CHARACTERIZATION OF STRYCHNINE BINDING SITES IN THE RODENT SPINAL CORD BY VINCENT MAURICE O’CONNOR UNIVERSITY COLLEGE LONDON A thesis submitted for the degree of Doctor of Philosophy from the University of London, 1992. I ProQuest Number: 10608898 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10608898 Published by ProQuest LLC(2017). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 This thesis is dedicated to the select band o f people, including the most recent arrivals and the sorely missed departed, that I hold closest in my affections. "At the end of the day" they make It all worthwhile. Remember, in the words of the famous actor whose name at present escapes me; "Nobody said it would be easy". Although my own feeling is that Nobody was probably wrong. II Thesis abstract The convulsant alkaloid strychnine is a selective and highly potent antagonist at postsynaptic receptor for the inhibitory neurotransmitter glycine . These properties have led to the extensive use of strychnine as a ligand to probe the postsynaptic glycine receptor. Despite the recent increased understanding of the molecular structure of this receptor protein there is still much dispute as to the nature of the interaction between glycine and strychnine. In an attempt to more clearly define this interaction the experiments described, combine the techniques of protein modification and ligand binding. These investigations also revealed a novel [3H]-strychnine binding site in the rodent CNS and attempts were made to characterize this phenomenon. The results described suggest that glycine is a fully competitive inhibitor of [3H]- strychnine binding and its reported action as a partial competitive inhibitor is an artefact of the assay conditions. The disruption of [3H]-strychnine binding by residue selective protein modifying reagents suggests some overlap in the strychnine and glycine binding sites at the receptor. Protection studies confirm this and the results are best explained by overlapping yet conformationally distinct recognition sites for strychnine and glycine. Experiments which describe protein modification and ligand protection of strychnine binding antisera highlight possible congruence in the molecular recognition at the antisera and the receptor. This is of interest in the light of proposed models of the strychnine binding site at the postsynaptic glycine receptor. Modification of spinal cord membranes by the arginine selective reagent 2,3- butanedione (BD) reveals a low affinity and high capacity [3H]-strychnine binding site which is not detectable in untreated membranes. This binding site showed a similar distribution in the CNS as the high affinity site. However, experiments using affinity purified glