r . Clark had been aware of t h i s , but he had simply p o s t u l a t e d that i n these - 17 -cases more than one drug molecule had to bind to a receptor ( C l a r k , 1937) i n order to t r i g g e r a response. Stephenson took a more general approach to t h i s problem. He introduced the term '$' ( f o r stimu l u s ) that was the product of e f f i c a c y (e) and f r a c t i o n a l receptor occupany ( Y), and simply stated that the response to a drug was r e l a t e d to 'S' by some mathematical f u n c t i o n that could be e m p i r i c a l l y defined ( i . e . , response = f ( S ) ; S = eY; Y = A.K/(1 + A.K), where A = agonist c o n c e n t r a t i o n and K = a s s o c i a t i o n constant of agonist f o r the r e c e p t o r ) . Stephenson's m o d i f i c a t i o n came at a p a r t i c u l a r l y opportune time since many i n v e s t i g a t o r s were recognizing the l i m i t a t i o n s of C l a r k ' s o r g i n a l theory. I t had long been known that the blockade produced by a s l o w l y r e v e r s i b l e antagonist could be overcome by i n c r e a s i n g agonist concentra-t i o n s , even though very high concentrations of agon i s t s ( r e l a t i v e to t h e i r agonist potency) d i d not appear to speed recovery from the blockade ( C l a r k , 1926; Gaddum, 1937). The same phenomenon had been observed with antagonists that appeared to combine i r r e v e r s i b l y with receptors (Nickerson, 1949; Furchgott, 1954). Stephenson's idea of spare receptors ( i . e . , those remaining when a drug of high e f f i c a c y produced a maximal response) provided a ready explanation f o r these observations. Ariens and de Groot (1954) had p r e v i o u s l y observed the phenomenon of p a r t i a l agonism and had attempted to ex p l a i n i t i n terms of ' i n t r i n s i c a c t i v i t y ' which they defined as a constant that described the e f f e c t per u n i t pharmacon (drug) receptor complex. This concept recognized that maximal receptor occupancy need not always produce the same maximal response, but i t implied that a l l drugs producing the same maximal response had the same i n t r i n s i c a c t i v i t y . Stephenson (1957) had c l e a r l y shown that t h i s was not the case. - 18 -d) A pharmacological d e f i n i t i o n of the r e c e p t o r : (c 1962) Thus, by the beginning of the 1960s, even though there was no d i r e c t evidence f o r a drug r e c e p t o r , receptor theory was v e r s a t i l e enough to account f o r most of the known drug concentration response r e l a t i o n s h i p s . The q u a n t i t a t i v e agreement between theory and experiment was q u i t e good. Moreover, antagonist potency was in many cases s i m i l a r , r e g a r d l e s s of the t i s s u e where the p a r t i c u l a r type of receptor was found (Arunlakshana and S c h i l d , 1959). Although the drug receptor was t r e a t e d as a h y p o t h e t i c a l e n t i t y , the i n d i r e c t evidence f o r i t s existence was hard to e x p l a i n any other way. For example, o p t i c a l isomers of a drug could possess i d e n t i c a l chemical p r o p e r t i e s and yet have s t r i k i n g l y d i f f e r e n t potencies i n producing a response (Beckett, 1959). In many cases, the same drug could cause d i f -f e r e n t types of responses i n d i f f e r e n t t i s s u e s and the order of potencies of chemically r e l a t e d drugs could be q u i t e d i f f e r e n t f o r d i f f e r e n t types of responses (Dale, 1914; A h l q v i s t , 1948). In other s i t u a t i o n s , many d i f f e r e n t drugs could cause the same response, yet i t was p o s s i b l e to block s e l e c t i v e -l y the actions of each type of drug (Furchgott, 1954). Although most pharmacologists were convinced that receptors e x i s t e d , they were g e n e r a l l y uncertain as to what should be included under the head-ing of receptor. The panel d i s c u s s i o n on receptors recorded at the Ciba Foundation Symposium on 'Enzymes and drug a c t i o n ' i n London i n 1961 (Mongar and de Reudq, 1962) i l l u s t r a t e s t h i s point n i c e l y . B. B. Brodie asked whether s i t e s which bound drugs i n the course of metabolism or tr a n s p o r t or storage should be considered r e c e p t o r s , but the general r e a c t i o n was negative. This l e d Bernard Katz to suggest t h a t the - 19 -common ground f o r c a l l i n g a substance a receptor was th a t i t s chemical c o n s t i t u t i o n was unknown. He s t a t e d : "As soon as i t s nature becomes known, we give i t i t s proper name and cease c a l l i n g i t a re c e p t o r . " I t was general-l y agreed that there were analogies between receptors and enzymes and that the biochemical approaches that had to that time proven very s u c c e s s f u l in d e f i n i n g enzyme f u n c t i o n should be applied to the rece p t o r . I n t e r e s t i n g l y , many of the suggestions made at the meeting such as i s o l a t i o n of r e c e p t o r s , a f f i n i t y l a b e l l i n g , the use of biochemical reagent, and various spectrosco-p i c techniques are the basis of much of present day b i o c h e m i c a l l y - o r i e n t e d receptor research. At the same conference, S i r Hans Krebs pointed out that drug receptor i n t e r a c t i o n s were more s i m i l a r to enzyme c a t a l y s t r e a c t i o n s than enzyme substrate r e a c t i o n s , since the drug was g e n e r a l l y unchanged by i t s encounter with i t s r e c e p t o r . Although some emphasis was placed on d e f i n i n g the a c t i v e s i t e , i . e . , the s i t e which bound the drug, i t was recognized that the recep-t o r was a f u n c t i o n a l u n i t which not only bound the drug, but also i n i t i a t e d the pharmacological response. I t was g e n e r a l l y agreed t h a t , no matter what i s decided to be a recep-t o r , i t must be made up of an acceptor — the minimum pattern of f o r c e s on the receptor necessary f o r i n t e r a c t i o n with the drug and an e f f e c t o r — the por t i o n of the receptor r e s p o n s i b l e f o r t r i g g e r i n g the response. F a s t i e r (1964) l a t e r suggested that a l l those atoms neighbouring the acceptor and the e f f e c t o r which play a r o l e i n binding or c o n t r o l l i n g access to the acceptor, or modify e f f e c t o r f u n c t i o n should a l s o be considered part of the rec e p t o r . These could be s a i d to make up an intermediary component of the receptor since they must al s o provide a l i n k between the acceptor and e f f e c t o r components. - 20 -e) D i r e c t evidence f o r the existence of receptors The f i r s t d i r e c t evidence f o r receptors came i n the mid '60s with the use of r a d i o a c t i v e i s o t o p e s , incorporated i n t o drugs, to demonstrate the existence of s p e c i f i c binding s i t e s f o r f o r these drugs. In f a c t at the same symposium mentioned i n the l a s t s e c t i o n (Mongar and de Reuch 1962) Waser (1962) presented evidence, based on autoradiography, that binding s i t e s f o r curare are l o c a l i z e d to the end-plate region (see also Waser and Luthi 1957). Paton and Rang (1965) measured the uptake of t r i t i a t e d a tropine by t h i n s t r i p s of guinea pig ileum l o n g i t u d i n a l muscle. They were able to r e s o l v e the absorption curve i n t o several components, one of which was c o n s i s t e n t with a saturable binding s i t e w i t h an a f f i n i t y f o r atropine very c l o s e to that determined from i t s a b i l i t y to block, i n the same muscle, c o n t r a c t i o n s produced by muscarinic a g o n i s t s . At about the same time i n v e s t i g a t o r s (see Lee and Tseng, 1966; Changeux, Kasi and Lee, 1970) were beginning to tag n i c o t i n i c receptors with a-bungarotoxin. Since that time many s t r a t e g i e s have been developed to i n v e s t i g a t e d i r e c t l y the c h a r a c t e r -i s t i c s of drug binding s i t e s on t i s s u e responsive to the drug (see Yamamura, Enna and Kuhar, 1978). One of the most popular methods, e s p e c i a l l y with i n v e s t i g a t o r s i n t e r e s t -ed i n the a c t i o n of hormones or n e u r o t r a n s m i t t e r s , i s e s s e n t i a l l y a modi-f i c a t i o n of the p r i n c i p l e of i s o t o p i c d i l u t i o n (see Snyder, 1978; Cuatreca-sas and Hollenberg, 1975; 1976). Receptor ligands ( i . e . , compounds thought to be able to associate with the acceptor p o r t i o n of the receptor in a satu-r a b l e manner) are 'tagged' with a r a d i o a c t i v e isotope and incubated with t i s s u e thought to contain the receptor. Saturable or s p e c i f i c b i n d i n g i s - 21 -defined as that p o r t i o n of the drug that becomes associated with the t i s s u e and can be dis p l a c e d by an excess of u n l a b e l l e d l i g a n d (usualy greater than 50 times the expected d i s s o c i a t i o n constant). Simple absorption or mixture of the drug with the t i s s u e , so that i t i s not q u i c k l y washed away, _is c a l l e d n o n - s p e c i f i c binding and i s g e n e r a l l y not sat u r a b l e and not d i s p l a c e d by excess u n l a b e l l e d l i g a n d . The major l i m i t a t i o n of a l l binding studies i s that only drug acceptor i n t e r a c t i o n s are measured and i t i s often d i f f i c u l t to determine that the acceptor being s t u d i e d i s a part o f , or r e l a t e d t o , the receptor which mediates a drug response. Many i n e r t substances such as talcum powder and glass f i l t e r possess acceptor s i t e s which bind ligands with high a f f i n i t y and f o r which the r e l a t i v e a f f i n i t i e s of r e l a t e d ligands p a r a l l e l t h e i r b i o l o g i c a l a c t i v i t y . The same i s true of albumin and cerebroside sulphate (see Cuatrecasas and Hollenberg, 1979, f o r review). Thus, i t i s l i k e l y that many c e l l c o n s t i t u e n t s can act as acceptors. Cuatrecasas and Hollenberg (1979) r e f e r to these types of s i t e s as s p e c i f i c nonreceptor binding s i t e s . Where care has been taken to d i s t i n g u i s h between s p e c i f i c r e c e p t o r , s p e c i f i c nonreceptor, and n o n s p e c i f i c binding and extensive comparisons have been made between p h y s i o l o g i c a l l y and b i o c h e m i c a l l y measured a f f i n i t i e s of a v a r i e t y of agonists and a n t a g o n i s t s , i t has proved p o s s i b l e to produce f a i r l y convincing evidence that the binding s i t e being c h a r a c t e r i z e d i s indeed the acceptor s i t e of the receptor involved i n the pharmacological a c t i o n of the drug. However, these studies provide no information about the consequences of the binding — i . e . , the primary response brought about by the e f f e c t o r portion of the receptor f o l l o w i n g a c t i v a t i o n by ac t i o n of an agonist at the acceptor s i t e on the receptor - 22 -f ) Drug receptor i n t e r a c t i o n s - an a c t i v e process The h y p o t h e t i c a l nature of the receptor was not the only problem with the Stephenson-Clark receptor theory. The theory was mainly d e s c r i p t i v e and provided l i t t l e i n s i g h t i n t o how drugs which occupy the same binding s i t e can vary i n e f f i c a c y and potency. I t was obvious that d i f f e r e n c e s i n drug chemical s t r u c t u r e played some r o l e but the nature of the r o l e was not considered by the Stephenson-Clark occupation theory. The r a t e theory of Paton (1961) suggested that only the i n i t i a l binding of a drug to the receptor i s involved i n t r i g g e r i n g the elementary e f f e c t o r response. Drugs which because of the chemical s t r u c t u r e d i s s o c i a t e from the receptor r a t h e r s l o w l y might continue to occupy the receptor even a f t e r com-p l e t i o n of the u n i t response. Thus, e f f i c a c y would be determined by the r a t e of d i s s o c i a t i o n of the agonist r e l a t i v e to the time course of the u n i t response, and the slower the r a t e of d i s s o c i a t i o n , the lower the e f f i c a c y . Responses would be determined by r a t e s of drug receptor i n t e r a c t i o n r a t h e r than simple receptor occu-pancy. The only ^evidence i n support of the -theory was that antagonists g e n e r a l l y had a higher a f f i n i t y than agonists; however there were many exceptions to t h i s r u l e . The theory a l s o explained the phenomenon of desen-s i t i z a t i o n , where continued exposure of a t i s s u e to agonist leads to a fade of the steady s t a t e response to ago n i s t , since with prolonged exposure to drug there could be a rundown of t r i g g e r a b l e r e c e p t o r . However, desensi-t i z a t i o n could be j u s t as e a s i l y explained along more t r a d i t i o n a l k i n e t i c l i n e s (Katz and T h e s l e f f , 1957b). The theory d i d not have any advantage over a simple occupation theory. - 23 -Another ex p l a n a t i o n , of how drugs that occupied the same binding s i t e can have d i f f e r e n t e f f i c a c i e s , i s often r e f e r r e d to as the two-state or a l l o s t e r i c model (Karl i n , 1967; Changeux et a l . , 1967; Colquhoun, 1973). This model invokes a thermodynamic e q u i l i b r i u m between a c t i v e and i n a c t i v e r e c e p t o r s , even i n the absence of receptor ligands and proposes that agonist s h i f t e d the e q u i l i b r i u m towards the a c t i v e " s t a t e by p r e f e r e n t i a l binding to the a c t i v e s t a t e while at the same time slowing the r e v e r s i o n back to the i n a c t i v e s t a t e . Antagonists do j u s t the opposite by combining with the i n a c t i v e s t a t e , and p a r t i a l agonist can combine with both s t a t e s . However, i n most cases of neurotransmitter and hormone receptors there i s l i t t l e evidence that s u b s t a n t i a l numbers of receptors are a c t i v e in the absence of an agonist. When few receptors are a c t i v e i n the absence of a g o n i s t , the two-state model i s i n d i s t i n g u i s h a b l e from occupation models (Thron, 1973). In a l l of the models discussed so f a r , the drug plays a r e l a t i v e l y passive r o l e . Simply i n s e r t i n g the key i n the lock i s enough to t r i g g e r a response. Other t h e o r i e s considered the consequences of t u r n i n g the key i n the lock. Koshland (1958) showed that enzymes e x h i b i t a high degree of con-formational a d a p t a b i l i t y to substrates and i n h i b i t o r s . Belleau (1964) pro-posed that s i m i l a r conformational perturbations could occur i n receptors when they bind ligands and that the type of conformational change could determine the e f f i c a c y of the drug. Thus, a g o n i s t s , upon binding to the acceptor s i t e on the r e c e p t o r , would cause productive conformational perturbations leading to the a c t i v a t i o n of the e f f e c t o r portion of the receptor while antagonists would cause unproductive conformational p e r t u r -bations. P a r t i a l agonists would cause some combination of these two types of conformational change. - 24 -F r a n k l i n (1980) has r e c e n t l y suggested a theory very s i m i l a r to that of B e l l e a u . He drew a t t e n t i o n to the concept of Jencks (1975) of the c o n t r i -bution of substrate binding energy to enzymatic c a t a l y s i s . Jencks has pointed out t h a t with many enzymes the d i s s o c i a t i o n constants of h i g h l y s p e c i f i c substrates are s u r p r i s i n g l y high (range 0.1-.01 mM). I f t h i s were determined p r i m a r i l y by the i n i t i a l noncovalent i n t e r a c t i o n between enzyme and s u b s t r a t e , i t would imply that the high s p e c i f i c i t y l i g a n d binding to these enzymes was produced by r e l a t i v e l y weak i n t e r a c t i o n s . To r e s o l v e t h i s discrepancy, Jencks proposed that part of the gain i n f r e e energy associated with substrate enzyme binding i s used to 'pay' f o r the substrate-induced conformational changes i n the enzyme that leads to c a t a l y s i s . By analogy, F r a n k l i n proposed that part of the f r e e energy gained by i n t e r a c t i o n of an agonist with i t s receptor i s used to 'pay' f o r the conformational change in the receptor that a c t i v a t e s the e f f e c t o r . I n t e r a c t i o n of antagonists or p a r t i a l agonists with the receptor would y i e l d 'nonproductive' free energy that would be expressed p r i m a r i l y i n the t i g h t n e s s of b i n d i n g . - 25 -CHAPTER 3 The ACh receptor as a molecular e n t i t y a) Preface From the above d i s c u s s i o n , a general statement can be made that a recep-t o r i s a f u n c t i o n a l u n i t made up of an acceptor s i t e that recognizes agonist molecules, an e f f e c t o r p o r t i o n that produces a u n i t response when a c t i v a t e d , and an intermediary p o r t i o n that l i n k s the acceptor to the e f f e c t o r . In recent years i t has become p o s s i b l e to define t h i s f u n c t i o n a l u n i t in mole-c u l a r terms. In the case of the n i c o t i n i c receptor at the neromuscular j u n c t i o n , there has been a steady progress towards t h i s end since the l a t e 1960 1 s. This progress began with the development of a source and an assay f o r ACh r e c e p t o r s . Nachmansohn (1959) pointed out that e l e c t r i c organs or e l e c t r o p l a q u e s , which are evolved from s k e l e t a l muscle and found i n c e r t a i n f i s h (rays and e e l s ) could provide a convenient source of large q u a n t i t a t i e s of r e c e p t o r . The e a r l y work of Changeux, K a s a i , Huchet and Munier (1970) and M i l e d i , M o l i n o f f and Porter (1971) showed th a t t h i s was indeed the case. These studies followed the devlopment of a s e n s i t i v e assay f o r Ach receptors (see Lee and Tseng, 1966; Changeux, Kasai and Lee, 1970). This assay made use of the f a c t that a-BuTX bound to the ACh receptor with a high s p e c i f i c i t y and i n an e s s e n t i a l l y i r r e v e r s i b l e manner (Chang and Lee, 1963). Since the e a r l y 70's i t has proved p o s s i b l e to i s o l a t e the ACh r e c e p t o r , c h a r a c t e r i z e i t s composition and s t r u c t u r e , and show that a c t i v i t y i s dependent on the presence i n a membrane of a large p r o t e i n , 250,000 Daltons (D), made up of 5 subunits. For the sake of b r e v i t y the ACh recept-o r , s t u d i e d i n the absence of a pharmacological response, w i l l henceforth be r e f e r r e d to as the AChR. - 26 -b) The i s o l a t e d receptor from Torpedo e l e c t r i c organ In the Torpedo e l e c t r i c organ, the AChR i s an i n t e g r a l membrane pr o t e i n as opposed to a p e r i p h e r a l membrane p r o t e i n such as AChE. An i n t e g r a l mem-, brane p r o t e i n i n t e r a c t s t i g h t l y with the l i p i d b i l a y e r and i s released from the c e l l surface membrane only by detergent e x t r a c t i o n . P e r i p h e r a l membrane prot e i n s on the other hand, are part of a calyx of l o o s e l y associated pro-t e i n s that i n t e r a c t weakly with the b i l a y e r and i n t e g r a l p r o t e i n s . They can be e x t r a c t e d by high or low s a l t treatment or by the use of c h e l a t i n g agents such as EDTA (see Singer, 1974). G e n e r a l l y , AChR i s ext r a c t e d from e l e c -t r i c t i s s u e by the detergent T r i t o n X-100 and p u r i f i e d by a f f i n i t y chroma-tography (Schmidt and Ra f t e r y , 1972; K a r l i n and Cowburn, 1973; Olsen, Meunier and Changeux, 1972). Using t h i s method on Torpedo e l e c t r i c organ, a 9 Svedburg (S) and a 13S p r o t e i n are g e n e r a l l y found to contain the a-BuTX binding s i t e . These two prot e i n s show the same number of binding s i t e s per mass of pr o t e i n (McNamee, Weii1 and K a r l i n , 1975) and i d e n t i c a l peptide ' f i n g e r p r i n t s ' . f o l l o w i n g t r y p -s i n d i g e s t s (O'Brien, G i l s o n and Sumikawa, 1978). Hence the 13S pr o t e i n i s l i k e l y to be a dimer of the 9S p r o t e i n . The monomers are l i n k e d v i a a d i s u l f i d e bond since the 13S form i s el i m i n a t e d by treatment with d i t h i o -t h r e i t o l (DTT), which cleaves d i s u l f i d e b r idges, and s t a b i l i z e d by the presence of iodoacetamide, which binds to f r e e SH groups (Weitzmann and Raf-t e r y , 1977). The f a c t that iodoacetamide s t a b i l i z e s r a t h e r than el i m i n a t e s the 13S form suggest that the dimer i s not an a r t i f a c t that develops during the p u r i f i c a t i o n of r e c e p t o r s , and may wel l be the normal f u n c t i o n a l form of the AChR ( M i l l e r , Moore, H a r t i g and R a f t e r y , 1978). - 27 -In t h i s regard, i t i s notable that DTT treatment of e l e c t r o p l a x c e l l s changes the H i l l c o e f f i c i e n t of the log response-log agonist concentration curve, from 1.7 to 1.0, as i f c o o p e r a t i v i t y had been el i m i n a t e d ( K a r l i n , 1967). This implies that the p o s i t i v e c o o p e r a t i v i t y normally observed i n agonist a c t i o n may r e f l e c t i n t e r a c t i o n between the two 9S monomers. However, no change of the H i l l c o e f f i c i e n t i s observed i n avian or amphibian s k e l e t a l muscle t r e a t e d with DTT (Rang and R i t t e r , 1971; Ben Haim, Dreyer and Peper, 1975). Of course, i n these species, dimers may not be present; dimers are not found when receptors are e x t r a c t e d from eel electroplaques (see Lindstrom, Cooper and T z a r t o s , 1980). On the other hand, DTT also i s known to reduce the potency of n i c o t i n i c a g o n i s t s ; i f a f r a c t i o n of recep-t o r s remained unaffected by DTT, receptor heterogeneity would cause a reduc-t i o n of the H i l l c o e f f i c i e n t to be observed even i f both forms of the recep-t o r s had the same c o o p e r a t i v i t y as before DTT treatment (Lester et a l . , 1980). K a r l i n et a l . (1979) report that a f t e r treatment with 5 mM DTT and 5 mM d i t h i o b i s c h o l i n e , which r e o x i d i z e s the s u l f h y d r y l group i n the v i c i n i t y of the ACh binding s i t e but not the one between 9S monomers, r e s t o r e s normal agonist potency without generating dimers. The p o s s i b i l i t y that the 9S monomers continue to be associated and i n t e r a c t non-covalently was not r u l e d out. As with most i n t e g r a l membrane pro t e i n s the i s o l a t e d AChR i s associated with bound detergent. This has been estimated to be between 20-50 percent of the mass of the receptor ( K a r l i n e t a l . , 1979; E d e l s t e i n , Bayer and Edefrawi, 1975). When t h i s i s taken i n t o account the best estimate of the molecular weight of the 9S monomer i s g e n e r a l l y around 250,000 daltons. Since these preparations contain 8 umoles of a-BuTX per gram p r o t e i n t h i s i n d i c a t e s that each 9S monomer i s associated with 2 a-BuTX s i t e s . - 28 -c) The subunit s t r u c t u r e of the ACh receptor i s o l a t e d from  e l e c t r i c organ and s k e l e t a l muscle The subunit s t r u c t u r e of the AChR has been e x t e n s i v e l y i n v e s t i g a t e d . A v a r i e t y of subunit patterns f i r s t reported has been shown to be due to p r o t e o l y t i c degradation of receptor during i s o l a t i o n . Now that care i s taken to minimize p r o t e o l y s i s there i s considerable agreement (Raftery et a l . , 1979; K a r l i n et a l . , 1979; Wei land Frieman and T a y l o r , 1979). Membrane f r a c t i o n s that contain r e c e p t o r s , when subjected to sodium dodecyl s u l f a t e (SDS) polyacrylamide gel e l e c t r o p h o r e s i s , g e n e r a l l y y i e l d 4 major bands of a c i d i c g l y c o p r o t e i n with molecular weights 40,000, 50,000, 60,000 and 65,000 d a l t o n s , i n r a t i o s of approximately 2:1:1:1. These subunits are g e n e r a l l y r e f e r r e d to as a , B , Y , <5 r e s p e c t i v e l y . Only the a (40,000 D) subunits bind a-BuTX. Thus the composition of the 9S monomer of o ^ B y f i i s c o n s i s t e n t with binding s t o i c h i o m e t r y of 2 a-BuTX binding s i t e s per receptor monomer and molecular weight of 250,000 D. The 13S dimers appear to be l i n k e d v i a a d i s u l f i d e bridge between 6 (65,000 D) subunits (Hamilton, McGlauglin and K a r l i n , 1979). Subunits of the B (50,000 D) type can also be induced to form d i s u l f i d e bridges between monomers. Some i n v e s t i g a t o r s have claimed that only the a (40,0000 D) subunit i s necessary f o r a f u n c t i o n a l receptor (Sobel, Weber and Changeux, 1977; M e r l i e , Changeux and Gros, 1978). However, the work of Lindstrom et a l . (1980b) has shown that the evidence upon which t h i s conclusion i s based i s l i k e l y to be a r t i f a c t u a l . Lindstrom et a l . (1980b) have shown that proteo-l y t i c enzymes, e i t h e r endogenous or exogenous, can p a r t i a l l y d igest a l l of the four types of subunits of the AChR. The digested receptor r e t a i n e d most - 29 -o f t h e a n t i g e n i c s i t e s c h a r a c t e r i s t i c o f e a c h o f t h e o r i g i n a l s u b u n i t s s o t h e s e w e r e s t i l l p r o b a b l y r e l a t i v e l y i n t a c t . I n s p i t e o f t h i s , t h e m a j o r p r o t e i n b a n d s e e n w i t h SDS g e l e l e c t r o p h o r e s i s m i g r a t e d t o t h e same r e g i o n as d i d t h e a s u b u n i t b e f o r e p r o t e a s e t r e a t m e n t and t h o s e b a n d s c h a r a c t e r i s t i c o f t h e i n t a c t h e a v i e r b a n d s w e r e n o t s e e n . T h u s , i t i s l i k e l y t h a t t h e r e m a i n s o f t h e h e a v i e r b a n d s , i . e . , e y 6> w e r e m i g r a t i n g i n t h e same f a s h i o n as t h e o r i g i n a l a s u b u n i t . W h e r e v e r i t h a s b e e n i n v e s t i g a t e d , t h e s u b u n i t s t r u c t u r e o f t h e AChR i n o t h e r s p e c i e s o f v e r t e b r a t e s a p p e a r s t o be s i m i l a r t o t h a t o f T o r p e d o A C h R . E a r l y r e p o r t s on t h e s u b u n i t s t r u c t u r e o f AChR p u r i f i e d f r o m t h e f r e s h w a t e r t e l e o s t E l e c t r o p h o r u s e l e c t r i c u s ( e e l ) s u g g e s t e d t h i s r e c e p t o r c o n t a i n e d o n l y t h r e e t y p e s o f s u b u n i t s 2a ' s , le' and ly' c o r r e s p o n d i n g i n m o l e c u l a r w e i g h t t o t h e a, B , and y s u b u n i t s o f t h e T o r p e d o r e c e p t o r ( K a r l i n , W e i l l McNamee and V a l d e r r a m a , 1 9 7 6 ) . H o w e v e r L i n d s t r o m , C o o p e r , and T z a r t o s ( 1 9 8 0 ) h a v e shown t h a t a 6* ( 6 5 , 0 0 0 ) s u b u n i t e q u i v a l e n t t o t h e 6 s u b u n i t o f t h e T o r p e d o r e c e p t o r c a n b e o b s e r v e d p r o v i d e d i o d o a c e t a m i d e i s p r e s e n t d u r i n g t h e i n i t i a l s t a g e s o f i s o l a t i o n . T h i s p r e s u m a b l y p r e v e n t s t h e f o r m a -t i o n o f d i s u l f i d e b r i d g e s b e t w e e n t h e 61 s u b u n i t and o t h e r p r o t e i n c o m p o -n e n t s w h i c h w o u l d c a u s e t h e 6 s u b u n i t t o e l u t e w i t h t h e l a r g e p r o t e i n c o m p o -n e n t s and be m i s s e d . M a m m a l i a n s k e l e t a l m u s c l e AChR i s a l s o made up o f f i v e a c i d i c g l y c o -p r o t e i n s u b u n i t s , t w o o f w h i c h p o s s e s s n i c o t i n i c l i g a n d b i n d i n g s i t e s ( N a t h a n s o n and H a l l , 1 9 8 0 b ) . A g a i n t h e m o l e c u l a r w e i g h t o f t h e s e s u b u n i t s s u g g e s t s t h a t t h e y a r e e q u i v a l e n t t o t h e a, B, Y» 5 s u b u n i t s o f t h e T o r p e d o A C h R . The two a - l i k e s u b u n i t s a p p e a r t o be s l i g h t l y d i f f e r e n t f r o m o n e a n o t h e r i n m o l e c u l a r w e i g h t . H o w e v e r , p e p t i d e maps o f t h e two a s u b u n i t s - 30 -i n d i c a t e that they are almost i d e n t i c a l . I t seems l i k e l y t hat these two sublimits are coded f o r by the same gene. The d i f f e r e n c e in molecular weight may be a p o s t - t r a n s l a t i o n a l change or p o s s i b l y an a r t i f a c t of the e x t r a c t i o n procedure (Nathanson and H a l l , 1980b). E x t r a j u n c t i o n a l and embryonic AChR i n s k e l e t a l muscle d i f f e r s from junc-t i o n a l receptor i n several aspects. They have a longer open channel dura-t i o n (Dreyer, Peper and S t e r z , 1976) and a lower a f f i n i t y f o r dTC both i n  s i t u (Beranek and V y s c h o c i l , 1967) and a f t e r e x t r a c t i o n (Brockes and H a l l , 1975a). There are d i f f e r e n c e s i n i s o e l e c t r i c point (Brockes and H a l l , 1975b) and immunological p r o p e r t i e s (Weinberg and H a l l , 1979), a l l of which suggest that there may al s o be s t r u c t u r a l d i f f e r e n c e s . I t appears that i f such s t r u c t u r a l d i f f e r e n c e s e x i s t they are s u b t l e because the subunit s t r u c -ture and peptide maps of p a r t i c u l a r subunits of j u n c t i o n a l and e x t r a j u n c -t i o n a l AChR are i n d i s t i n g u i s h a b l e . There i s considerable s t r u c t u r a l homology between the four types of subunits making up the AChR i n Torpedo e l e c t r o p l a q u e s . Tzartos and Lindstrom (1980) have i s o l a t e d monoclonal antibodies some of which cross react with a (40,000 D) and B (50,000 D) subunits while others c r o s s - r e a c t with Y (60,000 D) and 6 (65,000 D) subunits. The amino acid sequence of a l l four subunits i s also q u i t e s i m i l a r ( R aftery et a l . , 1980) and the subunits may a l l have evolved from a s i n g l e p r i m o r d i a l peptide. Raftery et a l . (1980) have constructed a t h e o r e t i c a l genealogical t r e e f o r the subunits, by assuming minimal n u c l e o t i d e s u b s t i t u t i o n (see Fetch and Margoliash, 1968), that suggests a l l four types of subunits diverged at about the same time; they are a l l e q u a l l y d i f f e r e n t from one another. - 31 -In order to v e r i f y that the f i v e subunits are indeed associated i n s i t u and do not simply aggregate during e x t r a c t i o n , a number of i n v e s t i g a t o r s have examined the a b i l i t y to c r o s s - l i n k one subunit to another or la b e l the various subunits with a f f i n i t y l a b e l s d i r e c t e d at the a subunit. K a r l i n et a l . (1979) have reported r e s u l t s with a n o n s p e c i f i c cross l i n k i n g agent d i t h i o - b i s - s u c c i n i n y l p r o p i o n a t e . Following e x t r a c t i o n of the AChR and sepa-r a t i o n of c r o s s - l i n k e d subunits by SDS e l e c t r o p h o r e s i s , the cross l i n k s between subunits can be cleaved and by using two-dimensional e l e c t r o p h o r e s i s i n d i v i d u a l chains involved i n a c r o s s - l i n k e d complex can be i d e n t i f i e d . Using t h i s technique they found evidence that the a chain can be c r o s s -l i n k e d to y and 8 and the B chain can be c r o s s - l i n k e d to 6. Raft e r y et a l . (1979) have reported that a l i g h t a c t i v a t e d a l k y l a t i n g agent attached to a-BuTX can c r o s s - l i n k a chains to other a chains and a chains to 6 chain. Another l i g h t a c t i v a t e d a f f i n i t y l a b e l that i s a d e r i v a -t i v e of a-BuTX used by Nathanson and H a l l (1980a) l a b e l l e d a l l subunits (a, 8, Y» «) i n s i t u i n both Torpedo and r a t AChR. Rather than look f o r c r o s s -l i n k e d subunits they looked f o r c o v a l e n t l y l i n k e d a-BuTX-AChR subunit com-plexes a f t e r noncovalently l i n k e d a-BuTX had d i s s o c i a t e d from the receptor. Even though unmodified a-BuTX binds only to the a subunit, i t prevented l a b e l l i n g of a l l subunits by the a-BuTX l i g h t a c t i v a t e d a f f i n i t y l a b e l . Moreover, l a b e l l i n g was not prevented by high concentrations of p-amino-benzoic a c i d , a n i t r e n e scavenger, which should prevent unbound pho t o a c t i v a t a b l e a f f i n i t y l a b e l from a t t a c h i n g to rece p t o r . Azoethidium i s a l i g h t a c t i v a t e d a f f i n i t y l a b e l that g e n e r a l l y only binds to the a subunit. The binding i s blocked by a-BuTX and (+)-tubocura-r i n e (dTC) but not by ag o n i s t s , which i n f a c t , enhance the binding. An - 32 -i n c r e a s e , by carbachol, i n the l a b e l i n g of B and 6 subunits of the AChR i n d i c a t e s that carbachol has caused a conformational, change not only in the a subunit, but i n the e and 6 subunits as wel l (Raftery et a l . , 1979). Bis ( 3-aminopyridinium)l,10 decane (DAP), a l i g h t a c t i v a t e d a f f i n i t y l a b e l that i s s t r u c t u r a l l y s i m i l a r to decamethonium, l a b e l s both a and B subunits i n s i t u and a and y subunits in ext r a c t e d AChR (Weitzmann and Raf-t e r y , 1977) . Again l a b e l i n g of a l l subunits was blocked by a-BuTX. I t was not p o s s i b l e to determine whether DAP a c t u a l l y c r o s s - l i n k e d receptor sub-un i t s and the authors suggested that DAP may be a c t i v a t e d on the a subunit and simply d i f f u s e to the nearby B and 6 subunits. 1 However, another p o s s i -b i l i t y i s that the B and 6 subunits contain an onium binding s i t e . Given the s t r u c t u r e homologies found between subunits of the AChR, t h i s s i t e may even be s t r u c t u r a l l y s i m i l a r to the ACh binding s i t e on the a subunit. I t should be noted that only h a l f of the t o t a l a subunits were l a b e l e d . Binding to only h a l f of t o t a l s i t e has been observed with a number of other ligands and i s discussed i n greater d e t a i l i n chapter 3 s e c t i o n g. This r e s u l t could be explained i f the two a subunits are not symmetrically arran-ged in r e l a t i o n to the other subunits because the number of subunits. This l a t t e r p o s s i b i l i t y seems l i k e l y s i n c e the 9S monomer i s made up of an odd number of subunits. d) Receptor enriched m i c r o v e s i c l e s derived from Torpedo electroplagues Since the AChR i s an i n t e g r a l p r o t e i n i t i s l i k e l y that e x t r a c t i o n and p u r i f i c a t i o n of the receptor w i l l d i s r u p t i t s conformational s t r u c t u r e , as well as e l i m i n a t e the a b i l i t y of the receptor to perform i t s f u n c t i o n , which i s to permit ions to pass through a membrane. The development of a proce-- 33 -dure f o r o b t a i n i n g receptor enriched closed membrane fragments or microsacs (Cohen, Weber, Huchet and Changeux, 1972) was thus a major advance f o r b i o -chemists i n t e r e s t e d in the AChR. The o r i g i n a l procedure y i e l d e d fragments of which about 12 percent of the membrane bound p r o t e i n was AChR (Cohen e_t a l . , 1972). This procedure has been r e f i n e d to give higher y i e l d s . S t a r t i n g with 1 Kg of e l e c t r i c organ Sobel, Weber and Changeux (1977) were able to obtain membrane fragments c o n t a i n i n g 150 mg p r o t e i n and 3.4 x 10* 4 a-BuTX binding s i t e s or 4 pinoles of a-BuTX binding site/gm p r o t e i n . I f a l l of the p r o t e i n were 250,000 D receptor with two a-BuTX binding s i t e s , 8 umoles 0 f a-BuTX sites/gm p r o t e i n would be expected. The microsacs are apparently sealed, and the response of receptors to agonist can be measured i n terms of a pp 4. change i n p e r m e a b i l i t y to r a d i o l a b e l e d c Na ions. Thus, receptor enriched microsacs provide a preparation i n which both the biochemical and pharmacological p r o p e r t i e s of receptors can be s t u d i e d with r e l a t i v e ease. When these membranes are subjected to polyacrylamide gel e l e c t r o p h o r e s i s i t i s apparent that p r o t e i n fragments other than the four types thought to make up the AChR receptor are present; a 43,000 D p r o t e i n i s the most promi-nent of these. Recently, Neubig, K r o d e l , Boyd and Cohen (1979) have shown that these other peaks can be mostly e l i m i n a t e d by p r i o r treatment of the microsacs with a l k a l i (pH 11). This treatment i s known to s o l u b i l i z e p e r i -pheral membrane p r o t e i n s (Shanahan and Czeck, 1977 ) and i n the case of receptor enriched microsacs, apparently removes most of the 'nonreceptor' p r o t e i n . A f t e r exposure to a l k a l i , even though the microsacs only contain the pentameric p r o t e i n complex that can be e x t r a c t e d by a f f i n i t y chromato-graphy, they r e t a i n the a b i l i t y to respond to n i c o t i n e agonists. Moreover, - 34 -the pharmacological p r o p e r t i e s of the receptor mediated response seems unchanged by treatment with a l k a l i (Neubig et a l . , 1979). The only detect-able e f f e c t of t h i s procedure i s to increase the m o b i l i t y of the AChR in the membrane (Lo, Garland, Lamprecht and Barnard, 1980; Barrantes, Neugebauer and Zinqsheim, 1980). Thus, i t seems v a l i d to r e f e r to the pentameric p r o t e i n complex in these membranes as a receptor. A number of i n v e s t i g a t o r s have treated these receptor enriched microsacs with p r o t e o l y t i c enzymes to determine which subunits are exposed on the synaptic and cytoplasmic sides of the membrane ( K a r l i n et a l . , 1979; Huang, 1979; Lindstrom, G u l l i c k , Conti-Tronconi and E l l i s m a n , 1980b; Strader and R a f t e r y , 1980). The s y n a p t i c ; and cytoplasmic faces can be e a s i l y d i s t i n -guished by the presence of a-BuTX binding s i t e s which are only found on the sy n a p t i c face. There i s general agreement that a l l four subunits are expo-sed on both surfaces to degradation by pepsin and t r y p s i n . Chymotrysin appears to attack only the cytoplasmic side of the receptor (Huang, 1979). On the s y n a p t i c s i d e a l l four subunits would seem to be e q u a l l y exposed, as the r a t e of degradation by t r y p s i n i s s i m i l a r f o r a l l four subunits. This has l e d Strader and Raftery (1980) to suggest that i t i s the carboxyterminal end of these subunit that i s exposed to -the synaptic side since there are considerable s t r u c t u r a l homologies between the carboxytermini of the four subunits ( R a f t e r y , H u n k a p i l l e r , Strader and Hood, 1980). On the cytoplasmic side the subunits do not appear to be e q u a l l y exposed; the l a r g e s t subunit («) i s degraded much more than the smallest (a) subunit, which i s hardly degraded at a l l . - 35 -I t i s i n t e r e s t i n g that treatment with the proteases does not d i s r u p t the c h a r a c t e r i s t i c s i z e and doughnut shape of the receptor seen with the e l e c -tron microscope (Lindstrom e t a l . , 1980b; Strader and R a f t e r y , 1980) nor does i t appear to a f f e c t the a b i l i t y of carbachol to increase Na + f l u x across the membrane (Lindstrom et a l . , 1980b). Though a l l of the subunits span the membrane they may not be e q u a l l y exposed to the hydrophobic intramembrane environment. Ta r r a b - H a z e l a i , B e r c o v i c i , Goldfarb and Gelten (1980) have i n v e s t i g a t e d t h i s problem using 1 pc the l i p i d s o l u b l e reagent 5-( I) i o d o n a p h t h a l y l - l - a z i d e (INA). Upon p h o t o a c t i v a t i o n t h i s reagent w i l l c o v a l e n t l y bond to proteins i n contact with membrane l i p i d s . When INA was mixed with receptor enriched microsacs, and a c t i v a t e d , only the a (40.000D) subunit was l a b e l e d , however, a f t e r e x t r a c t i o n and s o l u b i l i z a t i o n of the AChR by the detergent T r i t o n X-100, a l l subunits were l a b e l l e d . Trypsin degradation of AChR i n the receptor e n r i c h -ed microsac leads to the fragmentation of the a subunit i n t o 30,000D and 10,000D polypeptides. The INA l a b e l was l o c a l i z e d to the 10.000D polypep-t i d e while the a-BuTX s i t e was l o c a l i z e d to the 30,000D polypeptide. Receptor enriched microsacs contain at l e a s t 60 l i p i d molecule per mem-brane face per AChR and these appear to be arranged i n the standard b i l a y e r s t r u c t u r e (Ross e t a l . , 1977). E l e c t r o n spin resonance probes i n d i c a t e that much of the l i p i d around the AChR i s immobilized (March and Barrantes, 1978). This may be due to immob i l i z a t i o n of the receptor (see Barrantes et^ a l . , 1980), which i n turn may be the r e s u l t of the presence of the 43,000D pr o t e i n mentioned above. When t h i s p r o t e i n i s ext r a c t e d from receptor enriched microsacs by treatement with a l k a l i , r eceptors become much more mobile (see Cartaud, Sobel, Rousselet, Devaux and Changueux, 1981) - 36 -The l i p i d surrounding the AChR appears to contain l i t t l e c h o l e s t e r o l (Bridgman and Nakajima, 1980). Thus the receptor may be located i n a sub-phase of the membrane that may have pr o p e r t i e s d i f f e r e n t from those of the bulk phase of the normal c e l l membrane e) Functional r e c o n s t i t u t i o n of p u r i f i e d receptors i n t o l i p i d b i l a y e r s of defined composition Soon a f t e r the f i r s t i s o l a t i o n of the AChR by a f f i n i t y chromatography attempts were made to re i n c o r p o r a t e i t i n t o a r t i f i c i a l membrane, i n order to v e r i f y that p r o t e i n was i n f a c t the f u n c t i o n a l u n i t mediating the act i o n of ACh. Although some success was reported, the procedures s u f f e r e d from lack of r e p r o d u c i b i l i t y (see Eldefrawi and E l d e f r a w i , 1977). Greater success was achieved when receptor-enriched membrane fragments were used as the s t a r t i n g m a t e r i a l f o r r e c o n s t i t u t i o n r a t h e r than p u r i f i e d receptors (Epstein and Raker, 1977). The l a t t e r technique has now been r e f i n e d with the development of s o - c a l l e d r e c o n s t i t u t e d v e s i c l e s where n a t i v e receptor enriched v e s i c l e s are fused with liposomes of known l i p i d composition so that the na t i v e l i p i d i s d i l u t e d (see Wu and Ra f t e r y , 1981). Both n a t i v e and r e c o n s t i t u t e d v e s i c l e s r e t a i n the a b i l i t y to respond to c h o l i n e r g i c 22 + agonist with an increased p e r m e a b i l i t y to Na but the question remains: how s i m i l a r i s the response to that which occurs i n the i n t a c t c e l l , and i s the pentameric p r o t e i n complex the f u n c t i o n a l u n i t that mediates a normal response? Several recent papers have t r i e d to answer these questions. Two of these papers re p o r t attempts to incorporate receptor from nativ e and recons-t i t u t e d v e s i c l e s , obtained from Torpedo e l e c t r o p l a x , i n t o a phospholipid planar b i l a y e r . The advantage of the l a t t e r technique i s that channels - 37 -incorporated i n t o these membranes can be e a s i l y monitored e l e c t r i c a l l y by measuring the conductance between chambers separated by the planar membrane. I f o n l y a few channels are incorporated these can be c h a r a c t e r i z e d by a d i s t i n c t steps i n conductance of f i n i t e d u r a t i o n . Schindler and Quast (1981) s t a r t e d with receptor enriched n a t i v e v e s i c l e s . These were f i r s t exposed to a phospholipid monolayer which presumably incorporated some of the AChR. This monolayer was then caused to form a b i l a y e r with another monolayer i n an adjacent chamber, by all o w i n g the two monolayers to come i n contact across a small aperture. This generated a b i l a y e r that responded to carbachol with an increase i n conductance that could be blocked by dTC and i n which s i n g l e channel events could a l s o be observed. In 1 M NaCl these s i n g l e channels had a conductance of 90 ps and a mean duration of 1.3 ms. (at -100 mV on the side i n s e n s i t i v e to a g o n i s t ) . The channel was e q u a l l y permeable to Na + and K + and r e l a t i v e l y impermeable to C l ~ . These c h a r a c t e r i s t i c s are very s i m i l a r to those of subsynaptic AChR i n i n t a c t c e l l s (see Under and Qua s t e l , 1978). However, there were a number of anomalies. For example, only 0.001 to 0.1 percent of t o t a l receptor present (as i n d i c a t e d by a-BuTX binding) appeared to be f u n c t i o n a l . Also the receptors were r e l a t i v e l y i n s e n s i t i v e to dTC; 200 uM reduced the response to 50 carbachol by only 60 percent. This e f f e c t may have been due e n t i r e l y due to the 'channel plugging' a c t i o n of dTC (see Katz and M i l e d i , 1978). Nelson, Anholt, Lindstrom and Montal (1981) s t a r t e d with r e c o n s t i t u t e d v e s i c l e s which were added to the chamber or one si d e of a preformed planar b i l a y e r . F ollowing t h i s procedure channels were observed that had a conduc-tance of 19 pS i n 0.1 M NaCl. However, these had (at -10 mV) a mean dura-t i o n of 32 ms . At the end of the paper there was a note that when the - 38 -method of Schindler and Quast (1980) was used to incorporate receptor i n t o a planar b i l a y e r , a channel duration of 5 ms was obtained. Thus AChR derived from n a t i v e v e s i c l e s and from p u r i f i e d r e c o n s t i t u t e d v e s i c l e s e x h i b i t s i m i -l a r channel p r o p e r t i e s when studied under i d e n t i c a l c o n d i t i o n s . Wu, Moore and Raf t e r y (1981) and Moore and Raftery (1980) have taken another approach i n order to compare receptor i n n a t i v e and r e c o n s t i t u t e d v e s i c l e s . They have designed an imaginative technique f o r measuring i o n i c f l u x w i t h i n a m i l l i s e c o n d time s c a l e . B r i e f l y they preload receptor con-t a i n i n g v e s i c l e s with fluorophore 8-aminonaphthalene-l,3,6-trisulfonate (ANTS). The fluorescence of t h i s molecule i s quenched by the ion t h a l l i u m (Tl ); the AChR channel i s twice as permeable to Tl than Na (Adams, Dwyer and H i l l e , 1980) and the v e s i c l e s are r e l a t i v e l y impermeable to T l + i n the absence of agonist (Wu et a l . , 1981). Thus, the increase i n permea-b i l i t y to T l + induced by an agonist can be monitored using stopped flow spectrophometry. Using t h i s approach they obtain agonist c o n c e n t r a t i o n -response curves to carbachol and ACh which are e s s e n t i a l l y the same i n both n a t i v e a l k a l i t r e a t e d v e s i c l e s and r e c o n s t i t u t e d v e s i c l e s . In both types of v e s i c l e the pentameric p r o t e i n complex ( o ^ B ^ ) i s the major p r o t e i n compo-nent and i t i s l i k e l y that t h i s complex i s s o l e l y r e s p o n s i b l e f o r the response produced by n i c o t i n i c a g onists. The a f f i n i t i e s f o r carbachol and ACh (500 and 44 UM, r e s p e c t i v e l y ) are the same as seen i n i n t a c t c e l l s when precautions are taken to avoid d e s e n s i t i z a t i o n (see chapter 4, s e c t i o n c , i i ) . However, i n both types of v e s i c l e s the maximal r a t e of i n f l u x , which presumably occurred when a l l receptor were a c t i v a t e d , was only 10 percent of expected. - 39 -The r a t e of i n f l u x of T l + (mole/second) i s r e l a t e d to the ex t e r n a l concentration ( m o l e s / l i t e r ) by the p e r m e a b i l i t y constant ( l i t e r / s e c o n d ) This constant i s a property of the receptor channel and i s r e l a t e d to the channel conductance by the f o l l o w i n g equation (see Linder and Quastel, 1978). p - conductance x (RT/F)  T ' * f < [ T l + ] o u t The conductance of T l + i s twice that of Na + (Adams, Dwyer and H i l l e , 1980) and thus the s i n g l e channel conductance should be around 10 pS when the outside [ T l + ] i s around 30 mM (28 mM, Wu, Moore and R a f t e r y , 1981); 35 mM, Moore and R a f t e r y , 1980). D 10 x 10 amps/volt x 25 x 10 v o l t s TI = ^3 : 5 30 x 10 m o l e s / l i t e r x 10 amps/second —1 fi = 10 l i t e r s / s e c o n d I f a l l of the receptor channels were opened by a s a t u r a t i n g concentration of agonist then the maximum f l u x r a t e per v e s i c l e should be given by the f o l l o w -ing equation Flux r a t e = PJ-J x no. of channels/volume In the case of a l k a l i n e e x t r a c t e d n a t i v e v e s i c l e s the i n t e r n a l volume i s 1 0 " ^ l i t e r s and each v e s i c l e contains about 2 x 10^ a-BuTX binding s i t e s or 10^ channels (Moore and R a f t e r y , 1980), the r a t e of i n f l u x w i l l be - 40 -about 1 0 4 / s / v e s i c l e . The measured maximal i n f l u x was 1.5 x 10 3/sec. With the r e c o n s t i t u t e d v e s i c l e s there are much fewer channels per v e s i c l e . Wu, Moore and Raftery (1981) estimate that there are only about 2 channels/-v e s i c l e . However, the v e s i c l e s are smaller with an i n t e r n a l volume of about —?n 3 x 10 L. Hence, 2 channels should allow a r a t e of i n f l u x of 3 2 6x10 / s / v e s i c l e . The measured value was about 6 x 10 /s f o r ACh and s l i g h t l y l e s s (5 x 10 Is) f o r carbachol. The discrepancy between the actual and predicted f l u x r a t e s may be a r t i -f a c t u a l since h i s t r i o n i c o t o x i n (HTX) was used to reduce the f l u x at high concentrations of agonist to a measurable range. Raftery and h i s colleagues assumed that HTX was a c t i n g as noncompetitive antagonist and reduced respon-ses to low agonist concentrations to the same extent as high agonist concen-t r a t i o n s . However, si n c e t h i s drug seems to bind p r e f e r e n t i a l l y to the a c t i v a t e d channel (see Albuquerque, Kuba and Daly, 1974) i t i s l i k e l y t hat responses to high agonist concentrations were i n h i b i t e d to a greater extent than responses to -low agonist concentrations. Another p o s s i b i l i t y i s that only 10 percent of the a-BuTX binding s i t e s i n these v e s i c l e s are associated with f u n c t i o n a l r e c e p t o r s , but t h i s seems u n l i k e l y s i n c e in the case of the r e c o n s t i t u t e d v e s i c l e , with only about 4 a-BuTX bindin g s i t e s per v e s i c l e , Wu, Moore and Raftery (1981) found t h a t most v e s i c l e s responded to ACh, i . e . most of the dye could be quenched. In c o n c l u s i o n , i n s p i t e of the many d i f -f i c u l t i e s t hat need to be r e s o l v e d , these r e c o n s t i t u t i o n experiments are h i g h l y suggestive that the pentameric p r o t e i n complex described above may contain a l l or most of the components of the f u n c t i o n a l u n i t that mediates the response to ACh, i . e . the ACh r e c e p t o r . - 41 -f ) Fine s t r u c t u r e of the AChR Recent st u d i e s on the e l e c t r o n microscope of the AChR give a p i c t u r e of the s t r u c t u r e of the AChR i n i n t a c t membranes. When receptor enriched microsacs are examined with the negative s t a i n i n g technique they are seen to be studded with doughnut shaped p a r t i c l e s with an 8.0 nm outer diameter and a 2.0 nm inner diameter. The d e n s i t y of the p a r t i c l e s can be as high as 10,000/uirr and the r a t i o of a-BuTX s i t e to p a r t i c l e s appears to be 2:1 (Cartaud, Benedetti, Sobel and Changeux, 1978). This i s j u s t what would be expected i f each of these p a r t i c l e s represent the 9S monomer, each of which contain two a-BuTX binding s i t e s . These p a r t i c l e s can be 'decorated' with a n t i r e c e p t o r a n t i b o d i e s , and f o l l o w i n g exposure to a-BuTX they can al s o be 'decorated' with anti-a-BuTX antibodies (Klymkowsky and Stoud, 1980). The synapti c side of the p a r t i c l e extends 5.5 nm above the 4.0 nm t h i c k l i p i d b i l a y e r w h i le the cytoplasmic s i d e extend only an a d d i t i o n a l 1.5 nm making f o r a t o t a l length of 11.0 nm. X-ray d i f f r a c t i o n studies i n p a r t i a l l y o r i e n t e d membranes suggest that these p a r t i c l e s are funnel-shaped with the synapti c end about 8.0 nm i n diameter and the cytoplasmic end about 6.0 nm (Ross, Klymkowski, Agard, and Stoud, 1977; Klymkowsky and Stoud, 1979). The volume of an i n d i v i d u a l p a r t i c l e was estimated to be around 350,000 A . This i s j u s t the volume expected f o r a 250,000 D AChR monomer since Changeux et a l . (1975) have estimated that the AChR has a p a r t i a l s p e c i f i c volume of 0.74 cm /gm. A f u r t h e r i n d i c a t i o n that these p a r t i c l e s are indeed the 9S monomer i s that very s i m i l a r doughnut shaped p a r t i c l e s are seen when these monomers are r e c o n s t i t u t e d i n t o phospholipid v e s i c l e s . A l s o , there i s a one to one c o r r e l a t i o n between the proportion of receptors r e c o n s t i t u t e d i n the dimeric form and the r e l a t i v e numbers of 'doublet' p a r t i c l e s seen i n these v e s i c l e s (Cartaud, Popot and Changeux, 1980). - 42 -Heuser and Salpeter (1979) have i n v e s t i g a t e d the f i n e s t r u c t u r e of AChRs using small pieces of f i x e d and unfixed Torpedo e l e c t r o p l a x . They found AChR were organized i n a loose array with no o v e r a l l s t r u c t u r e , though ordered rows of receptor dimers and tetramers were observed. The o v e r a l l d e n s i t y of AChR i n the subsynaptic region was about 10,000/u . Freeze f r a c t u r e s of the postsynaptic membrane showed that there were intramembrane p a r t i c l e s . As observed by others (see Rash et a l . , 1979) these were fewer in number than the surface p a r t i c l e s . However, with improved techniques Heuser and Salpeter (1979) were able to show that in a d d i t i o n to the obvious intramembrane p a r t i c l e s there was also a cobblestone l i k e pattern on both^ f r a c t u r e f a c e s . Heuser and Salpeter (1979) concluded that many of the membrane p a r t i c l e s were f r a c t u r e d at the same time as the membrane. Indeed the d e n s i t y of a l l of the bumps and depressions i n the f r a c t u r e faces matched the d e n s i t y of the surface p a r t i c l e s and as with the surface p a r t i c l e s rows of cobblestone l i k e bumps two or four wide could be seen. They concluded that a l l of the surface p a r t i c l e s (presumably ACh receptors) span the membrane. Heuser and Salpeter (1979) also noted that they could not see any obvious s t r u c t u r a l changes produced by e i t h e r short or prolonged exposures to agonists and antagonists. g) Binding c h a r a c t e r i s t i c s of the AChR ( i ) Are a l l acceptor s i t e s on the AChR the same a-BuTX binds to receptors in a monophasic manner i n d i c a t i n g that the two binding s i t e s associated with the 9S monomer have equal a f f i n i t i e s f o r t h i s l i g a n d (Damle and-Karli'n, 1978). Sine and Taylor (1980) have determined the r e l a t i o n s h i p between the f r a c t i o n of receptors occupied by a-BuTX and the response to carbachol ( i n f l u x of "Na) i n c u l t u r e d s k e l e t a l muscle BC3H-1 c e l l s . This c e l l l i n e , w h i l e o r i g i n a l l y derived from a b r a i n tumour, has many c h a r a c t e r i s t i c s s i m i l a r to s k e l e t a l muscle, i n c l u d i n g the presence of n i c o t i n i c receptors (see Schubert et a l . , 1974). The e x p e r i m e n t a l deter-mined r e l a t i o n was nonlinear (convex); when 50 percent of the s i t e s were occupied by a-BuTX the response was 20 percent of c o n t r o l . The r e l a t i o n agreed w e l l with the p r e d i c t i o n s of a model where there are two binding s i t e s per receptor and both have to be occupied by an agonist in order f o r the receptor to be a c t i v a t e d ; occupation of e i t h e r s i t e by a-BuTX i s s u f f i -c i e n t to prevent receptor a c t i v a t i o n . Lindstom, Anholt, Einarson, Engel, Osame and Montal (1980a) c a r r i e d out a s i m i l a r experiment with receptors r e c o n s t i t u t e d i n t o phospholipid v e s i c l e s . They concluded that the two bind-ing s i t e s were d i f f e r e n t and that one of these s i t e s played a much l a r g e r r o l e i n receptor a c t i v a t i o n than the other. Lindstrom et a l . (1980a) found what appeared to be a l i n e a r r e l a t i o n between the f r a c t i o n of binding s i t e s occupied by a-BuTX and the f r a c t i o n of the response to carbachol remaining; when 50 percent of the s i t e s were occupied the i n f l u x of Na was 40 percent of c o n t r o l . Such a r e s u l t implies that one of the binding s i t e s can be occupied by the t o x i n without preventing that receptor from being a c t i v a t e d . However, i t i s r e a d i l y apparent from t h e i r data that the r e l a -t i o n s h i p was not l i n e a r , but rat h e r concave at the beginning and convex at the end; 60 percent occupancy of binding s i t e s by a-BuTX was associated with only 20 percent rather than 40 percent of the c o n t r o l response to carba-c h o l . This type of r e l a t i o n i s expected i f the f u n c t i o n a l receptor u n i t i s associated w i t h four b i n d i n g s i t e s and can s t i l l be a c t i v a t e d i f only one of these s i t e s i s associated with t o x i n . This model i s not unreasonable s i n c e these v e s i c l e s contained dimeric r e c e p t o r s . Thus, the r e s u l t s of Lindstrom et a l . (1980a) do not prove t h a t the acceptor s i t e s on the two a subunits play d i f f e r e n t r o l e s i n receptor a c t i v a t i o n . - 44 -Neubig and Cohen (1979) studied the binding of t r i t i a t e d antagonist to receptor enriched Torpedo microsacs. They found that the receptor had high and low a f f i n i t y binding s i t e s f o r dTC in approximately equal amounts and with d i s s o c i a t i o n constants of 33 ± 6 nM and 7.7 ± 4.6 yM, r e s p e c t i v e l y . ACh and carbachol could d i s p l a c e dTC bound to both s i t e s and the assumption that these agonists d i d not d i s t i n g u i s h between the s i t e s f i t the data reasonably w e l l . The t o t a l number of binding s i t e s was estimated and the r e l a t i v e numbers of a-BuTX/ACh/dTC s i t e s was 1/0.93/0.93. Thus, i t appears that h a l f of the ACh binding s i t e s in the postsynaptic membrane have a high a f f i n i t y f o r dTC and h a l f have a low a f f i n i t y . The d i f f e r e n c e in a f f i n i t y was seen with other bisonium antagonists, but v a r i e d i n degree (Table 1 ). Table 1. D i s s o c i a t i o n constants f o r a two s i t e model d e s c r i b i n g  the r e l a t i o n between s p e c i f i c binding and 1igand concentration Ligand h (yM) *2. (yM) Bisonium dTC 0.04 8 200 dimethylcurare 0.5 10 20 gallamine 2.0 100 50 decamethonium 0.1 16 20 Mononium dihydro-B-e r y t h r o i d i n e 1.0 40 40 t r i e t h y l - ( 4 - p h e n y l b u t y l ) -ammonium iodide 50 50 1 - 45 -Dihydro-e-erythoidine, which contains only one onium group, showed the d i f f e r e n c e in a f f i n i t y but triethyl-(4-phenylbutyl)-ammonium i o d i d e , another antagonist with only one onium group, d i d not. This may suggest that the antagonist molecule i t s e l f introduces the s t e r i c hindrance that reduces the a f f i n i t y of the adjacent binding s i t e on the adjacent a subunit. This i s conceivable s i n c e Michelson and Zeimal (1973) have argued from s t r u c t u r e a c t i v i t y s t u d i e s that the two binding s i t e are q u i t e c l o s e (< 1.6 nm) to one another (see chapter 4, s e c t i o n b ) . A l s o , Neubig and Cohen (1979) found evidence that dTC that i s bound to one binding s i t e could hinder binding of a-BuTX to the adjacent s i t e ; concentrations of dTC intermediate between i t s d i s s o c i a t i o n constants were more potent than expected i n reducing the i n i t i a l r a t e of bindi n g of a-BuTX. This would suggest that h a l f of the ACh binding s i t e s have a lower a f f i n i t y f o r dTC because of negative c o o p e r a t i v i t y i n dTC b i n d i n g . Sine and Taylor (1980) have argued against t h i s p o s s i b i l i t y because they f i n d that h a l f of the binding s i t e s continue to have a lower a f f i n i t y f o r dTC even a f t e r a large f r a c t i o n of the binding s i t e s have bound a-BuTX and most of the s i t e s that remain f r e e and can i n t e r a c t with dTC are there-f o r e adjacent t d a s i t e occupied by a-BuTX. However, the observation that dTC bound to one a subunit can i n t e r f e r e with the binding of a-BuTX to ano-ther subunit (see Neubig and Cohen, 1980) suggests that the converse may al s o be t r u e ; a-BuTX may i n t e r f e r e w i t l \ the binding of dTC i n the adjacent a subunit. The f a c t that only one of the s i t e s adjacent to the one occupied by a-BuTX has a lower a f f i n i t y f o r dTC may simply r e f l e c t a d i f f e r e n c e in the way that the two s i t e s on a 9S receptor monomer are arranged r e l a t i v e to the other subunits or i t may i n d i c a t e that these two binding s i t e s are indeed d i f f e r e n t . - 46 -Non-equivalence of the two a subunits i s also seen when the binding of a f f i n i t y l a b e l s i s i n v e s t i g a t e d . A number of a f f i n i t y l a b e l s have been reported to l a b e l only h a l f of the a subunits, f o r example maleimide benzyl-t r i m e t h y l ammonium iodide (MBTA) and bromoacetylcholine (BAC) (Karl i n , W e i l l , McNamee and Valderrama, 1975; Damle, McLaughlin and K a r l i n , 1978; Moore and R a f t e r y , 1979). Wolosin, L y d d i a t t , D o l l y and Bernard (1980) have shown that the" l i g a n d binding s i t e on both a subunits can be lab e l e d with BAC. They showed t h i s to be true of both Torpedo el e c t r o p l a q u e and s k e l e t a l muscle r e c e p t o r s . One of the s i t e s i s lab e l e d more s l o w l y , and i s l i k e l y to have been missed in e a r l i e r s t u d i e s . Very r e c e n t l y Young, Oshiki and Sigman (1981) have found that incubation of receptor enriched membrane fragment from Torpedo electroplaques with short chain alkanols or v o l a t i l e anaesthetics such as halothane w i l l give r i s e two equal populations of s i t e s that bind a-BuTX at rates d i f f e r i n g by a f a c t o r of 10. Without preincubation with these agents only a s i n g l e popula-t i o n of a-BuTX binding s i t e s e x i s t e d . In c o n s t r u c t i n g k i n e t i c models of drug receptor i n t e r a c t i o n s a common r u l e i s to assume the simplest case c o n s i s t e n t with the data. Up to now most i n v e s t i g a t o r s who have approached the problem have assumed that the two s i t e s are e q u i v a l e n t . From the above d i s c u s s i o n i t i s evident t h i s may not be a v a l i d assumption. Under c e r t a i n c o n d i t i o n s the two a subunits are not equivalent and the f u n c t i o n a l s i g n i f i c a n c e of t h i s f a c t i s u n c e r t a i n . Nor i s i t c e r t a i n that such a d i f f e r e n c e i s always present. Though the inherent asymmetry of the AChR may be s u f f i c i e n t to e x p l a i n the observed non-equiva-lence of a subunits, t h i s might e q u a l l y a r i s e as the r e s u l t of d e s e n s i t i z a -t i o n or some other l i g a n d induced conformational change. Even antagonists such as dTC produce conformational changes that can change the binding c h a r a c t e r i s t i c s of HTX. Moreover t h i s change i s s i m i l a r to that associated with agonist induced d e s e n s i t i s a t i o n (Cohen 1978; Medinsky and Cohen, 1981). - 47 -( i i ) How many acceptor s i t e s per ACh r e c e p t o r ? The biochemical evidence (see chapter 3, s e c t i o n c and d) i n d i c a t e s that the major l i g a n d binding s i t e s on the AChR are labe l e d by a-BuTX and that there are two of these binding s i t e s per receptor monomer. However, i t i s p o s s i b l e that the receptor r e s p o n s i b l e f o r the elementary event i s made up of more than one 9S monomer . I t i s c l e a r that under normal c o n d i t i o n s adjacent monomers are l i n k e d by s u l f h y d r y l bonds to form dimers. Though not in t e r p r e t e d as such by the authors, the r e s u l t s of Lindstom et a l . (1980a) discussed i n the l a s t s e c t i o n are c o n s i s t e n t with such i n t e r a c t i o n s t a k i n g place. Removal of the covalent coupling between monomers does not cause major changes i n the responsiveness of the receptors but noncovalent l i n k -ages may s t i l l be p o s s i b l e (see K a r l i n e t . a l . , 1979). Some i n v e s t i g a t o r s have indeed suggested that the 13S dimer form of the receptor i s the f u n c t i o n a l receptor u n i t (see M i l l e r , Moore, H a r t i g and Raftery , 1978). Evidence i n support of t h i s p o s s i b i l i t y came p r i m a r i l y from s t u d i e s on the binding of HTX, which binds with high a f f i n i t y to a s i t e on the receptor that has an a f f i n i t y f o r l o c a l anaesthetics but not f o r c h o l i n e r g i c l i g a n d s . Since both HTX and l o c a l anaesthetics appear to 'plug' channels which have been opened by n i c o t i n i c agonists i t has been suggested that HTX might be a marker f o r the channel p o r t i o n of the receptor ( E l d e f r a w i , Elde-f r a w i , Mansour, Doly, Wiktop and Albuquerque, 1978). E a r l y measurements of the r e l a t i v e numbers of h i s t r i o n i c o t o x i n (HTX) binding s i t e s compared to a-BuTX s i t e s suggested a r a t i o of 1:4 ( E l l i o t and Ra f t e r y , 1977; K r o d e l , Beckmann and Cohen, 1978). Sobel, Heidman, Cartaud and Changeux (1980) reported a r a t i o of 1:1.5. S t i l l more r e c e n t l y Medynsky and Cohen (1981) - 48 -have detected a problem i n e s t i m a t i n g the s p e c i f i c a c t i v i t y of ( H) HTX and now report that the r a t i o i s c l o s e to 1:1. I f t h i s i s so, i t suggests that the HTX binding s i t e i s not a marker of the channel as p r e v i o u s l y b e l i e v e d . I t i s important to note that a-BuTX may not i n f a c t l a b e l a l l of the s i t e s with which ACh can i n t e r a c t . This i s suggested by the pharmacological evidence f o r a CIO s t r u c t u r e (see Michelson and Zeimal 1973; chapter 4 s e c t i o n b) that i s too small (1.4 nm) to span the distance between monomers (9.0 nm center to center) and by the observation that a number of a f f i n i t y l a b e l s that contain a quaternary ammonium group (such as azoethedium and bis-(3-aminopyridinium)l,10 decane) can bind to the receptor at s i t e s other than the a-BuTX binding s i t e ( R a ftery et a l . , 1979). Though c a r e f u l binding s t u d i e s suggest that f o l l o w i n g d e s e n s i t i z a t i o n (see below) there are as many high a f f i n i t y ACh binding s i t e s as there are dTC or a-BuTX binding s i t e s (Neubig and Cohen 1979) these studies do not r u l e out the p o s s i b i l i t y of a d d i t i o n a l low a f f i n i t y b i n d i n g s i t e s f o r ACh and other c h o l i n e r g i c l i g a n d s . Indeed, given the s t r u c t u r a l s i m i l a r i t i e s between the receptor s u b u n i t s , each of -the 5 subunits may posses a s i t e that can i n t e r a c t with ACh and i t s analogues (see Heidemann and Changeux, 1981) - 49 -CHAPTER 4 Diverse types of drug - receptor i n t e r a c t i o n . a) Preface A receptor contains an acceptor component, an e f f e c t o r component, and an intermediary component. I t i s perhaps not s u p r i s i n g that each of these components i n the ACh receptor can be inf l u e n c e d by drugs. Those that act at the acceptor component g e n e r a l l y have a we l l defined s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p . This i s e s p e c i a l l y t r u e of n i c o t i n i c a g o n i s t s . The e f f e c t of drugs on the intermediary component i s harder to define and often secondary to an a c t i o n at the acceptor s i t e . These actions are expressed i n the phe-nomena of p o s i t i v e c o o p e r a t i v i t y , where occupancy of more than one acceptor s i t e i s necessary to i n i t i a t e a u n i t response by the e f f e c t o r component and d e s e n s i t i z a t i o n where the agonist binding to the acceptor s i t e no longer i n i t i a t e s a response i n the e f f e c t o r component. F i n a l l y , many drugs appear to i n t e r a c t d i r e c t l y with i o n i c channels opened by n i c o t i n i c agonist. This r e s u l t s i n a reduction of i o n i c p e r m e a b i l i t y or a complete plugging of the channel. The s t r u c t u r a l requirements f o r t h i s i n t e r a c t i o n are minimal yet are not alt o g e t h e r non-existent. The 'channel plugging' (Katz and M i l e d i , 1978) a c t i o n i s thus an i n t e r e s t i n g example of a n o n s p e c i f i c receptor mediated event. - 50 -b) Drug receptor i n t e r a c t i o n s at the acceptor s i t e on the ACh receptor ( i ) N i c o t i n i c agonists The e f f i c a c y and potency of ACh ( I ; roman numerals r e f e r to s t r u c t u r a l formula given i n appendix to t h i s chapter) a c t i o n on n i c o t i n i c receptors appears to be determined p r i m a r i l y by the ammonium group ana the carbonyl oxygen (see Michelson and Zeimal, 1973). 3,3-Dimethyl a c e t y l acetate ( I I ) i s i s o s t e r i c with ACh (I) yet has l i t t l e or no n i c o t i n i c a c t i v i t y (Banister and Whittaker, 1951). E t h y l a c e t a t e ( I I I ) can cause some d e p o l a r i z a t i o n of the neuromuscular j u n c t i o n (Auerbach, del C a s t i l l o and Specht, 1980) but again i t i s much l e s s e f f e c t i v e than ACh. Tetramethylammonium (IV) i n con t r a s t i s a f u l l n i c o t i n i c agonist (Adams, 1975b), though 50 times less potent than ACh. Hence, the e t h y l a c e t a t e p o r t i o n of the ACh molecule does not appear to be necessary f o r f u l l e f f i c a c y but l i k e l y plays a r o l e in determining the potency of ACh. Because there i s a s i g n i f i c a n t d i p o l e along the carbonyl bond, the carbonyl oxygen probably makes a hydrogen bond with an e l e c t r o n acceptor i n the AChR. This bond may s t a b i l i z e the i n t e r a c t i o n of the onium group at the anio n i c s i t e . Thus, carbachol (V) i s about 10 times less potent than ACh. The amine of the carbonate w i l l tend to reduce the dipol e at the carbonyl oxygen. Removal of the carbonyl oxygen of ACh (see VI) reduces potency f o r c o n t r a c t i o n of f r o g rectus by 4 0 - f o l d (Barlow 1964). ' The carboxyl oxygen also plays an important r o l e in reducing the l i k e l i h o o d of non-productive binding (see F r a n k l i n , 1980; B e l l e a u , 1964). This i s exe m p l i f i e d by c h o l i n e (VII) and pentyltrimethylammonium ( V I I I ) which are both p a r t i a l a g o nists. ~ - 51 -I t i s l i k e l y that the ammonium group i n t e r a c t s with an anio n i c group i n the a c e t y l c h o l i n e r e c e p t o r . The potency of dimethyl aminoethylacetate (IX) which has a pK g of 8.35, changes with pH in accordance with the change i n percentage of i o n i z a t i o n (Michelson and Frugentov 1963; c i t e d i n Michaelson and Zeimal, 1973). I t i s notable that even i n the f u l l y i o n i z e d form the potency of t h i s analog i s l e s s than 1 percent of that of ACh; thus, i t appears that the anionic s i t e of the AChR has a s t r i c t l y defined s t e r i c con-f i g u r a t i o n . Increasing the s i z e of the charged group by r e p l a c i n g the methyl group with an e t h y l group reduces potency 5000-fold. Replacing the nitrogen with a l a r g e r atom such as sulphur reduces potency 15-f o l d ( M i c h e l -son and Zeimal, 1973). T r i g g l e and T r i g g l e (1976) have argued that the f a l l i n potency seen when the trimethylammonium group of ACh i s replaced by a t r i e t h y l ammonium group i s too large to be explained simply by an increased i n t e r i o n i c d i s t a n c e . They suggest that the methyl group bind around the ani o n i c s i t e through Van der Waal's i n t e r a c t i o n s and form a low d i e l e c t r i c 'tent' i n which i o n i c i n t e r a c t i o n s w i l l be g r e a t l y strengthened. Replacing N-methyl groups with N-ethyl groups abolished agonist e f f i c a c y . This may be due to an increase i n the distance between the ammonium and the receptor a n i o n i c group, but also may allow the i n t r u s i o n of water i n t o the i o n i c b inding locus. Whatever the case, the e f f e c t of N-ethyl s u b s t i t u t i o n sug-gests that the onium group f i t s i n t o a pocket i n the AChR. E a r l y c a l c u l a t i o n s of the d i s t r i b u t i o n of e l e c t r o n density on the ACh molecule (Pullman and Pullman, 1963) suggested that there might be a net p o s i t i v e charge on the ether oxygen. However, more recent c a l c u l a t i o n s (Pullman, C o u r r i e r e and C o u r b e i l s , 1971) suggests that there i s i n f a c t a considerable negative charge on the ether oxygen. I t i s s t i l l uncertain - 52 -what r o l e i s played by t h i s oxygen (see Michelson and Zeimal, 1973). The two carbons l i n k i n g the acetate moiety of ACh to the onium moiety also do not appear to play much of a r o l e i n determining the potency of agonists. The potency of TMA (IV) i s about the same as pentyl-TMA ( V I I I ) (Adams, 1975b). This suggests a two point i n t e r a c t i o n with the receptor which i n turn i s c o n s i s t e n t with the general lack of s t e r e o s e l e c t i v i t y of the n i c o t i n i c receptor ( T r i g g l e and T r i g g l e , 1976). Methylation of these carbons can, however, reduce potency presumably through s t e r i c hinderance. a Methyl a c e t y l c h o l i n e i s h a l f as potent as ACh and 6 methylcholine i s only 1 percent as potent (Simonart, 1932). The n i c o t i n i c receptor appears to pr e f e r the gauche conformation of ACh. In s o l u t i o n , the gauche c o n f i g u r a t i o n of ACh i s more s t a b l e . Replace-ment of the e s t e r i n ACh with an amide (X) causes the trans c o n f i g u r a t i o n to be more s t a b l e and reduces potency 1000-fold. Methylation of the amide (XI) causes a f u r t h e r 3 f o l d reduction of potency and maintains the trans conformation. P i p e r a z i n e r i n g c l o s u r e (see XII) however, s t a b i l i z e s the gauche conformation and increases potency to 10 percent of ACh or a 300-fold increase i n potency. This occurs without any a d d i t i o n to the molecule and implies that the increased potency i s due s o l e l y to the s t a b i l i z a t i o n of the gauche c o n f i g u r a t i o n . The distance between the H-bond acceptor group of the receptor and the onium group of ACh i n the gauche conformation i s 0.59 nm. This distance i s a c h a r a c t e r i s t i c of n i c o t i n i c agonists (Beers and Reich, 1979; Chothia and P a u l i n g , 1970). N i c o t i n e ( X I I I ) i t s e l f does not possess a carbonyl oxygen but one of the nitrogens i n the p y r i d i n e r i n g i s electroneg-a t i v e , capable of H-bond formation — and at a distance of 0.59 nm from the c a t i o n i c n i t r o g e n . With muscarine (XIV) the distance between the c a t i o n i c nitrogen and the H-bond acceptor would be 0.44 nm, and muscarine has l i t t l e or no n i c o t i n i c a c t i v i t y . However, muscarone (XV), i n which an alcohol group has been replaced by a keto group, i s capable of making an H-bond such that the H-bond acceptor i s 0.59 nm from the c a t i o n i c n i t r o g e n , and i s twice as potent as ACh as a n i c o t i n e agonist (Givers and Rademacher, 1974). ( i i ) Mutual d i s p o s i t i o n of acceptor s i t e s on n i c o t i n i c receptors The a c t i v i t y of decamethonium (CIO; see XVI) as an agonist and a d e p o l a r i z i n g blocker seems to be r e l a t e d to the 1.4 nm interonium spacing found in t h i s molecule (Paton and Zaimis, 1948). Since the discovery of decamethonium, many other molecules c o n t a i n i n g inter-onium separations of 1.4 nm have been found to be agonists; f o r example, s u c c i n y l c h o l ine (XX). Another peak of agonist potency i s seen with molecules such as carbolinium ( X V I I I ) , s u b e r y l d i c h o l i n e (XIX), and hexadecamethonium (C16; see XVII) the i n t e r onium separation i n these compounds i s equivalent to 16 methylene groups or 2.0 nm. This has led to the concept of 'CIO' and 'C161 s t r u c t u r e s i n the r e c e p t o r , i . e . s t r u c t u r e s that recognize decamethonium- and .hexa-decamethonium-1ike compounds (Khromov-Borisov and Michelson, 1966), and has given r i s e to the idea that the s t r u c t u r a l s p e c i f i c i t y of n i c o t i n i c agonists may r e f l e c t the way i n which ACh acceptor s i t e s on the receptor are arranged r e l a t i v e to each other, i . e . t h e i r mutual d i s p o s i t i o n . Much of the o r i g i n a l work concerning the 'CIO' and 'C161 s t r u c t u r e has been published i n Russian and can be found i n a book by Michelson and Zeimal (1973). The information in the next few paragraphs, are taken from t h i s book and the o r i g i n a l a r t i c l e s are not c i t e d . - 54 -Decamethonium (XVI) and s u c c i n y l c h o l ine (XX) are about e q u a l l y potent i n the r a b b i t head droop t e s t f o r neuromuscular blockade. These agents cause a form of neuromuscular blockade that i s dependent on d e p o l a r i z a t i o n of the end-plate region (see al s o Zaimis and Head, 1976). Moreover, removal of one of the onium groups i n s u c c i n y l c h o l i n e (see XXI) causes a 100-fold drop i n potency. Even though s u c c i n y l c h o l ine (XX) and XXI have H-bond acceptor groups, these appear to play l i t t l e r o l e i n governing potency. Potency appears to be p r i m a r i l y dependent on the onium groups. These observations suggest that the H-bond acceptor group i n the AChR i s not ali g n e d with the CIO s t r u c t -ure. The space between the two an i o n i c s i t e s i n the CIO s t r u c t u r e appears to be a s y m e t r i c a l . Thus, adding one 8 methyl group to s u c c i n y l c h o l ine (see XXI) increases potency 3 0 - f o l d w h ile making the molecule symmetrical by adding another 6 methyl group (see XXII) reduces potency about 100-fold. A h i n t about the alignment of the group r e l a t i v e to the CIO s t r u c t u r e comes from compounds of the type XXIV where two ACh molecules are l i n k e d at the ammonium group by an alkane chain of n carbonyl res i d u e s . When n = 10, t h i s compound i s equal i n potency to decamethonium. Unlike the s i t u a t i o n with decamethonium analogues, shortening the polymethylene chain by 1 carbon (to n = 9 ) , or i n c r e a s i n g i t by one carbon (to n = 11), produce a d r a s t i c change i n a f f i n i t y . Again, t h i s suggests that the H-bond acceptor groups are not ali g n e d with the CIO s t r u c t u r e . The C16 s t r u c t u r e on the other hand does appear to be aligned with the anion i c s i t e s and H-bond acceptors i n groups of two ACh acceptor s i t e s . Carbolinium (XIX) i s much more potent than hexadecamethonium ( X V I I ) . Removing one of the ammonium groups i n carbolinium does not reduce potency - 55 -very much, presumably because the a d d i t i o n a l carboxyl oxygen maintains the appropriate alignment on the receptor. Barlow (1960) o r i g i n a l l y pointed,out that the C16 and CIO s t r u c t u r e s could represent the diagonal and the side of a tetrameric array of ACh acceptor s i t e s . However, at the time the only evidence i n favour of t h i s hypothesis was from the methonium compounds and he d i d not f e e l t h i s p o s s i b i l i t y was very l i k e l y . Later evidence that the C16 s t r u c t u r e d i d appear to be aligned with the c h o l i n o r e c e p t i v e u n i t (see X V I I I , XIX) led Khromov-Borisov and Michelson (1966) to r e v i v e t h i s i d e a . Khromov-Borisov (1979) suggested that when the receptor i s a c t i v a t e d the C16 s t r u c t u r e decreases from 2.0 to 1.2 nm and the C14 s t r u c t u r e decreases from 1.4 to 0.8 nm. In support of t h i s idea he has shown that a d e p o l a r i z i n g b l o c k e r , i . e . an agonist, with a 2.0 nm i n t e r onium distance (XXV) can be converted to a nondepolarizing antagonist by s l i g h t l y changing the s t r u c t u r e so that the molecule becomes r i g i d (XXVI). The only c l a s s of r i g i d bisonium agonists that has so f a r been described c o n s i s t s of analogues of 3,3'-bis [a-(trimethylammonium)- methyl] azobenzene simply, or Bis Q (XXVII). This i s a symmetrical and planar molecule (see Wasserman et a l . , 1979) with a v a r i e t y of isomers; the trans c o n f i g u r a t i o n of the 3,3' isomer i s the most potent of these. The potency of Bis Q analogues i s very s e n s i t i v e to the c o n f i g u r a t i o n of the molecule; the c i s c o n f i g u r a t i o n i s 100 times less potent than the t r a n s . In both halves of t h i s molecule the distance between the onium group and the electonegative azo nitrogen i s 0.5 nm. In order to i n t e r a c t with the C16 s t r u c t u r e the onium and azo nitrogens of both halves have to be a l i g n e d , which only occurs i n the trans c o n f i g u r a t i o n . In t h i s c o n f i g u r a t i o n the interonium distance i s 1.1 nm, which i s close to the distance (1.2 nm) p r e d i c t e d by the hypothe-s i s of Kromov-Borisov (1979). - 56 -Bis Q has the i n t e r e s t i n g property that the rat e s of in t e r c o n v e r s i o n between the c i s and trans c o n f i g u r a t i o n i s p h o t o s e n s i t i v e . The photosensi-t i v e property of Bis Q has been used by Lester and h i s colleagues to produce the equivalent of a near instantaneous jump i n the agonist c o n c e n t r a t i o n . The subsequent response r e l a x a t i o n allows k i n e t i c a n a l y s i s of the agonist receptor i n t e r a c t i o n (Lester et a l 1980b). ( i i i ) A f f i n i t y l a b e l s f o r the acceptor s i t e Many pr o t e i n s contain d i s u l f i d e bonds which can be reduced by d i t h i o -t h r e i t o l (DTT) and a l k y l a t e d by N-ethyl maleimide (XXVIII). These agents i n f l u e n c e the ac t i o n of ACh on the n i c o t i n i c receptor (see chapter 3, sec-t i o n b). Karl i n (1969) synthesized quaternary ammonium maleimide d e r i v a -t i v e s i n the hope th a t a r e a c t i v e s u l f y d r y l group might be i n the v i c i n i t y of the ACh binding s i t e so that such agents could be used as a f f i n i t y l a b e l s . He found that one of these compounds 4-(N-maleimide) b e n z y l t r i -methy1 ammonium (XXIX; MBTA) reacted on average 1000X f a s t e r with a reduced d i s u l f i d e i n the region of the binding s i t e than with other p r o t e i n s u l f y -d r y l groups. Conversely N-ethyl maleimide reacted with the AChR 1000X slow-er than MBTA. The t e r t i a r y d e r i v a t i v e of MBTA (see XXX) was no more e f f i c a -cious than N-ethyl maleimide and the r e a c t i o n of MBTA with the receptor was completely blocked by a-BuTX. A l l of these data suggest that MBTA i s a c t i n g as an a f f i n i t y l a b e l . K a r l i n (1969, 1973) conluded that i t was s e l e c t i v e l y l a b e l l i n g a d i s u l f i d e bond i n the v i c i n i t y of the ACh binding s i t e . MBTA i s a p a r t i a l agonist. A f t e r i n t e r a c t i o n with the receptor and removal of f r e e MBTA, the bound MBTA can s t i l l open channels. This a c t i o n i s expressed as an a d d i t i o n a l conductance that can be blocked by dTC (prob-- 57 -ably as the r e s u l t of i t s 'channel plugging' a c t i o n ; see Lester et a l . , 1980a) and a d d i t i o n a l ' n o i s e 1 . The noise i s unusual i n that the power spectrum i s b i p h a s i c and the s i n g l e channel conductance i s q u i t e small (see Cox et a l . , 1980). The a b i l i t y to produce such ' i r r e v e r s i b l e a c t i v a t i o n ' a l s o occurs with agents l i k e bromoacetylcholine (XXXII) where the distance between the r e a c t i v e carbon atom and the quaternary ammonium group i s 0.66 nm. Adding a methylene group between the quaternary nitrogen and the maleimide moiety of MBTA produces an i r r e v e r s i b l e antagonist (XXXI) with a 0.9 nm separation between the r e a c t i v e carbon atom and the quaternary amonium. K a r l i n (1973) pointed out that i n a s e r i e s of a f f i n i t y a l k y l a t i n g agents, the optimum separation was about 0.6 nm f o r agonist a l k y l a t o r s and 0.9 nm f o r antagonist a l k y l a t o r s . A l l of t h i s data i s c o n s i s t e n t with the a c t i v a t i o n of the receptor i s associated with a shortening of the acceptor region of the receptor (see Kromov-Borisov, 1979). In the case of t h i s c l a s s of compounds, the exact s i g n i f i c a n c e of these distances i s uncertain since the receptor has to be pretreated with DTT, before the receptor can be a l k y l a t e d . This i s known to change the binding c h a r a c t e r i s t i c s of the receptor and the apparent dimensions of the C10 s t r u c t u r e . Thus, hexamethonium i s an antagonist before treatment with DTT and a p a r t i a l agonist a f t e r exposure to DTT. In f a c t , with the methonium s e r i e s of compounds DTT changes the a c t i v i t y as though each of these compounds was lengthened by one methylene group (Rang and R i t t e r , 1971). The f a c t that bromoacetylchol ine i s a good a l k y l a t o r suggests that the r e a c t i v e s u l f h y d r y l group i s c l o s e to the hydrogen-bond acceptor portion of the ACh b i n d i n g s i t e . At f i r s t glance, another good agonist a l k y l a t o r , QBr (XXXIII) seems to be an exception , i t i s most a c t i v e i n the trans c o n f i g u r -- 58 -a t i o n where the distance between the r e a c t i v e carbon and the quaternary nitrogen i s 1.0 nm. However, i t i s an analog of Bis Q which, as discussed above, probably i n t e r a c t s with the C16 s t r u c t u r e . Hence, i t may be able to i n t e r a c t with the a n i o n i c s i t e i n one h a l f of the C16 s t r u c t u r e , and the r e a c t i v e s u l f h y d r y l i n the other h a l f . ( i v ) Competitive antagonists that act at the acceptor s i t e of the n i c o t i n i c  receptor Many of the c l i n i c a l l y used nondepolarizing neuromuscular blockers are b i s onium s a l t s with an i n t e r onium distance of about 1.1 nm. This i s true of dTC, pancuronium, t o x i f e r i n e I and I I , and gallamine (Pauling and Fetcher, 1973). There are exceptions, f o r example: tercuronium, 1.4 nm (see Khromov-Borizov, 1979); stercuronium, 1.01 nm, ( B u s f i e l d , C h i l d s , C l a r k , Davis and Dodds, 1968); alocuronium, 0.97 nm and HH-8165, 0.75 nm ( P o i n t e r , W i l f o r d and Bishop, 1972). Moreover, i n the case of dTC, pancuronium and stercuronium one of the onium groups can be t e r t i a r y without much loss of a c t i v i t y (see Stenlake, 1979). Some compounds such as norcoralydine methio-dide contain only one onium group yet are only s l i g h t l y l e s s potent than dTC. Stenlake (1979) has compared the a c t i v i t i e s of isomers of n o r c o r a l y -dine and dTC and has concluded that the t e r t i a r y nitrogen of dTC i s i n the most favoured p o s i t i o n to bind to the r e c e p t o r . Thus, i s o t u b o c u r a r i n e , where the p o s i t i o n s of the t e r t i a r y and quaternary amonium groups are the reverse of dTC, i s two times more potent than dTC and i s equipotent with the bisquaternary compound chondocurarine S i m i l a r l y , i n the case of pancuronium analogues, although the second nitrogen i s e s s e n t i a l f o r a c t i v i t y the lack of a second quaternary group does not a f f e c t potency (Buckett, Hewett and Savage, 1973). The newly - 59 -introduced t e r t i a r y d e r i v a t i v e of pancuronium, norcuron (ORG NC-45), i s about twice as potent as pancuronium i n humans (Fahey, M o r r i s , M i l l e r , Sohn, Crownelly and G o n c a r e l l i , 1981) or on the i s o l a t e d r a t diaphragm preparation (see Lee-Son, Waud and Waud, 1981). The potency i s increased during r e p i r a -t o r y a c i d o s i s ( M a r s h a l l , Agoston, B o o i j , Durant and Foldes, 1980) suggesting that at p h y s i o l o g i c a l pH the second nitrogen i s not completely protonated. Thus, i t i s u n l i k e l y that the second nitrogen i s i n t e r a c t i n g with an anio n i c s i t e s i m i l a r to that i n the main ACh acceptor s i t e since as mentioned above, dimethylamino e t h y l a c e t a t e i s 100 times l e s s potent than ACh even i n the f u l l y protonated form. B e r y t h r o i d i n e , l i k e n o r c o r a l y d i n e , has only one onium group. I t i s notable that the apparent a f f i n i t y of e e r y t h r o i d i n e increases with concen-t r a t i o n . This has been taken to i n d i c a t e that two an i o n i c s i t e s have to be occupied to produce blockade (Van Maanum, 1950). However, an e q u a l l y l i k e l y explanation i s that t h i s antagonist has an ac t i o n on the e f f e c t o r p o r t i o n of the receptor (see chapter 4, s e c t i o n d , i i i ) . Though the interonium spacing may not be c r i t i c a l the b i s onium s t r u c -t u r e does appear to play some r o l e i n producing a nondepolarizing competi-t i v e blockade. Thus, hexamethonium i s about as potent as h e p t y l t r i m e t h y l -ammonium (Adams, 1975b) or f o r that matter tetramethylammonium i n a s s o c i a t i n g with the receptor but hexamethonium i s a competitive antagonist (see Paton and Zaimis, 1948) while hexyltrimethylammonium i s a p a r t i a l agonist (Adams, 1975b). The two c a t i o n i c groups at e i t h e r end of the bis-onium compound may serve to o r i e n t the interonium group along the surface of the hydrophytic surface of the receptor and keep the spacing groups of the molecule out of tro u b l e ( i . e . out of the way of conformational changes that occur during channel a c t i v a t i o n ) . - 60 -v) Appendix (1) S t r u c t u r a l formulae of drugs that i n t e r a c t with the AChR acceptor s i t e 0 II I. ( C H 3 ) 3 N C H 2 C H 2 0 C — C H 3 0 II I I . (CHg^C C H 2 C H 2 0 C — C H 3 0 1/ I I I . CH 3 CH 2 0 C CH 3 IV. (C^J^N - 61 -0 + II V. ( C H 3 ) 3 N C H 2 C H 2 0 C — N H 2 VI. (CHgJgN CH 2 CH 2 0 CH 2 CH 3 V I I . (CH-^N CH 2 CH 2 — OH V I I I . ( C ^ N CH 2 CH 2 CH 2 — CH 3 - 62 -IX. (CH3)*NH — CH 2 CH 2 0 C CH 3 0 X. ( C H 3 ) 3 N — C H 2 C H 2 NH — C CH 3 XI. (CH 3) 2N -CH-CH, CH, N -I CH-// C CH-XII. (CH 3) 2N \ CHo " CH-CHo CH-N C CH-j - 63 -XIII. , 0 . 5 9 nm XVI. ( CV3 N XVII. < C H 3)3 N 0 + II XV I I I . (CH ) N CH CH 2 0 C - N.. 0 X I X * (CH-^N CH 2 CH 2 0 C ( C H 2 ) 1 0 _ N + ( C H 3 ) 3 ( C H 2 ) 1 6 - N + ( C H 3 ) 3 ( C H 2 ) g - NH - C 0 CH 2 CH 2  N + ( C H 3 ) 3 CTl ( C H 2 ) 6 0 II C 0 CH 2 CH 2 N ( C H 3 ) 3 ( eH0) N ZH3 H3 0 3 ZH3 + II 0 £ ( e H 3 ) N ZH3 ZH3 0 3 2H3 II 0 i 2 £ ( e H 3 ) N ZH3 ZH3 0 3 ZH3 II 0 e ( e H 3 ) + N 2H3 0 II 0 £H3 2H3 3 0 H3 ZH3 N £( £H3) 'IIIXX tH3 ZH3 3 0 H3 ZH3 HZ{£H3) +v ' *IIXX 0 ZH3 3 0 ZH3 £H3 *IXX 2H3 3 ^ - 0 ZH3 ZH3 HZ{£H3) .YY II + 0 \CH N (CH ) 2 3 } - 68 -XXVIII. 0 \\ CH CH // XXIX, 0 w xxx, (CH ) NH 3 2 0 \\ r V // XXXI (CH ) N CH 3 3 2 W r // XXXII. (CH ) N CH CH — 0 C CH Br 3 3 2 2 2 - 69 -c) Pharmacological phenomena i n v o l v i n g the intermediary component of the receptor ( i ) Preface Between the primary acceptor s i t e on the receptor and the u l t i m a t e e f -f e c t o r component ( i . e . the channel), there must e x i s t a p o r t i o n of the receptor that t r a n s m i t s , to the e f f e c t o r component, information about the status of the acceptor s i t e ( i . e . whether or not i t i s occupied by an ago-n i s t ) . In the case of the ACh receptor simple occupancy of the acceptor s i t e , even by f u l l a g onist, does not always lead to a u n i t response by the e f f e c t o r . I t has already been pointed out that the ACh receptor has two main agonist binding s i t e s both of which have to be occupied in order to induce the e f f e c t o r component to produce a response. This phenomenon i s known as c o o p e r a t i v i t y . With prolonged exposure to n i c o t i n i c a g o n i s t s , the acceptor s i t e can become uncoupled from the e f f e c t o r component and the response to agonist fades. This phenomenon i s known as d e s e n s i t i z a t i o n . ( i i ) D e s e n s i t i z a t i o n of the ACh receptor by n i c o t i n i c - agonists (1) Fast and slow d e s e n s i t i z a t i o n . I t i s now apparent that d e s e n s i t i z -a t i o n develops i n at l e a s t two stages ( F e l t z and Trautmann, 1980; Clark and Adams, 1981). There i s f a s t d e s e n s i t i z a t i o n that develops w i t h i n seconds and perhaps even f r a c t i o n s of seconds a f t e r exposure to agonist, and slow d e s e n s i t i z a t i o n which develops over a period of seconds to minutes, depend-ing on the type and concentration of the agonist. i - 70 -With bath a p p l i c a t i o n of agonist slow d e s e n s i t i z a t i o n has an onset at a r a t e of about 500/M/s (Adams, 1975a) with a maximum r a t e of 0.2/s (see Adams, 1981); f a s t d e s e n s i t i z a t i o n develops at a rate about 20 times f a s t e r ( F e l t z and Trautmann, 1980). Recovery from slow d e s e n s i t i z a t i o n takes place with a time constant of around 3 min and i s independent of agonist concen-t r a t i o n or l e v e l of d e s e n s i t i z a t i o n (Rang and R i t t e r , 1970; Scubon-Mulieri and Parsons, 1977). There i s also a f a s t phase of recovery with a time constant of about 3 s (Adams, 1975a) which may represent recovery from the f a s t d e s e n s i t i z e d s t a t e . With i o n t o p h o r e t i c a p p l i c a t i o n of agonist the time constant of recovery i s a l s o several seconds (see Katz and T h e s l e f f , 1957). Recently, Sakmann, Patlak and Neher (1980) have provided more d i r e c t evidence f o r f a s t and slow d e s e n s i t i z a t i o n using the patch clamp technique which allows the r e s o l u t i o n of square pulses of current that are thought to r e f l e c t the opening and c l o s i n g of i n d i v i d u a l channels coupled to recept-o r s . In the e x t r a j u n c t i o n a l areas of the muscle membrane, patches c o n t a i n -ing a s i n g l e channel can be examined and i n 10 percent of these patches the channel has pr o p e r t i e s s i m i l a r to those of j u n c t i o n a l receptors. With low concentrations of ACh channel opening i s randomly d i s t r i b u t e d i n time. How-ever, i n the presence of d e s e n s i t i z i n g concentrations of ACh (5-50 yM), channels opening occurs i n bu r s t s . At 20 pM ACh, -130 mV and 11°C, during bursts the average duration of i n d i v i d u a l channel openings was 10 ms and equal to the average i n t e r v a l between channel opening. Under these same cond i t i o n s the bursts themselves had a mean duration of about 500 ms and an i n t e r b u r s t i n t e r v a l of about 200 ms. Thus, an i n d i v i d u a l receptors could be, on average, a c t i v a t e d about 25 times before converting i n t o a r e f r a c t o r y s t a t e . The b u r s t s , i n t u r n , occurred i n c l u s t e r s with a mean duration of about 5 seconds and mean i n t e r c l u s t e r i n t e r v a l of about 30 seconds. Thus, - 71 -f a s t and slow r e f r a c t o r y s t a t e s , presumably corresponding to f a s t and slow d e s e n s i t i z a t i o n , could be detected at the l e v e l of i n d i v i d u a l r e c e ptors. The existence of f a s t d e s e n s i t i z a t i o n may e x p l a i n the lack of agreement between the maximal responses to n i c o t i n i c agonists as measured by several authors and the value expected from the number of a-BuTX s i t e s present on the c e l l and from s i n g l e channel conductance measurements. Dreyer, Peper and Sterz (1978) estimated the maximal response i n d i r e c t l y using a q u a n t i t a -t i v e iontophoresis approach. In the f r o g , the motor nerve terminal branches are not t i g h t l y clumped, rather they are spread out i n a few r e l a t i v e l y long (300 um) s t r i p s along the muscle f i b e r . By p o s i t i o n i n g an i o n t o p h o r e t i c electrode at a known p o s i t i o n over the end of one of these branches, the d i s t r i b u t i o n of i o n t o p h o r e t i c a l l y released ACh along the branch can be c a l c u -l a t e d from d i f f u s i o n equations. One of the constants that can be estimated from these equations i s the maximal number of a c t i v a t e d receptors per ym of nerve t e r m i n a l . With b r i e f i o n t o p h o r e t i c pulses (5 ms) the response reached a peak w i t h i n 100 ms; the estimated maximal response to ACh was 170 nS/ym. With carbachol the maximum was s l i g h t l y l e s s , 150 nS/ym. Assuming a s i n g l e channel conductance of 25 pS and 2.7 ym of subsynaptic membrane per ym of nerve terminal branch (see Matthews-Bellinger and S a l p e t e r , 1978) t h i s i s equi v a l e n t to a maximal open channel d e n s i t y of about 2500/yirr or 1/8 of the estimated d e n s i t y of a-BuTX s i t e s (see chapter 1). Dionne, Steinbach and Stevens (1978) using a s i m i l a r approach but longer (up to 5 s) a p p l i c a -t i o n of agonists obtained a value of only 54 nS/ym or 800 channels/ynr. Adams (1975a) stu d i e d responses to bath a p p l i e d agonist that developed - 72 -w i t h i n a f e w s e c o n d s . Adams e s t i m a t e d a maximum r e s p o n s e o f 8 0 y S f o r t h e e n t i r e e n d - p l a t e . A g a i n a s s u m i n g a s i n g l e c h a n n e l c o n d u c t a n c e o f 25 pS t h i s r e p r e s e n t s t h e a c t i v a t i o n o f 3 . 2 x 10^ r e c e p t o r s o r 1/12 t h e e x p e c t e d number ( s e e M a t t h e w s - B e l l i n g e r and S a l p e t e r , 1 9 7 8 ) . I t i s u n l i k e l y t h a t t h e s m a l l r a t i o s o f a c t i v a t e d c h a n n e l s t o a - B u T X s i t e s c a n be e x p l a i n e d b y s l o w d e s e n s i t i z a t i o n s i n c e t h i s d e v e l o p s a t a m a x i m a l r a t e o f 0 . 2 / s ( s e e A d a m s , 1 9 8 1 ) . L e s t e r , K o b l i n and S h e r i d a n ( 1 9 7 8 ) i n v e s t i g a t e d t h e d o s e r e s p o n s e r e l a t i o n s h i p f o r ACh a c t i o n on a s i n g l e c e l l i s o l a t e d f o r m t h e e e l e l e c t r o -p l a q u e and v o l t a g e - c l a m p e d t r a n s c e l l u l a r l y . T h e s e w e r e h e l d b e t w e e n two c h a m b e r s s o t h a t t h e h o l d i n g c u r r e n t p a s s e d t h r o u g h t h e c e n t e r , n o n - i n n e r v a -t e d f a c e and 3mm o f t h e i n n e r v a t e d f a c e ( s e e S h e r i d a n and L e s t e r , 1 9 7 7 ) . T h e y a t t e m p t e d t o m i n i m i z e d e s e n s i t i z a t i o n b y h o l d i n g t h e t r a n s m e m b r a n e p o t e n t i a l a t a p o s i t i v e v a l u e (+40 mV) w h i l e t h e a g o n i s t was w a s h i n g i n t o t h e b a t h , t h e n ' j u m p i n g ' t o more n e g a t i v e p o t e n t i a l s ( u p t o - 1 7 5 mV) t o m e a s u r e a r e s p o n s e . The" c o n d u c t a n c e ( g ) c h a n g e d w i t h ACh c o n c e n t r a t i o n , [ A C h ] , s u c h t h a t 1/Vg* was l i n e a r w i t h l / [ A C h ] s u g g e s t i n g a c o n c e r t e d model o f c o o p e r a t i v i t y ( s e e c h a p t e r 4 , s e c t i o n c , i i i ) . The a p p a r e n t d i s s o c i a t i o n c o n s t a n t ( c a l c u l a t e d a s s u m i n g a c o n c e r t e d m o d e l ) was 5 0 yM a t - 8 5 mV. T h i s was i n good a g r e e m e n t w i t h t h e d i s s o c i a t i o n c o n s t a n t p r e d i c t e d f r o m t h e r a t i o o f t h e r a t e s o f o p e n i n g and c l o s i n g o f c h a n n e l s e s t i m a t e d f r o m v o l t a g e jump r e l a x a t i o n s ( S h e r i d a n and L e s t e r , 1 9 7 7 ) . The maximum r e s p o n s e was 1 3 0 ci 2 -mS/cm . In t h e e e l e l e c t r o p l a q u e , a b o u t 2 p e r c e n t o f t h e p o s t s y n a p t i c membrane i s s u b s y n a p t i c w i t h a d e n s i t y o f N a j a N a j a t o x i n b i n d i n g s i t e s o f 5 x 10 / y m . T h e e x t r a s y n a p t i c r e c e p t o r d e n s i t y i s 4 x 10 /ym , t h u s t h e r a t i o o f s u b s y n a p t i c t o e x t r a s y n a p t i c r e c e p t o r s i s 1 . 5 ( B o u r g o i s e , Popot, Rytan and Changeux, 1978). These values add up to about 3 x 10 a-toxin s i t e s per c e l l , a number which agrees we l l with other estimates. D i v i d i n g the maximum current by the number of channels (assuming two t o x i n binding s i t e s per channel) gives a s i n g l e channel conductance of about 2 pS or 1/10th of the value determined by d i r e c t measurement. Lester et a l . (1978) a l s o measured the maximum current that could be e l i c i t e d by nerve s t i m u l a t i o n (with frequency f a c i l i t a t i o n i n the presence of B a + + ) . This was g e n e r a l l y about 1.2 times the maximal response produced by bath a p p l i c a t i o n of ACh. Since presumably t h i s only r e f l e c t s a c t i v a t i o n of subsynaptic r e c e p t o r s , and probably not a l l subsynaptic recep-t o r s at that ( i . e . « 60 percent of t o t a l r e c e p t o r s ) , t h i s experiment i n d i -cates that in s p i t e of t h e i r precautions there was probably some f a s t desen-s i t i z a t i o n whith bath a p p l i c a t i o n of agonist. Thus, the maximal response that develops w i t h i n seconds of exposure to agonist and i n the absence of appreciable slow d e s e n s i t i z a t i o n has generaly been found to be about 10 percent of that expected from the numbers of receptors present. I f t h i s i s due to f a s t d e s e n s i t i z a t i o n then i t may i n d i -cate that in the presence of a n i c o t i n i c agonist the e q u i l i b r i u m between the undesensitized and f a s t d e s e n s i t i z e d s t a t e i s such that when maximum f a s t d e s e n s i t i z a t o n has developed 10 percent of the receptors remain in the unde-s e n s i t i z e d s t a t e . Fast d e s e n s i t i z a t i o n and the d e s e n s i t i z a t i o n observed when agonist i s applied i o n t o p h o r e t i c a l y can, at low agonist c o n c e n t r a t i o n s , have an onset slower than the o f f s e t (see Katz and T h e s l e f f , 1957b). Hence, one can con-clude that the presence of agonist can slow the recovery from f a s t desensi-t i z a t i o n . This becomes apparent when simple models of d e s e n s i t i z a t i o n are - 74 -considered. I f a c t i v a t a b l e receptor ( R ) i s i n e q u i l i b r i u m with desensi-t i z e d receptor ( R' ) and the r a t e of formation of R1 i s a f u n c t i o n of agon-i s t c o n c e n t r a t i o n , f ( A ) ; k o n f ( — ) k o f f Then, f o l l o w i n g a jump i n agonist concentration R'(t) = R ( 0 ) [ l - e x p ( - t / T)] Where T, the time constant of the system, i s < ko„ f< * ' + k o f f r 1 The only way f o r the time constant of d e s e n s i t i z a t i o n to be greater i n the presence of A than i n the absence of A i s f o r k ^ to be reduced by A. Katz and T h e s l e f f (1957b) pointed out that the f o l l o w i n g c y c l i c model i s one of the simplest models i n which t h i s i s the case. K l A + R \ * AR -2 A + R' ^ * AR' K 3 To account f o r f a s t d e s e n s i t i z a t i o n with t h i s model i t i s necessary t o assume t h a t : 1) d e s e n s i t i z a t i o n i s associated with an increase i n the a f f i -n i t y of the receptor f o r agonist (K^ > K 3),, 2) the presence of agonist on the receptor favours the i s o m e r i z a t i o n to the d e s e n s i t i z e d s t a t e (k^ > k ^ ) , and 3) the absence of agonist on the receptor favours the isomeriz-ation to the non-desensitzed s t a t e (k_^ > k_ 2) - 75 -With slow d e s e n s i t i z a t i o n by f u l l agonist such as ACh or carbachol the data i s more equivocal as to whether or not the onset r a t e can be l e s s than the o f f s e t r a t e (Rang and R i t t e r , 1970; Adams, 1981). This would suggest that k_2 = k_ 4 (see Boyd and Cohen 1980a). Nevertheless there i s b i o -chemical evidence suggesting that the c y c l i c model i s a l s o a p p l i c a b l e to slow d e s e n s i t i z a t i o n . (2) Biochemical measures of d e s e n s i t i z a t i o n . E a r l y attempts to measure the a f f i n i t y of n i c o t i n i c receptors f o r agonists using techniques such the displacement of r a d i o l a b e l e d ligands suggested that agonists such as carba-chol and ACh could bind to n i c o t i n i c receptors with q u i t e high a f f i n i t i e s ; 0.02 uM and 0.5 yM r e s p e c t i v e l y (Cohen and Changeux, 1973). I t was recogn-ized that such a high a f f i n i t y f o r ACh was incompatible with the time course of postsynaptic p o t e n t i a l s generated by nerve s t i m u l a t i o n , and the sugges-t i o n was made that i t was the a f f i n i t y of d e s e n s i t i z e d receptor that was in f a c t being measured. This was subsequently confirmed by measuring the e f -f e c t of duration of exposure to agonist on the i n i t i a l r a t e of binding of a-BuTX. This technique can generate estimates of receptor occupancy with r e l a t i v e l y short incubation times (= 15 s ) . Weber, David-Pfeuty and Chan-geux (1975) showed that i f fragments from Torpedo el e c t r o p l a q u e were p r e i n -cubated with agonist f o r several minutes, the a f f i n i t y derived from the a b i l i t y of agonist to slow the i n i t i a l r a t e of b i n d i n g of a-BuTX was the same as the a f f i n i t y derived from the displacement of r a d i o l a b e l e d l i g a n d . However, i f there was no period of preincubation much lower a f f i n i t i e s were found. - 76 -Wei land and Tay l o r (1979) have determined that the change i n the a b i l i t y 1 pc of agonist to compete with the i n i t i a l r a t e of ( I ) l a b e l e d a-BuTX bind -ing can be f u l l y accounted f o r by the c y c l i c model of d e s e n s i t i z a t i o n . Sine and Taylor (1979) found very s i m i l a r r e s u l t s with the nonfusing c u l t u r e d muscle c e l l l i n e , BC3H-1, with which they were able to measure simultaneous-00 l y the change i n Na p e r m e a b i l i t y produced by a c t i v a t i o n of re c e p t o r s . They showed that the change i n a f f i n i t y did i n f a c t p a r a l l e l the conversion of a c t i v a t a b l e receptor to nonactivatable receptor. In both Torpedo mem-branes and BC3H-1 c e l l s t h i s conversion developed at a r a t e of 500/M/s and recovered at a r a t e of 0.01/s. These rates are very s i m i l a r to those seen f o r slow d e s e n s i t i z a t i o n of the conductance response at the f r o g end-plate (see Adams, 1981). Thus, i t i s l i k e l y that the increase in a f f i n i t y of the receptor f o r agonist r e s u l t s from the same s t a t e t r a n s i t i o n as d e s e n s i t i z a -t i o n . Sine and Taylor (1979, 1980) noted that even with complete d e s e n s i t i z a -t i o n there remained a response equal to 10-20 percent of the maximum. The same r e s i d u a l response i s al s o seen at the voltage clamped fro g end-plate, i . e . even with high concentrations and 'complete' d e s e n s i t i z a t i o n there remains a plateau response equivalent to 5-10 percent of the peak response, which i s in turn i s 10 percent of that expected from the numbers of recep-t o r s present. In the study of Sine and Taylor (1980) the maximal Na i n f l u x r a t e was 2 percent that expected from the den s i t y of a-BuTX binding s i t e s on the c u l t u r e d muscle c e l l s . As with f a s t d e s e n s i t i z a t i o n t h i s may i n d i c a t e that i n the steady s t a t e the r a t e constants are such that a s i g n i -f i c a n t f r a c t i o n of normal receptors remain, even at maximal d e s e n s i t i z a -t i o n . A l t e r n a t i v e l y , d e s e n s i t i z e d receptors might r e t a i n some a b i l i t y to - 77 -o p e n i o n i c c h a n n e l s . I f t h i s i s t h e c a s e , t h e s i n g l e c h a n n e l c o n d u c t a n c e c o u l d b e a s l o w as 1 p e r c e n t o f n o r m a l . B o t h p o s s i b l i t i e s seem u n l i k e l y s i n c e s i n g l e c h a n n e l c o n d u c t a n c e e s t i m a t e d f r o m n o i s e a n a l y s i s i s u n c h a n g e d b y d e s e n s i t i z a t i o n ( s e e A n d e r s o n and S t e v e n s , 1 9 7 3 ) e v e n t h o u g h t h e e x i s t e n c e o f v e r y s m a l l c h a n n e l s w o u l d i n c r e a s e t h e mean r e s p o n s e w i t h o u t a f f e c t i n g t h e v a r i a n c e o f t h e ' n o i s e ' . Boyd and Cohen ( 1 9 8 0 a , b ) h a v e m e a s u r e d t h e a g o n i s t i n d u c e d c h a n g e i n 3 3 a f f i n i t y d i r e c t l y b y m e a s u r i n g t h e k i n e t i c s o f [ H] ACh and [ H] c a r b a -c h o l b i n d i n g t o r e c e p t o r e n r i c h e d T o r p e d o e l e c t r o p l a x m e m b r a n e s . T h i s was d e t e r m i n e d u s i n g an u l t r a f i l t r a t i o n a s s a y c o u p l e d w i t h an a p p a r a t u s f o r r a p i d m i x i n g f o l l o w e d b y q u e n c h i n g o f b i n d i n g . M e a s u r e m e n t c o u l d be made w i t h i n 4 0 0 msec o f m i x i n g a g o n i s t and r e c e p t o r . T h e y f o u n d t h a t 2 0 p e r c e n t o f t h e r e c e p t o r i n t h e s e membranes p r e - e x i s t e d i n t h e h i g h a f f i n i t y s t a t e e v e n i n t h e a b s e n c e o f a g o n i s t . Upon m i x i n g t h e membranes w i t h v e r y l o w c o n c e n t r a t i o n o f a g o n i s t ( 5 - 5 0 nM ACh) t h i s p o p u l a t i o n o f r e c e p t o r s became r a p i d l y a s s o c i a t e d w i t h a g o n i s t . The b i m o l e c u l a r a s s o c i a t i o n c o n s t a n t f o r t h i s p r o c e s s was 5 x 1 0 ^ / M / s and l i n e a r w i t h a g o n i s t c o n c e n t r a t i o n ( i . e . a p p a r e n t l y n o t c o o p e r a t i v e ) . The d i s s o c i a t i o n c o n s t a n t was 2 . 5 n M . W i t h p r o l o n g e d e x p o s u r e t o a g o n i s t a l m o s t a l l o f t h e r e c e p t o r s w e r e c o n v e r t e d t o a h i g h a f f i n i t y s t a t e , i n d i c a t i n g t h a t a t e q u i l i b r i u m f e w i f a n y n o r m a l r e c e p t o r s r e m a i n . S c a t c h a r d p l o t s i n d i c a t e d t h a t t h e s e h i g h a f f i n i t y r e c e p -t o r s a r e h a l f o c c u p i e d a t 8 nM f o r ^ACh and w e r e p r e s e n t i n a c o n c e n t r a t i o n i d e n t i c a l t o t h e t o t a l number o f a - B u T X b i n d i n g s i t e s . A t low a g o n i s t c o n -c e n t r a t i o n s t h e r a t e o f a c c u m u l a t i o n o f b o u n d ACh on t h e s e h i g h a f f i n i t y r e c e p t o r s w i l l r e f l e c t a m o p p i n g up o f t h e s e h i g h a f f i n i t y s i t e s as t h e y o c c u r s p o n t a n e o u s l y . I n t e r m s o f t h e c y c l i c m o d e l ( s e e a b o v e ) t h e r a t e o f - 78 -accumulation w i l l r e f l e c t the i s o m e r i z a t i o n between R and R1 and w i l l have a l i m i t i n g r a t e equal to k 4 + k_ 4; since k 4 » k_ 4, t h i s r a t e w i l l be s * 4 . The l i m i t i n g r a t e observed at lOnM ACh was 0.0035/s at 4°C f o r both ACh and carbachol. At higher concentrations (> 50 nM f o r ACh ;> 1 pM f o r carbachol) the accumulation r e f l e c t s generation of d e s e n s i t i z e d receptor by agonist and occurred at a r a t e of 4000/ M/ s f o r ACh and 400/ M/ s f o r car-bachol. This compares very w e l l with the onset r a t e s of slow d e s e n s i t i z a -t i o n measured e l e c t r o p h y s i o l o g i c a l y of 3000/ M/ s f o r ACh (Wray 1981) and 500/ M/ s f o r carbachol (Adams 1975a). When di s p l a c e d by high concentrations of c o l d l i g a n d ACh d i s s o c i a t e d from the high a f f i n i t y receptor at a rate of 0.04/s at 4°C and 0.23/ s at 23°C. This was independent of the concentration or type of d i s p l a c i n g l i g a n d ( e i t h e r ACh, carbachol or dTC). The high a f f i n i t y s t a t e r e l a x e d back to the normal s t a t e at a r a t e of 3 x 10~ 3/s at 4°C and. 9 x 10~ 3/s at 23°C. This r a t e should equal k^ + k_ 4 and as expected was almost iden-t i c a l to the r a t e of accumulation of the high a f f i n i t y s t a t e with low agonist c o n c e n t r a t i o n . At 23°C the r a t e was c l o s e to the r a t e of recovery from slow d e s e n s i t i z a t i o n measured e l e c t r o p h y s i o l o g i c a l y (see Scubon-Meulie-r i and Parsons, 1978). The f a c t that the rate of recovery i s much l e s s than the r a t e of d i s s o c i a t i o n of ACh from the d e s e n s i t i z e d receptor i s most e a s i l y explained by the c y c l i c model. Moreover, by f i t t i n g the c y c l i c model to the data, Boyd and Cohen (1980a) estimated that the low a f f i n i t y form had an ACh d i s s o c i a t i o n constant of 1 pM and a carbachol d i s s o c i a t i o n constant of 30 pM. This i n turn suggested that k^ and k_^ were .2/s and 0.5x10 /s r e s p e c t i v e l y f o r both ACh and carbachol. Again these values agree well with those expected from e l e c t r o p h y s i o l o g i c a l data on slow desen-- 79 -s i t i z a t i o n ; the rates are independent of the type of agonist and the maxi-mum r a t e of slow d e s e n s i t i z a t i o n ( i . e . k,, + k_ 2) i s about 0.2/ s (see Adams, 1981). With the r a p i d mixing technique, Boyd and Cohen (1980b) were able to determine that 1 pM ACh accumulates on the low a f f i n i t y receptor with a r a t e of 2/s. This r a t e i s s i m i l a r to that observed f o r the r a p i d quenching of pr o t e i n fluorescence by pM concentrations of ACh (Bonner, Barrantes and Jovin 1976) and the r a p i d increase i n quinacrine or ethidium fluorescence produced by c h o l i n e r g i c agonists (Grunhagen, Iwatsubo and Changeux, 1977; Quast, Schemerlik and Raftery , 1979). But the r a t e measured by Boyd and Cohen (1980b) i s much slower than the r a t e of development of increased Na + p e r m e a b i l i t y measured i n the same apparatus and the same preparation (see Neubig and Cohen, 1980). Thus, i t i s l i k e l y that t h i s 'low a f f i n i t y ' s t a t e does not represent the a c t i v a t a b l e form of the receptor and may represent the f a s t d e s e n s i t i z e d s t a t e . As with the high a f f i n i t y s t a t e the r a t e of accumulation of the 'low a f f i n i t y ' or f a s t d e s e n s i t i z e d s t a t e w i l l r e f l e c t the spontaneous develop-ment of the f a s t d e s e n s i t i z e d s t a t e and should approximate the rate at which the f a s t d e s e n s i t i z e d s t a t e r e v e r t s back to the normal s t a t e . The r a t e of 2/s i s in agreement with e l e c t r o p h y s i o l o g i c a l data (see Sakmann et a l . 1980). (3) Molecular mechanisms of f a s t and slow d e s e n s i t i z a t i o n . The two a-BuTX binding s i t e s associated with the receptor monomer may have d i f f e r e n t p r o p e r t i e s (see chapter 3, s e c t i o n g , i ) i n terms of t h e i r a f f i n i t i e s f o r agonists and antagonists. This suggests the p o s s i b i l i t y that the process of d e s e n s i t i z a t i o n may not be e x a c t l y the same at these two s i t e s . Indeed t h i s may be the basis of the two components of d e s e n s i t i z a t i o n . - 80 -Sine and Taylor (1980) have shown that the change i n a f f i n i t y thought to correspond to d e s e n s i t i z a t i o n can occur independently at the two binding s i t e s and p o s s i b l y at d i f f e r e n t r a t e s These i n v e s t i g a t o r s generated a popu-l a t i o n of receptors that only had one binding s i t e by incubating c u l t u r e d muscle c e l l s with a-BuTX f o r a l i m i t e d period of time. I f a-BuTX binds inde-pendently at the two s i t e s , and Y i s the t o t a l f r a c t i o n a l occupancy at the end of t h i s p e r i o d , then the f r a c t i o n of receptors with zero, one, and two bound a-BuTX molecules w i l l be (1-Y)2, 2Y(1-Y), and Y2, r e s p e c t i v e l y . I f Y= 50 percent then 25 percent of the receptors w i l l be f r e e of t o x i n , 50 percent w i l l be associated with only one t o x i n molecule and 25 percent w i l l have both s i t e s blocked. They found that i n the absence of t o x i n and a f t e r 15 s incubation with agonist the occupancy curve f o r carbachol (estimated by competition with a-BuTX) had a H i l l c o e f f i c i e n t of 1.3 and an apparent d i s -s o c i a t i o n constant of 60 uM. When binding was measured under s i m i l a r condi-t i o n s a f t e r treatment with t o x i n i t was evident that there was a population of low a f f i n i t y binding s i t e s . Even when 80 percent of the remaining f r e e binding s i t e s were on receptors with only one f r e e binding s i t e , h a l f of the f r e e b i n d i n g s i t e s appeared to have a low a f f i n i t y f o r carbachol (about 500 uM) and the other h a l f had a higher a f f i n i t y , around 50 pM. This r e s u l t can be explained i f the two binding s i t e s normally found on t h i s receptor can d e s e n s i t i z e at d i f f e r e n t r a t e s . Fast d e s e n s i t i z a t i o n could develop w i t h i n the 15 s of incubation required to t i t r a t e the acceptor s i t e s and may be produced by the generation of a s t a t e of the receptor where only one of the primary acceptor s i t e s on the receptor i s d e s e n s i t i z e d . - 81 -Sine and Taylor (1980) also noted that when r e a c t i o n rates are slowed by lowering temperature to 3.5°C the onset of slow d e s e n s i t i z a t i o n becomes b i p h a s i c . The f a s t e r component of t h i s slow d e s e n s i t i z a t i o n becomes domi-nant as the carbachol concentration i s increased from 30 to 100 yM carbachol (see t h e i r F i g . 2 ) . One p o s s i b l e i n t e r p r e t a t i o n of t h i s r e s u l t i s that the slow d e s e n s i t i z e d s t a t e develops from the f a s t d e s e n s i t i z e d s t a t e and t h i s process i s f a s t e r when the f a s t d e s e n s i t i z e d s t a t e i s mostly associated with agonist ( i . e at concentrations of carbachol > 30 yM). This model would be c o n s i s t e n t with the H i l l c o e f i c i e n t of 1.7 f o r the steady s t a t e l e v e l of d e s e n s i t i z a t i o n (see Sine and T a y l o r , 1980) and the f a c t that the onset r a t e of d e s e n s i t i z a t i o n i s n e a r l y l i n e a r with carbachol concentrations between 20 yM and 500 yM (see Adams, 1975b). I t i s notable that the r a t e of a s s o c i a t i o n of ACh with the high a f f i n i -t y , and presumably d e s e n s i t i z e d , s t a t e of the receptor has a low temperature s e n s i t i v i t y . The change i n r a t e with a 10°C increase i n temperature ( i . e . Q^Q) i s about 1.4 (see Boyd and Cohen, 1980a). The r a t e of opening of channels by ACh on the other hand, probably has a much higher Q^Q (= 4.4) s i n c e the steady s t a t e response increases with temperature (Q^Q = 1.4) even though the channel c l o s i n g r a t e also increases with temperature ( Q 1 0 = 3; see L e s t e r et a l . , 1980a). The s i g n i f i c a n c e of these observa-t i o n s i s best seen by d e r i v i n g the thermodynamic parameter d e s c r i b i n g these processes. This can be done i f the r a t e constants and Q^Q are known (see Dixon and Webb, 1956; Plowman, 1972). Table 2 i s derived from the work of Lester et a l . (1980a) and Sheridan and Lester (1977) on the rates and tempe-r a t u r e s e n s i t i v i t y of ACh receptor-channel opening and c l o s i n g i n the eel - 82 -e l e c t r o p l a q u e , and the work of Cohen and Boyd (1980a) on ACh a s s o c i a t i o n with receptor i n membrane fragments of the Torpedo e l e c t r o p l a q u e . Table 2. Thermodynamic parameters d e s c r i b i n g the r a t e l i m i t i n g s t a t e s in ACh i n t e r a c t i o n with normal and d e s e n s i t i z e d receptors Normal (10°C) Desensitized (4°C) Opening C l o s i n g A s s o c i a t i o n D i s s o c i a t i o n Rate constant 5 x 10 6M" 1s~ 1 0.25 ms - 1 3 x 10 7M" 1s~ 1 0.041 s - 1 « 1 0 * 4.4 3.0 1.4 2.5 AH* (Kcal/M) 24 18 5 15 AS* (cal/M/°K) 60 18 2 -10 AG*- (Kcal/M) 7 12 6 17 Estimated from of steady s t a t e response. ( AH^ i s the change i n enthalpy necessary to reach the r a t e l i m i t i n g s t a t e ; AS* i s the change i n entropy; AG* i s the f r e e energy d i f f e -rence between the i n i t i a l s t a t e and the r a t e l i m i t i n g s t a t e ) . The high enthalpy of the channel opening r a t e i n d i c a t e s that t h i s process involves more breaking than formation of bonds; the r e a c t i o n goes forward because the combination of ACh with the normal receptor causes a large increase i n entropy. Entropy i s a measure of the number of states i n which a substance can e x i s t . This concept of m u l t i p l e c o e x i s t i n g s t a t e s i s defined by the thermodynamic p r o b a b i l i t y (P) (see Weast, 1969) which i s r e l a t e d to entropy by the equation: S = k l n ( P ) Thus, the large increase of entropy when ACh i n t e r a c t s with a normal receptor i n d i c a t e s that ACh increases the number of s t a t e s in which the - 83 -receptor can e x i s t . One of these s t a t e s w i l l spontaneously isomerize to a s t a b l e s t a t e t h a t i s associated with an open channel. Binding energy i s used to pay f o r the generation of the disordered s t a t e that precedes channel opening. When the receptor i s d e s e n s i t i z e d , and channel opening cannot occur, the enthalpy of the r a t e l i m i t i n g s t a t e i n a s s o c i a t i o n of ACh with r e c e p t o r , i s much l e s s . Moreover, entropy i s a c t u a l l y reduced, as one might expect s i n c e the ACh molecule i s no longer f r e e . The net r e s u l t of desensi-t i z a t i o n i s that much more of the energy of bindin g (see F r a n k l i n , 1980; Jencks, 1975) i s expressed i n the agonist a f f i n i t y . This would account f o r the high a f f i n i t y of the d e s e n s i t i z e d receptor f o r ACh. Agonist binding appears to cause the same l o c a l conformational change independent of the receptor s t a t e . Damle and Karl i n (1980) have measured the r a t e and extent of reduction of the binding s i t e s u l f h y d r y l group (see chapter 4, s e c t i o n b , i i i ) by measuring the extent of l a b e l i n g of receptor 3 ( i n receptor enriched microsacs) by [ H] MBTA a f t e r exposure to d i t h i o -t h r e i t o l . They report that ACh and carbachol decrease the r a t e of t h i s reduction by f a c t o r s of up to 60- and 2 0 - f o l d , r e s p e c t i v e l y . In general, agonists reduce the r a t e by at l e a s t 15 f o l d . In c o n t r a s t , antagonists such as dTC reduce the r a t e by about two f o l d while a p a r t i a l agonist such as decamethonium or c h o l i n e provides intermediate p r o t e c t i o n . Since bulky molecules such as dTC can be much l e s s e f f e c t i v e than small molecules l i k e tetramethylammonium, i t i s l i k e l y that p r o t e c t i o n i s secondary to a l o c a l conformational change i n the receptor. The d i f f e r e n c e i n the rates of reduction i n the absence and presence of agonist i s s i m i l a r to the d i f f e r -ence between the r a t e of reduction of s u l f h y d r y l groups i n proteins exposed and not exposed to polar s o l v e n t s . Thus, the conformation change that p r o t e c t s the s u l f h y d r y l group may do so by t r a n s f e r r i n g t h i s group to a l e s s a c c e s s i b l e , l e s s polar region of the p r o t e i n (Damle and K a r l i n , 1980). The maximal extent of p r o t e c t i o n i s the same whether or not the recep-to r i s i n the high a f f i n i t y d e s e n s i t i z e d s t a t e ; only the agonist potency i s changed. Thus, when the receptor has been exposed to agonist f o r only 10 seconds, 50 yM carbachol i s needed to produce a h a l f maximal p r o t e c t i o n while a f t e r 30 minutes exposure to agonist only 1 yM carbachol i s needed to produce an equivalent degree of p r o t e c t i o n . In detergent e x t r a c t e d receptor 1 mM carbachol i s required f o r the same degree of p r o t e c t i o n . The binding of agonist to the receptor causes a l o c a l conformational change i n the v i c i -n i t y of the agonist binding s i t e that i s s i m i l a r in most st a t e s of the recept o r . However, t h i s l o c a l conformational change can only t r i g g e r a global .change i n receptor conformation leading to receptor a c t i v a t i o n in the nondesensitized s t a t e . Thus, d e s e n s i t i z a t i o n appears to r e s u l t from an uncoupling of the acceptor s i t e from the e f f e c t o r component of the receptor. ( i i i ) C o o p e r a t i v i t y i n a c t i v a t i o n of the ACh receptor In many b i o l o g i c a l systems the response to a drug i s sigmoid with respect to agonist c o n c e n t r a t i o n . This has been i n t e r p r e t e d in terms of cooperative drug receptor i n t e r a c t i o n but could e q u a l l y well r e f l e c t a non-l i n e a r r e l a t i o n between the stimulus produced by a c t i v a t i o n of receptors and the measured response (see Rang, 1971). The assumption of l i n e a r i t y between the l e v e l of a c t i v a t e d receptor and response becomes more v a l i d when the measured response i s clo s e to the primary event that s i g n a l s the a c t i v a t i o n of a rece p t o r . In the case of the a c e t y l c h o l i n e receptor the primary event i s the opening of i o n i c channels that are permeable to Na + and K + (see Linder and Quastel, 1978) and which can be measured as a change in permea-b i l i t y using l a b e l e d ions or as a change i n the cu r r e n t necessary to main-- 85 -t a i n a constant voltage across the membrane c o n t a i n i n g the ACh receptor. Even i f the measured response i s not the primary response associated with receptor a c t i v a t i o n i t can often be r e l a t e d by a known mathematical f u n c t i o n to the primary response. For example, i n the muscle f i b e r , changes i n membrane p o t e n t i a l can be converted to values p r o p o r t i o n a l to the changes i n conductance by means of Martin's c o r r e c t i o n ( M a r t i n , 1955). In a l l of the cases where the primary response has been measured e i t h e r d i r e c t l y or i n d i r e c t l y , an 'S' shaped r e l a t i o n between response and n i c o t i n i c agonist concentration has been observed. This suggests that a c t i v a t i o n of the a c e t y l c h o l i n e receptor i s indeed a cooperative drug receptor i n t e r a c t i o n . The 'S' shaped r e l a t i o n was f i r s t observed by Katz and T h e s l e f f (1957b) using m i c r o i o n t o p h o r e t i c a p p l i c a t i o n of ACh. A number of other i n v e s t i g a -t o r s have subsequently found a sigmoidal r e l a t i o n between conductance and amount of charge (Q) passed during an i o n t o p h o r e t i c a p p l i c a t i o n of agonist ( F e l t z and M a l l a r t , 1971; Norman 1976; Dreyer, Peper, S t e r z , Bradley and M u l l e r , 1979) Non-integral H i l l c o e f f i c i e n t s between 2-3 are g e n e r a l l y reported f o r agonists such as ACh and carbachol. The H i l l c o e f f i c i e n t determined from i o n t o p h o r e t i c studies can be decreased to between 1 and 2 by ac i d (pH 5 ) , high C a + + (20 mM), ethanol (0.4 M) and edrophonium ( S t e r z , Dreyer and Peper, 1976; 1977). The conclusion that the H i l l c o e f f i c i e n t r e f l e c t s the number of coope-r a t i n g agonist molecules r e l i e s on the assumption that the concentration i n the v i c i n i t y of the receptor at the peaks of the response to i o n t o p h o r e t i c -a l l y a p p lied agonist i s l i n e a r l y r e l a t e d to Q. This i s not always the case and the actual r e l a t i o n between agonist e j e c t e d and Q i s influenced by a v a r i e t y of f a c t o r s i n c l u d i n g shape of the i o n t o p h o r e t i c electrode t i p , - 86 -e l e c t r o d e r e s i s t a n c e , backing current and the shape and duration of the current pulses. These various f a c t o r s a l s o i n f l u e n c e the time lag between the onset of the current pulse and the steady s t a t e e j e c t i o n of drug (see Purves 1979). Changes i n t h i s lag may account f o r some of the e f f e c t s of pH and C a + + ions on the H i l l c o e f f i c i e n t and the lag i t s e l f may generate some of the n o n l i n e a r i t y i n the r e l a t i o n between Q and response. Katz and T h e s l e f f (1957b) suggested another p o s s i b l e c o m p l i c a t i n g f a c t o r . I f a s i g n i f i c a n t amount of high a f f i n i t y binding s i t e s ( i . e . d e s e n s i t i z e d recep-t o r s ) e x i s t under r e s t i n g c o n d i t i o n s (20 percent according to Boyd and Cohen, 1980a) these may be able to 'mop up' ACh e j e c t e d by low currents as f a s t as the ACh d i f f u s e s i n t o the c l e f t . This would lead to an overestimate of the H i l l c o e f f i c i e n t . Nevertheless, these p o s s i b l e a r t i f a c t s cannot e n t i r e l y account f o r the power f u n c t i o n r e l a t i o n between dose and response because a sigmoidal r e l a t i o n s h i p i s also seen when carbachol i s bath applied to e i t h e r the neuromuscular j u n c t i o n (Oenkinson, 1960) or c e l l s from the e l e c t r i c organ (Higman, Podleski and B a r t e l s , 1963); with bath a p p l i c a t i o n H i l l c o e f f i c i e n t s between 1.3 and 2.0 are reported (Rang, 1971; Changeux and P o d l e s k i , 1968; see also Colquhoun, 1978). The existence of c o o p e r a t i v i t y in ACh receptor a c t i v a t i o n i s a l s o supported by the observation that the r a t e of opening of channels determined from noise a n a l y s i s (Sakmann and Adam, 1978) and actual observation of i n d i v i d u a l channels (Neher, Patlak and Sakmann, 1980) show a sigmoidal r e l a t i o n with agonist concentration. In a l l of these cases the H i l l c o e f f i c i e n t determined g e n e r a l l y has a non-integral value. This has been taken as evidence against the independent subunit explanation f o r the sigmoidal r e l a t i o n s h i p . The l a t t e r model propo-ses that there i s no i n t e r a c t i o n between subunits, but more than one subunit - 87 -has to be occupied i n order f o r an elementary response to occur ( P o d l e s k i , 1973; Colquhoun, 1973); t h i s p r e d i c t s i n t e g r a l H i l l c o e f i c i e n t s . Neverthe-l e s s , when precautions are taken to minimize d e s e n s i t i z a t i o n H i l l c o e f f i -c i e n t s of e x a c t l y 2.0 are found (see f o r example Neubig and Cohen, 1980). The independent subunit model p r e d i c t s that the response (E) r e l a t i v e to the maximal response ( E m a x ) produced by a agonist concentration (A) with a d i s s o c i a t i o n constant K g i s given by the f o l l o w i n g r e l a t i o n E = 1 Emax < 1 + K a / ™ " where n i s the number of subunits that need to be occupied i n order to produce a response. Thus, the inverse of the n root of the response w i l l be p r o p o r t i o n a l to the inverse of the agonist concentra-t i o n ; i . e . n^r - n T F d + V ™> V E V E m a x With the cooperative subunit model where there i s a high degree of i n t e r a c t i o n , then: E = 1 W " 1 + ( K a/[A]) n For a given E m a x and n, the r e l a t i o n p r e d i c t e d by the coopera-t i v e subunit model i s steeper at s a t u r a t i n g concentrations than that p r e d i c t e d by the independent subunit models (see Dudel, 1976). In general, the data i s b e t t e r f i t by a cooperative subunit model than by an independent subunit model (Dryer, Peper and S t e r z , 1978; Dionne, Steinbach and Stevens, 1978; Adams, 1975b); t h i s i s also true with the glutamate receptor at the c r a y f i s h neuromuscular j u n c t i o n (Dudel 1976). - 88 -The s t r u c t u r e a c t i v i t y s t u d i e s described in chapter 4, s e c t i o n b, i i , suggest t h a t at l e a s t two acceptor s i t e s are involved i n receptor a c t i v a t i o n . Bis-onium compounds such as s u b e r y l d i c h o l i n e and Bis Q seem to owe t h e i r high potency to the f a c t t h a t they have a s t r u c t u r a l component analogous to two ACh molecules attached t a i l to t a i l . Yet both of these compounds show a sigmoidal r e l a t i o n between concentra-t i o n and response (Adams 1977a; Lester et a l . , 1980b). I t was sugges-ted i n Chapter 4, s e c t i o n b , i i , that molecules which recognise the C16 s t r u c t u r e i n v o l v e s both a subunits on a receptor monomer. P o s s i b l y occupancy of the ACh acceptor s i t e on one monomer may f a c i l i t a t e occu-pancy of a subunits on adjacent monomers. The receptor or f u n c t i o n a l u n i t i n t h i s case would then be a dimer. A l t e r n a t i v e l y , there may be accessory s i t e s on the receptor monomer, p o s s i b l y on e and y subunits (see Chapter 3, s e c t i o n c ) , that can bind ACh and f a c i l i t a t e binding of ACh to the a subunits. Neubig and Cohen (1979) have shown t h a t , when standard e q u i l i b r i u m techniques are used to assess the number of bindin g s i t e s f o r ACh and dTC i n membrane fragments f o r Torpedo e l e c t r o p l a x , these turn out to be equal i n number to a-BuTX binding s i t e ( i . e . only 2 per receptor monomer). However, i t i s p o s s i b l e that d e s e n s i t i z a t i o n masks the binding s i t e on the other subunits, or a l t e r n a t i v e l y , that these s i t e s are only exposed when the receptor i s a c t i v a t e d or i n the process of being a c t i v a t e d . I t i s l i k e l y that a c t i v a t i o n and d e s e n s i t i z a t i o n i n v o l v e major conformational changes that i n f l u e n c e a l l subunits (see K i s t l e r et a l . , 1982; Weitzman and Raf t e r y , 1978; Rubesamen et a l . , 1978). Eigen (1974) has discussed how accessory s i t e s can increase the r a t e capture of l i g a n d by recep-- 89 -t o r s i n c e these w i l l increase the number of l i g a n d molecules i n the v i c i n i t y of the primary binding s i t e . I f such accessory s i t e s i n f a c t e x i s t on the ACh r e c e p t o r , they could account f o r the e f f e c t of dTC on the r a t e of r e l a x a t i o n currents produced by a c t i v a t i o n of n i c o t i n i c receptors without having to p o s t u l a t e very high r a t e s of a s s o c i a t i o n of dTC with the receptor (see Sheridan and Colquhoun, 1982). Pennefather and Quastel (1981) (see Chapter 10) have estimated t h a t the maximum number of channels that can be a c t i v a t e d by an MEPC i s about 2,500 and that most of the ACh i n a quantum i s bound at the peak of an MEPC. A number of authors using a v a r i e t y of techniques have presented evidence that each quantum represents the r e l e a s e of about 10 4 molecules ( K u f f l e r and Yoshikami, 1975; Guioro et a l . , 1978; Tauc, 1980). Thus i t i s p o s s i b l e that as many as four ACh mole-cules are bound per receptor a c t i v a t e d by a quantum. d) A c t i o n of drugs at the e f f e c t o r component of the r e c e p t o r ( i ) Preface Drugs which act as s p e c i f i c ligands f o r the receptor acceptor s i t e have q u i t e d e f i n a b l e s t r u c t u r e a c t i v i t y r e l a t i o n s h i p s . As a r e s u l t , the drug receptor i n t e r a c t i o n i s s a i d to be s p e c i f i c . However, drug receptor i n t e r a c t i o n s at s i t e s other than the acceptor s i t e need not be very s p e c i f i c i f the f i n a l consequence of such i n t e r a c t i o n i s simply blockade of receptor f u n c t i o n s . Many of the neuromuscular blockers discussed i n t h i s t h e s i s appear to have such n o n s p e c i f i c actions on receptor f u n c t i o n , but t h i s does not r u l e out the p o s s i -- 90 -b i l i t y that these agents act at a dist inct acceptor of their own. This poss ib i l i ty is best i l lustrated by the action of membrane stabi -l i zers on the electrosensitive Na channels responsible for the action potential . Since many of the nonspecific neuromuscular blockers that wi l l be discussed are also c lass i f ied as membrane s tab i l i zers , i t is useful to describe in detail the actions of membrane stabi l izers on the electrosensitive sodium channel. ( i i ) Action of membrane stabi l izers on the electrosensitive Na+  channel of nerve The term membrane stabi l i zer refers to a large and diverse group of drugs that can reduce ce l l membrane exc i tabi l i ty without having any appreciable action on resting membrane potential . This group include local and general anaesthetics, as well as alcohols, steroids, non-ionic detergents and even noble gases such as xenon. The term was f i r s t coined by Bishop (1932) to distinguish these agents from agents that interfered with ce l l exc i tabi l i ty by changing the resting membrane potential . Membrane stabi l izers stabi l ized the resting membrane potential . Shanes (1958a,b) concluded from a review of the l i terature that membrane stabi l izers acted to block a l l membrane ionic permeability systems, but that some systems were less sensitive to membrane stabi l izers than others. This idea was supported by experiments on the effects of general and local anaesthetics on ionic channels responsible for nerve action potential generation (Shanes, 1958; Taylor, 1959) and on those channels responsible for ACh respon-ses at the neuromuscular junction (Thesleff, 1956). - 91 -I t i s c l e a r that the hydrophobic nature of membrane s t a b i l i z e r s p lays an important r o l e i n producing t h e i r l o c a l anaesthetic a c t i o n (see Seeman, 1966). Most membrane s t a b i l i z e r s can also cause n a r c o s i s . I t has long been recognized t h a t there i s a good c o r r e l a -t i o n between the a b i l i t y of agents to cause n a r c o s i s and t h e i r o i l -water p a r t i t i o n c o e f f i c i e n t . Indeed the Meyer-Overton r u l e (see Meyer, 1906) formulated at the turn of the century, suggested that equal n a r c o t i c e f f e c t s can be produced by very d i f f e r e n t substances as long as t h e i r molar concentrations i n c e l l l i p i d s are i d e n t i c a l (around 30-60 mM f o r n a r c o s i s ) . Mull ins (1954) noted that d e v i a t i o n from the l i p i d s o l u b i l i t y r u l e often e x i s t s i n a s e r i e s of homologous compounds such as the n-alkanols, and that these d e v i a t i o n s are reduced when n a r c o t i c potency i s c o r r e l a t e d with the estimated molar volume of the n a r c o t i c i n the c e l l l i p i d s . Thus, Mull i n s ' r u l e s t a t e s t h a t i t i s the volume of anaesthetic in the l i p i d , r ather than the amount, that determines the n a r c o t i c e f f e c t (= 1 ml/kg membrane) f o r n a r c o s i s . L i p i d s o l u b i l i t y and hydrophobicity has al s o been i m p l i c a t e d i n the l o c a l anaesthetic a c t i o n of membrane s t a b i l i z e r s . Lofgren (1948) noted that the p a r a l l e l between potency and p a r t i t i o n c o e f f i c i e n t e x i s t e d f o r the l o c a l anaesthetic a c t i o n of uncharged n a r c o t i c s . Seeman (1966) followed up the o l d observation (see Jacobs and Papart, 1932) that membrane s t a b i l i z e r s p r o tect e r t h r o c y t e s against osmotic haemolysis and that t h i s e f f e c t i s c o r r e l a t e d with l o c a l anaesthetic potency. Seeman l a t e r extended t h i s work to a wide v a r i e t y of mem-brane s t a b i l i z e r s (see Roth and Seeman, 1972; Seeman, 1972) and deter-mined exp e r i m e n t a l l y the concentrations of drug i n the membrane asso-c i a t e d with an antihaemolytic e f f e c t . This e f f e c t obeyed Mull ins r u l e and, under the c o n d i t i o n s chosen, 50 percent antihaemolysis occured when about 2-4 ml/kg membrane was occupied by membrane s t a b i l i z e r and there was a one to one c o r r e l a t i o n between the ED^g f o r blockade of the f r o g s c i a t i c nerve compound a c t i o n p o t e n t i a l and the concentration producing 50 percent antihaemolysis. Since they determined that there was a one to one c o r r e l a t i o n between the membrane:buffer p a r t i t i o n c o e f f i c i e n t of a v a r i e t y of agents f o r erythrocyte ghost membranes and that f o r e x c i t a b l e membranes such as nerve or muscle, they concluded that l o c a l anaesthesia i s associated with a c r i t i c a l volume of about 2-4 ml/kg membrane. The l o c a l anaesthetic potency i s about 1/5-1/10 the general anaesthetic potency f o r many of these drugs even though the c r i t i c a l volume (2-4 ml/kg) i s s i m i l a r to that proposed by M u l l i n s (1954) from o i l : w a t e r p a r t i t i o n data. Seeman (1972)'has pointed out that membrane:buffer p a r t i t i o n c o e f f i c i e n t s are g e n e r a l l y 1/5-1/10 the o i l : w a t e r p a r t i t i o n c o e f f i c i e n t s . None of t h i s information e x p l a i n s how membrane s t a b i l i z e r s cause l o c a l anaesthesia; M u l l i n s ' r u l e i s a r u l e , not a hypothesis. Theor-ies concerning the a c t i o n of membrane s t a b i l i z e r s can be l o o s e l y d i v i -ded i n t o two b a s i c types; (1) that s t a b i l i z e r s i n t e r a c t d i r e c t l y with i o n i c channels or (2) that they d i s s o l v e i n the l i p i d b i l a y e r region of the plasmalemma and perturb the l i p i d environment of i o n i c , channels (see Richards et a l . , 1978; P r i n g l e , Brown and M i l l e r , 1981). In the l a t t e r case, the membrane s t a b i l i z e r s w i l l be a c t i n g i n a s t r i c t l y n o n s p e c i f i c manner. However, i n the former case, the i o n i c channel could be considered a n o n s p e c i f i c receptor f o r the membrane s t a b i l i -z e r s . - 93 -Such l i p i d t h e o r i e s are s a i d to be u n i t a r y because they propose that a s i n g l e a c t i o n on l i p i d b i l a y e r membranes can account f o r the actions of membrane s t a b i l i z e r s on both e l e c t r i c a l l y e x c i t a b l e systems such as nerves and n o n - e l e c t r i c a l l y e x c i t a b l e systems such as er y t h r o -cytes or p r o t e i n f r e e a r t i f i c i a l membranes. Their basis i s the obser-v a t i o n that i n many cases the potency of membrane s t a b i l i z e r s i n af-f e c t i n g simple membrane systems c o r r e l a t e s with t h e i r l o c a l anaesthe-t i c potency. In most cases the 'membrane s t a b i l i z e r s ' seem to a f f e c t simple membrane systems i n d i r e c t l y by d i s s o l v i n g i n the l i p i d b i l a y e r and d i s o r d e r i n g the membrane s t r u c t u r e . I f t h i s type of a c t i o n were the basis of the e f f e c t of membrane s t a b i l i z e r s on e x c i t a b l e membranes then Mull i n s 1 r u l e could be e a s i l y explained, since a given volume of any i n e r t compound i n the membrane should d i s o r d e r the packing of the membrane l i p i d s to an equivalent extent. Such an e f f e c t would a l s o account f o r the observation that the e f f e c t s of some membrane s t a b i l i -zers can be reversed by i n c r e a s i n g h y d r o s t a t i c pressure ( n a r c o s i s : Johnson and F l a g e r , 1951; M i l l e r , Paton, Smith and Smith, 1973; l o c a l anaesthesia: Kendig, 1980). I t i s reasoned that increased pressure makes i t harder f o r the anaesthetic to expand the membrane and reach a c r i t i c a l volume i n the membrane. These t h e o r i e s are.not very c l e a r i n explaining, how perturbation of the l i p i d environment d i s r u p t s the f u n c t i o n of i o n i c channels or what i s the c r i t i c a l p e r t u r b a t i o n . Much of the work involves e x p e r i -ments on a r t i f i c i a l y formed b i l a y e r s of l i p i d . Most general and l o c a l anaesthetics w i l l d i s o r d e r these b i l a y e r s and the potency f o r producing t h i s e f f e c t c o r r e l a t e s w e l l with anaesthetic potency (see - 94 -Met c a l f e , Seeman and Burgen, 1968; Boggs, Young and Hsia , 1976; Papahadjopoulos, Jackobson, Poste and Shepard, 1975) although, i n the case of b a r b i t u r a t e s and t e r t i a r y amine l o c a l a n a e s t h e t i c s , choles-t e r o l has to be added to the b i l a y e r f o r t h i s to be true (see M i l l e r and Pang, 1976). 1 At l o c a l anaesthetic concentrations the d i s o r d e r i n g e f f e c t i s small but i t has been argued that these small changes might be import-ant i f they can be a m p l i f i e d ; f o r example, i f the d i s o r d e r i n g e f f e c t occurs at c r i t i c a l areas i n the membrane such as at l a t e r a l phase t r a n s i t i o n s ( T r u d e l l , 1977) or at boundary l i p i d surrounding i n t r i n s i c membrane proteins (see Hesketh, Smith, Housley, M c G i l l , B i r d s a l l , Metcalfe and Warren, 1976; Lee, 1976). A major problem with these t h e o r i e s i s that an increase i n temperature a small as a few °C can cause perturbations equivalent to those produced by r e l a t i v e l y high concentrations of anaesthetic (see Pang, B r a s w e l l , Chang, Sommer and M i l l e r , 1980; Frank and L e i b , 1981). Yet the l o c a l anaesthetic a c t i o n of butanol, f o r example, i s r e l a t i v e l y unaffected over a temperature range of 20-45°C (Richards et a l . , 1978). In the case of n a r c o s i s , the c o r r e l a t i o n of hydrophobicity and potency may be misleading. These c o r r e l a t i o n s are g e n e r a l l y based on octanol:water p a r t i t i o n c o e f f i c i e n t s , yet octanol i s a rather polar s o l v e n t , compared to the l i p i d core of the membrane. Benzene:water p a r t i t i o n c o e f f i c i e n t s c o r r e l a t e poorly with anaesthetic potency even though benzene provides a more hydrophobic environment than octanol (Franks and L e i b , 1978). Katz and Simons (1977) have noted that the r e l a t i o n between a v a r i e t y of physical-chemical p r o p e r t i e s and anaes-- 95 -t h e t i c potency suggest that the s i t e at which anaesthetics act to produce n a r c o s i s has both hydrophobic and h y d r o p h i l i c p r o p e r t i e s . This may be a boundary l i p i d phase or a p r o t e i n phase but not the hydrophobic -core of the b i l a y e r , which most l i p i d t h e o r i e s imply i s the s i t e of a c t i o n of membrane s t a b i l i z e r s . Thus co n t r a r y to the non-s p e c i f i c nonreceptor hypothesis, these f i n d i n g s suggest t h a t i t i s the anaesthetic concentration or volume at a s p e c i f i c s i t e r ather than i n the membrane as a whole that i s c r i t i c a l i n producing anaesthesia. In the case of amphipathic l o c a l a n a e s t h e t i c s , Conrad and Singer (1980) have r e c e n t l y questioned whether these agents can even d i s s o l v e in b i o l o g i c a l membranes. They i n v e s t i g a t e d the i n t e r a c t i o n of c h l o r -promazine and methylchlorpromazine, a permanently charged analogue, with a v a r i e t y of model membranes both b i o l o g i c a l ( e r y t h r o c y t e , lymph-oma c e l l and v e s i c l e s from sarcoplasmic reticulum) and a r t i f i c i a l ( l a r g e u n i l a m e l l a r phospholipid v e s i c l e s ) . They measured the p a r t i -t i o n c o e f f i c i e n t of the drugs f o r these membranes using the standard c e n t r i f u g a t i o n method (see Roth and Seeman, 1972) and a new method that they developed c a l l e d hygroscopic desorption. In both methods, the membranes are incubated with l a b e l l e d drug but i n the c e n t r i f u g a l method, f r e e drug i s removed by c e n t r i f u g a t i o n while i n the hygroscop-i c desorption method the incubating medium i s sucked up across a s p e c i a l l y designed sie v e by a wad of c e l l u l o s e . The l a t t e r method does not produce a p e l l e t so that drug which i s only l o o s e l y a s s o c i a t -ed with the membranes and can be removed; i t does not become trapped w i t h i n a p e l l e t . The two methods give i d e n t i c a l r e s u l t s when a r t i f i -c i a l membranes are used and i n d i c a t e that both chlorpromazine and - 96 -methylchlorpromazine have a high membrane .'buffer p a r t i t i o n c o e f f i c i e n t (= 1000). Both methods agree that methylchlorpromazine has a very low p a r t i t i o n c o e f f i c i e n t when a r t i f i c i a l membranes are used (=0.1). However, the two methods disagree on the p a r t i t i o n c o e f f i c i e n t f o r chlorpromazine with b i o l o g i c a l membranes. The c e n t r i f u g a l method i n d i c a t e s that i t remains high (= 1000) while the hygroscopic desorp-t i o n technique i n d i c a t e s t h a t , as i s the case f o r methylchlorproma-z i n e , the p a r t i t i o n c o e f f i c i e n t i s much smaller with b i o l o g i c a l mem-brane (= 0.1). They conclude that the p a r t i t i o n c o e f f i c i e n t determi-ned by the c e n t r i f u g a l method i s a r t i f a c t u a l and suggest chlorproma-z i n e i s only l o o s e l y associated with b i o l o g i c a l membranes. They point out that methylchlorpromazine has a c r i t i c a l m i c e l l e concentration that i s 20 times that of chlorpromazine and suggest that chlorproma-z i n e molecules may be able to form some s o r t of m i c e l l e or hemimicelle on the surface of b i o l o g i c a l membranes. Whatever the case, the r e s u l t s of Conrad and Singer (1980) suggest that amphipathic molecules cannot s t i c k t h e i r hydrophobic t a i l s i n t o the core of the l i p i d b i l a y e r of b i o l o g i c a l membranes. This makes i t d i f f i c u l t to account f o r the n a r c o t i c and l o c a l anaesthetic actions of amphipathic membrane s t a b i l i z e r s using b i l a y e r p e r t u r b a t i o n t h e o r i e s . Also the 1000-fold d i f f e r e n c e i n p a r t i t i o n c o e f f i c i e n t of methylchlor-promazine f o r b i o l o g i c a l , as opposed to a r t i f i c i a l membrane b i l a y e r s , leads one to question the use of a r t i f i c i a l membranes as models of b i o l o g i c a l membranes. - 97 -Perhaps the most important c r i t i c i s m of l i p i d t h e o r i e s i s that a l l of the evidence used to support these t h e o r i e s can also be used to support the p r o t e i n theory. Anaesthetics enter a r t i f i c i a l l i p i d b i l a y e r s because they get i n the way of hydrogen bonds between water molecules (see Hildebrand, 1979; Sandorsky, 1980). In energetic terms, they cause l e s s d i s r u p t i o n of the l i p i d than the water. Yet some d i s r u p t i o n of the l i p i d s t r u c t u r e must nevertheless e x i s t and i t i s not su p r i s i n g . that t h i s can be detected. The a b i l i t y to d i s r u p t water to a greater degree than the l i p i d b i l a y e r s i s dependent on intermolecular i n t e r a c t i o n s that are c h a r a c t e r i s t i c of a v a r i e t y of processes other than penetration of a l i p i d b y l a y e r . As a r e s u l t , each of these processes can be c o r r e l a t e d to n a r c o t i c potency. Seeman (1966) c i t e s a number of examples such as the d i s s o c i a t i o n pressure of hydrates of anaesthetic gases ( P a u l i n g , 1961), the a b i l i t y to coagu-l a t e c o l l o i d a l s o l u t i o n s of mastic (Sekera and Vrba, 1959) and absorp-t i o n of anaesthetics onto charcoal (Buchi and P e r l i a , 1962). A l l of these processes i n v o l v e e x c l u s i o n of anaesthetic from s t r u c t u r a l water at i n t e r f a c i a l r egions. In b i o l o g i c a l membranes, t h i s e x c l u s i o n may occur at the surface of the membrane d r i v i n g the n a r c o t i c i n t o the membrane core, but i t may e q u a l l y w e l l occur at the surface of a p r o t e i n . Aequorin i s a s o l u b l e p r o t e i n that f l u o r e s c e s when i t combines with Ca . Membrane s t a b i l i z e r such as d i e t h y l ether (100 mM), urethane (100 mM), chloroform (50 mM), ethanol (100 mM), procaine (25 mM), dibucaine (2.5 mM), and a l t h e s i n increase the amount of l i g h t emitted by s o l u t i o n of the p r o t e i n i n the presence of constant b u f f e r -ed concentrations of C a + + i n the p h y s i o l o g i c a l range. Thus, the - 98 -membrane s t a b i l i z e r must be able to i n t e r a c t d i r e c t l y with the aequor-i n p r o t e i n and change i t s s t r u c t u r e i n such a way th a t i t has a higher a f f i n i t y f o r C a + + (Baker and Schapiro, 1980). Membrane s t a b i l i z e r s w i l l a l s o a s s o c i a t e with haemoglobin. The consequences of t h i s absorption has been stud i e d using nuclear magnet-i c resonance (Brown, Halsey and Richards, 1976; Brown and Halsey, 1980). At low concentrations of general a n a e s t h e t i c s , i n the c l i n i c a l range, only one s i t e i n the haemoglobin molecule i s a f f e c t e d and the potency of general anaesthetics to produce t h i s e f f e c t c o r r e l a t e s w e l l with hydrophobic s o l u b i l i t y (and hence, n a r c o t i c potency). This sug-gests a mechanism whereby a r e l a t i v e l y n o n s p e c i f i c a c t i o n can be con-verted i n t o a r e l a t i v e l y s p e c i f i c e f f e c t . In the case of nerve con-duction blockade, one need only postulate s i t e s on or near the e l e c t r o s e n s i t i v e sodium channel, capable of i n t e r a c t i n g hydrophobic-a l l y with membrane s t a b i l i z e r s , and capable of d i s r u p t i n g the f u n c t i o n of the sodium channel when associated with a membrane s t a b i l i z e r . A s p e c i f i c d i s r u p t i o n w i l l occur i f one such s i t e has a higher a f f i n i t y f o r a membrane s t a b i l i z e r than other s i t e s on the receptor. There i s now convincing evidence that such a s i t e mediates the l o c a l anaesthe-t i c a c t i o n of membrane s t a b i l i z e r s (see Armstrong, 1981). The f i r s t i n d i c a t i o n s that l o c a l anaesthetics may act at a s p e c i -f i c s i t e r a t h e r than simply occupying a c r i t i c a l membrane volume comes from attempts to determine whether the charged or uncharged species of t e r t i a r y amine l o c a l anaesthetics i s the a c t i v e form. Skou (1954) was the f i r s t to demonstrate t h a t pH i n f l u e n c e d the a c t i v i t y of t e r t i a r y amine l o c a l a n a e s t h e t i c s , and had no e f f e c t on the a c t i o n of uncharged - 99 -analogues such as benzocaine. Since i n c r e a s i n g pH increased potency, he concluded that the uncharged form of amine l o c a l anaesthetics was the a c t i v e form. This conclusion was l a t e r challenged by R i t c h i e and Greengard (1961) who found t h a t , when they removed the myelin sheath that surrounds the nerve, i n c r e a s i n g pH decreased l o c a l anaesthetic potency. This suggested t h a t the charged form was a c t i v e and the r e s u l t s of Skou were due to the presence of p e r m e a b i l i t y b a r r i e r s to the charged form, keeping i t from accumulating at i t s s i t e of a c t i o n . Narahashi, F r a z i e r , and Yamada (1970a) r e s o l v e d the question f o r l i d o c a i n e by studying the e f f e c t of pH on the a c t i o n of a s e r i e s of l i d o c a i n e d e r i v a t i v e s using the i n t e r n a l l y perfused squid axon prepa-r a t i o n . The e f f e c t s of changing i n t e r n a l or external pH on l o c a l a naesthetic potency were c o n s i s t e n t with the charged form being a c t i v e . When the i n t e r n a l concentration of one of the l i d o c a i n e d e r i -v a t i v e s was increased but pH adjusted so that the l e v e l of charged l o c a l anaesthetic remained the same, the uncharged form of t h i s d e r i -v a t i v e s had l i t t l e i f any a c t i v i t y ; i n c r e a s i n g the l e v e l of the uncharged form to 8X that of the charged form produced only a 26 per-cent increase i n blockade ( F r a z i e r , Murayama and Narahashi, 1972). Thus, the uncharged form was more than 10 times less potent then the charged form. Quaternary l i d o c a i n e d e r i v a t i v e s cannot d i s s o l v e i n t o the l i p i d b i l a y e r s and are i n e f f e c t i v e when ap p l i e d outside the nerve, but these agents are j u s t as e f f e c t i v e as l i d o c a i n e when applied i n s i d e the nerve ( F r a z i e r , Yamada and Narahashi, 1970b). - 100 -The exact r o l e of the quaternary or p o s i t i v e l y charged nitrogen i n producing a l o c a l anaesthetic a c t i o n i s u n c e r t a i n . Quaternary ammo-nium compounds, c o n t a i n i n g triethylammonium groups i d e n t i c a l to those in quaternary l i d o c a i n e , and a l i p o p h i l i c t a i l ( i . e . alkane t r i e t h y l -ammoniums), do not act l i k e quaternary l i d o c a i n e d e r i v a t i v e s when app l i e d i n s i d e squid axons (see Armstrong and H i l l e , 1972), while 2 , 6 - x y l i d i n e (the other end of l i d o c a i n e ) i s a l o c a l anaesthetic (see Takman, 1975). Benzocaine i s a procaine analog that lacks the terminal alkylamine, but i s about as potent as procaine i t s e l f ( R i t c h i e and R i t c h i e , 1968) and thus much more potent than the uncharged form of procaine. Morse and R i t c h i e (1978) have suggested that benzocaine and l i d o c a i n e act at separate s i t e s because t h e i r a ctions on the compound a c t i o n p o t e n t i a l appeared to be s y n e r g i s t i c . However, a c a r e f u l study by Schmidtmayer and U l b r i c h t (1980) on the Na + currents at the f r o g node has f a i l e d to confirm t h i s r e s u l t ; t h e i r data was e n t i r e l y c o n s i s t e n t with l i d o c a i n e and benzocaine i n t e r a c t i n g at the same s i t e . The charged nitrogen probably plays l i t t l e d i r e c t r o l e i n the binding of the l o c a l anaesthetic to the Na + channel. Blockade of sodium channels by l o c a l anaesthetics seems to be determined p r i m a r i l y by hydrophobic i n t e r a c t i o n s (see S t r i c h a r t z , 1976; Courtney, 1980). The polar nature of the amide or ester bond may also i n f l u e n c e a c t i v i t y since procaine d e r i v a t i v e s i n which the ester oxygen i s replaced with'the more e l e c t r o n e g a t i v e Se or S atoms are more potent than procaine (Rosenberg and Mauthner, 1967). - 101 -The charged ammonium group appears to play a more i n d i r e c t r o l e by preventing access when the channel i s closed and loss from t h i s s i t e when the channels i s i n a c t i v a t e d (Courtney, 1980). S t r i c h a r t z (1973) presented evidence from experiments on the voltage clamped node of Ranvier that Na + channels had to open in order f o r quaternary l i d o -caine d e r i v a t i v e s such as QX-314 to cause blockade. Recovery from blockade was a l s o dependent on how often channels were open. Further-more, when a l l of the Na + channels were open to begin w i t h , the degree of blockade increased with d e p o l a r i z a t i o n . A l l of t h i s suggested that QX-314 i n t e r a c t e d with a s i t e i n s i d e the Na channel. Courtney (1975) and H i l l e (1977a,b) showed that many l o c a l anaes-t h e t i c s i n c l u d i n g QX-314 caused a s h i f t i n the voltage dependence of i n a c t i v a t i o n to more negative p o t e n t i a l . They suggested that binding to the Na + channel by the l o c a l anaesthetics s h i f t e d the e q u i l i b r i u m between r e s t i n g , open and i n a c t i v a t e d states ( i . e . as in the c y c l i c scheme discussed i n the l a s t s e c t i o n the l o c a l anaesthetics have a higher a f f i n i t y f o r the i n a c t i v a t e d s t a t e ) . This was c a l l e d the 'modulated receptor' hypothesis. Because the binding of charged agents re q u i r e s channel opening, the e f f e c t on the d i s t r i b u t i o n of the various states b u i l d s up with r e p e t i t i v e s t i m u l a t i o n g i v i n g r i s e to a frequency dependent drug a c t i o n . This modulated receptor hypothesis may provide an explanation f o r the observation that the uncharged procaine d e r i v a t i v e , benzocaine, i s j u s t as potent as the charged form of procaine. The a c t i o n of benzo-caine i s not frequency dependent presumably because i t can reach i t s s i t e of ac t i o n through a hydrophobic pathway ( H i l l e , 1977b). Benzo-- 102 -caine acts to s h i f t the steady r e s t i n g l e v e l of i n a c t i v a t i o n but t h i s e f f e c t occurs much more r a p i d l y than with procaine, and the r a t e of onset of t h i s e f f e c t i s not pH s e n s i t i v e ( H i l l e , 1977a). The low potency of the uncharged form of procaine may i n d i c a t e that i t i s not as t i g h t l y bound to the binding s i t e i n the channel, although in i t s uncharged form i t can, presumably, reach the s i t e by the same pathway as benzocaine. The charged form of procaine may i n t e r a c t with the bindi n g s i t e j u s t as weakly but, because i t i s charged, cannot escape as r e a d i l y from the region of the binding s i t e . The e f f e c t to t r a p p o s i t i v e l y charged molecules i n the channel may e x p l a i n why the reg r e s s i o n l i n e r e l a t i n g the l o c a l anaesthetic potency of p o s i t i v e l y charged l o c a l anaesthetic to membrane:buffer p a r t i t i o n c o e f f i c i e n t i s 1 log-^Q u n i t f u r t h e r to the l e f t of the l i n e f o r uncharged agents (see Seeman, 1975). The above d i s c u s s i o n suggests that the charged ammonium group of amine l o c a l anaesthetics i s not very important i n modifying Na + channel f u n c t i o n . Theories of l o c a l anaesthesia which emphasized the binding of t h i s group to negative s i t e s on the nerve (see B l a u s t e i n and Goldman, 1966) have mostly been discounted (see Narahashi, F r a z i e r and Takino, 1976; Ueda et a l . , 1980). The f a c t that c l i n i c a l l y used l o c a l anaesthetics u s u a l l y have an ammonium group probably stems from the f a c t that t h i s group w i l l improve water s o l u -b i l i t y and reduce t o x i c i t y by keeping the molecule away from hydropho-b i c s i t e s on other membrane p r o t e i n s . - 103 -In s p i t e of general lack of s t r u c t u r a l requirements f o r the l o c a l a n aesthetic e f f e c t , these agents appear t o act d i r e c t l y on the Na channel. Since t h i s i s the f u n c t i o n a l u n i t that mediates the response produced by l o c a l a n a e s t h e t i c s , i t can be considered a l o c a l anaesthe-t i c r e c e p t o r . More recent work on gating currents suggest that l o c a l anaethetics are a c t i n g on a s p e c i f i c p o r t i o n of the Na + channel (see Armstrong, 1981; Cahalan, 1980) and t h a t the s t r u c t u r e of l o c a l anaes-t h e t i c s can modify t h i s a c t i o n . Thus, the acti o n of l o c a l anaesthe-t i c s on e l e c t r o s e n s i t i v e Na + channels i s not, s t r i c t l y speaking, n o n s p e c i f i c (see F a s t i e r , 1964) i n that p h y s i c a l presence i s not the only f a c t o r i n v o l v e d . The act i o n can be modified by the s t r u c t u r e of the agent, yet the s t r u c t u r a l requirement f o r the bas i c a c t i o n i s minimal and a wide v a r i e t y of agents can block f u n c t i o n s of the Na + channel i n a s i m i l a r manner. Thus, the ac t i o n of membrane s t a b i l i z e r s on the e l e c t r o s e n s i t i v e Na + channel can be considered a n o n s p e c i f i c receptor-mediated a c t i o n . In the r e s u l t s s e c t i o n , evidence w i l l be presented that membrane s t a b i l i z e r s a l s o have d i r e c t though r e l a t i v e l y n o n s p e c i f i c a c t i o n on the n i c o t i n i c receptor. i i i ) Steady s t a t e k i n e t i c s ; acceptor vs. e f f e c t o r blockade Much of the mathematical formulation f o r q u a n t i t a t i n g the e f f e c t s on a pharmacological response of receptor blockade i s borrowed from enzyme k i n e t i c s (see Dixon and Webb, 1956). C l a s s i c a l enzyme k i n e t i c s puts much emphasis on the 'active s i t e ' of the enzyme where enzyme both binds substrate and transforms i t i n t o enzyme product. Three bas i c forms of i n h i b i t i o n of the process are recognized. These are - 104 -c o m p e t i t i v e , noncompetitive and uncompetitive which d i s t i n g u i s h between s i t u a t i o n s where the a n t a g o n i s t i c a c t i o n on the enzyme i s pre-vented by, independent o f , or dependent on the presence of the sub-s t r a t e at the a c t i v e s i t e r e s p e c t i v e l y . Drug receptors on the other hand, g e n e r a l l y have separate acceptor and e f f e c t o r components both of which can be i n f l u e n c e d by drugs. Moreover, the r e l a t i o n between drug concentration and response may be d i s t o r t e d when the measured response i s several steps subsequent to the event produced by a c t i v a t i o n or i n h i b i t i o n of the primary e f f e c t o r ( i . e . the e f f e c t o r w i t h i n the receptor f u n c t i o n a l u n i t ) . In s p i t e of these d i f f i c u l t i e s i t i s p o s s i b l e to apply the concepts of c o m p e t i t i -ve, noncompetitive and uncompetitive antagonism to drug receptor blockade. Competitive blockade would a r i s e when the antagonist binds s p e c i -f i c a l l y and r e v e r s i b l y to the primary acceptor s i t e of the receptor This type of blockade can be overcome simply by i n c r e a s i n g agonist c o n c e n t r a t i o n . Hence, such a blockade w i l l increase the EC50 f o r the agonist. Blockade of the e f f e c t o r component w i l l only give r i s e to noncompetitive antagonism i f i n t e r a c t i o n with the e f f e c t o r component has no e f f e c t on the acceptor s i t e and conversely i n t e r a c t i o n of the agonist with the acceptor s i t e has no e f f e c t on the a f f i n i t y of the receptor f o r the antagonist. Such an antagonism w i l l reduce the maxi-mum primary response without changing the EC50. However, i t i s becoming i n c r e a s i n g l y c l e a r that pure noncompetitive antagonists are r e a l l y q u i t e rare (see Pennefather and Quastel, 1982) and that the s t a t e of the receptor in f l u e n c e s the drug a f f i n i t y f o r both acceptor - 105 -and e f f e c t o r components of the rece p t o r . For example, t h i o p e n t a l ap-pears to block the e f f e c t o r component of the ACh receptor only a f t e r the receptor has been a c t i v a t e d . Indeed i t has been suggested that i t binds to a s i t e w i t h i n i o n i c channels that i s opened by ACh-receptor i n t e r a c t i o n (Adams, 1976). This type of blockade gives the appearance of uncompetitive antagonism. The f r a c t i o n of receptors that become blocked increases with the f r a c t i o n of receptors that are a c t i v a t e d . Thus, such a blockade involves a much greater reduction of the maximal response than the response produced by low l e v e l s of receptor occupan-cy. As a r e s u l t , the apparent EC50 f o r the primary response can actu-a l l y be reduced. Various types of mixed blockade, intermediate between pure compe-t i t i v e and pure uncompetitive, can e x i s t depending on the r e l a t i v e a f f i n i t y of the various s t a t e s f o r the e f f e c t o r antagonist. This becomes apparent when a simple four s t a t e , c y c l i c system i s considered. A + R + D 7 AR + D A + RD K 7 ARB At e q u i l i b r i u m , one has: K a 1[AR] = [A ] [ R ] ; K H 1[ARD] = [AR][D] K,p[ARD] = [A][RD]; K H ?[RD] = [R][D] And thus, [AR]/[R t] = { l + [ D ] / K d i + (1 + [ D ] / K d 2 ) K a l / [ A ] ] - l - 106 -I f the drug (D) has a higher a f f i n i t y f o r the i n a c t i v e s t a t e of the receptor (R) ( i . e . K d 2 « K ^ ) , then blockade can be p a r t i a l l y overcome by i n c r e a s i n g the agonist c o n c e n t r a t i o n . This gives the blockade more of the appearance of a competitive type. Uncompetitive aspects of the blockade are enhanced when the converse i s t r u e . I t i s also apparent that at e q u i l i -brium i f the antagonist has d i f f e r e n t a f f i n i t i e s f o r the nonactivated and a c t i v a t e d s t a t e s and i f the a c t i v a t e d s t a t e e x i s t s only when agonist i s associated with the re c e p t o r , then binding of the antagonist w i l l i n f l u e n c e the a f f i n i t y of the agonist such that K ^ K ^ = K a 2 ^ d 2' Thus, ^ t n e antagonist has a high a f f i n i t y f o r the a c t i v a t e d s t a t e , the agonist receptor complex w i l l be s t a b i l i z e d by the presence of antagonists. In a sense, the antagonist traps the agonist on the receptor. The actual s i t u a t i o n i s l i k e l y to be even more complicated s i n c e i t i s now recognised that receptors can e x i s t i n st a t e s other than a c t i v a t a b l e and a c t i v a t e d . In the case of receptor a c t i v a t i o n by ACh, binding of at l e a s t two ACh molecules i s required (see chapter 4 s e c t i o n c , i i i ) so that there must e x i s t a monoliganded s t a t e which precedes receptor a c t i v a t i o n . Expo-sure of the receptor to agonist f o r more than a few seconds gives r i s e to the development of d e s e n s i t i z e d states where agonist binding to the receptor no longer leads to a response (see chapter 4, s e c t i o n c , i i ) ) . Moreover, there seems to be at l e a s t two types of d e s e n s i t i z e d s t a t e s . - 107 -Many of the r a t e constants involved i n the bindin g and d i s s o c i a t i o n of antagonists and i n the in t e r c o n v e r s i o n between receptor s t a t e s are q u i t e slow r e l a t i v e to the time course of events involved i n synaptic transmis-s i o n . Thus s t a t e s which may have important e f f e c t s on the e q u i l i b r i u m or steady s t a t e response to agonist may have l i t t l e or no e f f e c t on synaptic t r a n s m i s s i o n . Steady s t a t e k i n e t i c s are the basis of much of q u a n t i t a t i v e pharmacology and are useful i n d e s c r i b i n g i n t e r a c t i o n s of drugs and b i o l o -g i c a l systems where steady s t a t e e x i s t s . However, t h i s i s not the case with s y n a p t i c transmission and new approaches are r e q u i r e d . One of the aims of the work described i n t h i s t h e s i s was to develop such new approaches. - 108 -CHAPTER 5 Methods f o r analyzing the k i n e t i c s of ACh receptor a c t i v a t i o n . a) Preface A c t i v a t i o n of ACh r e c e p t o r s , at the end-plate, i s associated with the opening of i o n i c channels permeable to Na , K and a number of other s m a l l , p o s i t i v e l y charged and ne u t r a l molecules, the l a r g e s t of which are about 0.6-0.7 nm i n diameter (Maeno, Edwards and Anraku, 1977; Huang, Catte-r a l l and Ehrenstein, 1978; Under and Quastel, 1978; Adams, Dwyer and H i l l e , 1980). In the presence of a voltage across the muscle membrane t h i s permea-b i l i t y change can be measured as a flow of c u r r e n t . A number of e l e c t r o -p h y s i o l o g i c a l methods are p r e s e n t l y being used to analyze the rates at which these channels open and c l o s e . These methods in c l u d e : noise a n a l y s i s , r e l a x a t i o n a n a l y s i s , s i n g l e channel a n a l y s i s , and a n a l y s i s of the time course of MEPCs. Only the l a s t method does not r e q u i r e a steady s t a t e r e s -ponse to exogeneous agonist and most of the work i n t h i s t h e s i s makes use of the l a s t method. Nevertheless, information can be obtained from the other methods and they merit some d i s c u s s i o n s i n c e the present r e s u l t s must be i n t e r p r e t e d i n l i g h t of r e s u l t s using these other methods. - 109 -b) Noise a n a l y s i s The elementary ACh receptor event was f i r s t s u c c e s s f u l l y measured by Katz and M i l e d i i n 1970. Fatt and Katz (1952) had p r e v i o u s l y r u l e d out that the MEPP i t s e l f represented the elementary event s i n c e the response to bath applied ACh was continuously graded even at an amplitude of an order of magnitude smaller than the MEPP. However, Katz and M i l e d i (1970) l a t e r recognized that during a steady a p p l i c a t i o n of agonist, the molecular bom-bardment of receptors might be discernable as an increase i n membrane noise superimposed on the maintained average d e p o l a r i z a t i o n . I f these elementary events occur at random with frequency n and each i s described by the func-t i o n f ( t ) , then the mathematical a n a l y s i s of random noise (Rice 1944) i n d i -cates t h a t : — the mean steady s t a t e response u = n f ^ * f ( t ) dt 5.1 0 — and the mean e x t r a variance Var = n j f ( t ) dt 5.2 0 I f the elementary response i s a square pulse with an amplitude 'a', and duration T", then: u = n a T and Var(u) = na^T Hence, the variance to mean r a t i o w i l l give an estimate of ' a 1 , the amplitude of the elementary event. Katz and M i l e d i (1970, 1971, 1972) found - 110 -that the e x t r a noise was indeed measurable and the variance to mean r a t i o s i n d i c a t e d that the amplitude of the elementary event was less than 0.1 per-cent of the MEPP height. Another way of looking at t h i s r e l a t i o n s h i p i s to consider N independent channels that can be e i t h e r in the open or closed s t a t e , with p r o b a b i l i t i e s P and (1-P), r e s p e c t i v e l y . I f channels open and c l o s e at random the mean number open at any moment w i l l be Y where: Y = NP = u/a Thus, Var(Y) = NP(l-P) Var(u) = Na 2P(l-P) 5.3 Var(u)/u = a ( l - P ) 5.4 The equation s,howss that as P becomes l a r g e , with i n c r e a s i n g agonist con-c e n t r a t i o n , the variance to mean r a t i o decreases. Though in most case t h i s i s an a r t i f a c t to be avoided, i t can be u t i l i z e d to determine the f r a c t i o n of n i c o t i n i c receptors a c t i v a t e d by a given concentration of agonist and used to construct dose response curves. Using t h i s approach Sakmann and Adams have estimated that channels are open h a l f of the time at 20 uM ACh (-80 mV, 10°C) (see Adams, 1981). This corresponds almost e x a c t l y to the value determined by d i r e c t observation of s i n g l e channel events with a patch clamp-(see Sakmann et a l . , 1980). A s i m i l a r approach has been taken in the i n v e s t i g a t i o n of the dose response r e l a t i o n s h i p of responses produced by a c t i v a t i n g muscarinic receptor in the heart S.A node (see Noma, Peper, and Trautwein 1979). - I l l -A n a l y s i s of the frequency components of the noise can be used to deter-mine the time course of the elementary event. Much of t h i s type of a n a l y s i s i s based on Fou r i e r ' s theorem that n o ise, or f o r that matter, any temporal f u n c t i o n , can be expressed as the weighted sum of sine and cosine waves of d i f f e r e n t f r e q u e n c i e s . I f the elementary event i s an impulse f u n c t i o n that i s i n f i n i t e l y b r i e f then a l l frequencies can be found e q u a l l y i n the r e s u l -tant noise (by analogy t h i s i s c a l l e d white n o i s e ) ; the power density spec-trum (a p l o t of power de n s i t y vs. frequency) i s a s t r a i g h t h o r i z o n t a l l i n e . However, as soon as the elementary event l a s t s f o r some time, the power den-s i t y of higher frequencies i s reduced, i n e x a c t l y the same way as i f the impulse f u n c t i o n had been f i l t e r e d . As a r e s u l t the power de n s i t y spectrum f a l l s at higher frequencies. At one time these s p e c t r a were determined by repeatedly passing the same s t r e t c h of recorded noise through a narrow band pass f i l t e r , stepping the center frequency each time, and measuring the variance c o n t r i b u t e d by each frequency. The power de n s i t y of a given f r e -quency, W(f), i s the po r t i o n of the t o t a l variance due to that frequency. Thus More r e c e n t l y , an algorithm c a l l e d the Fast F o u r i e r Transform, that makes use of d i g i t a l computer processing of the s i g n a l , has g r e a t l y speeded the c a l c u l a t i o n of these spectra (see Bendat and P i e r s o l 1971) Noise can al s o be defined by the a u t o c o r r e l a t i o n f u n c t i o n . An estimate f o r the a u t o c o r r e l a t i o n between values at u(T) at time T and t + T may be obtained by t a k i n g the product of the two values and averaging over the observation time L. The r e s u l t i n g average product w i l l approach the exact a u t o c o r r e l a t i o n product as L approaches i n f i n i t y . Thus, W(f) = d Var(u)/df 5.5 5.6 oo 0 - 112 -The a u t o c o r r e l a t i o n f u n c t i o n and the power d e n s i t y spectra are c l o s e l y r e l a t e d ; the former describes those frequencies absent from the noise while the l a t t e r descibes those frequencies present i n the n o i s e . The F o u r i er transform of the a u t o c o r r e l a t i o n f u n c t i o n i s i d e n t i c a l to the power d e n s i t y spectrum f o r a given s t r e t c h of ' n o i s e 1 . In order to i n t e r p r e t e i t h e r of these f u n c t i o n s m e c h a n i s t i c a l l y , a model of the elementary event has to be proposed and the t h e o r e t i c a l curves gene-ra t e d by the model have to be f i t t e d to the data. With a f u l l agonist the a u t o c o r r e l a t i o n f u n c t i o n i s indeed found to be a s i n g l e e x p o n e n t i a l , sug-gesting that there are only two major s t a t e s of the channel — open and c l o s e d . In general, i f there are n states of the channel, the a u t o c o r r e l a -t i o n f u n c t i o n has (n-1) components (Colquhoun and Hawkes, 1977). Since at l e a s t 2 ACh molecules are necessary to a c t i v a t e the r e c e p t o r , and the chan-nel opening involves an i s o m e r i z a t i o n step, there must be intermediate s t a t e s between the ground s t a t e and the a c t i v a t e d s t a t e . I f there i s no cooperation i n binding before the i s o m e r i z a t i o n step and both binding s i t e s on the receptor are e q u i v a l e n t , then a simple model d e s c r i b i n g channel a c t i -v a t i o n would be 5.7 a - 113 -Since there i s only one component to the a u t o c o r r e l a t i o n f u n c t i o n or the power d e n s i t y spectrum, t h i s model must c o l l a p s e to one of the form e' 2 A + R ZZT A 2R* 5.8 a' That i s , c l o s i n g of a channel does not e n t a i l r e v e r s i o n of receptor to a form that has a high p r o b a b i l i t y of r e t u r n i n g to the open channel s t a t e . Sakmann and Adam (1978) have o u t l i n e d the three c o n d i t i o n s which can r e s u l t i n t h i s k i n e t i c 'degeneracy'. The f i r s t r e s u l t s from a steady s t a t e assumption; the r a t e l i m i t i n g step i n opening i s s e t t i n g up A 2R which i s o -merizes very r a p i d l y to the open form A 2R , while the r a t e l i m i t i n g step in c l o s i n g i s the i s o m e r i z a t i o n . Since the channel i s u n l i k e l y to reopen a f t e r c l o s i n g k_ 2 must be l a r g e . The second c o n d i t i o n i s one i n which the is o m e r i z a t i o n i s r a p i d ; here the channel f l i c k e r s open and closed too r a p i d -l y to be detected. The t h i r d p o s s i b i l i t y a r i s e s i f the binding of agonist reaches e q u i l i b r i u m q u i c k l y r e l a t i v e to the is o m e r i z a t i o n step. Table 3 summarizes these 3 p o s s i b i l i t i e s . In a l l cases e' i s a fu n c t i o n of [A] and at low agonist concentrations the time constant of the system i s equal to a'. Note that of these three simple models only the r a t e l i m i t i n g i s o m e r i z a t i o n assumption p r e d i c t s t h a t the time constant w i l l s aturate at high agonist concentrations. - 114 -TABLE 3. R e l a t i o n s h i p s between r a t e constants, determining the channel open-ing and c l o s i n g r a t e , that can give r i s e to s i n g l e components i n a u t o c o r r e l a t i o n f u n c t i o n s of current f l u c t u a t i o n s produced by ago-n i s t at the neuromuscular j u n c t i o n . Assumption R e l a t i o n of Rate Constants B' •a' Steady s t a t e k , k , 6 » k [ A ] ,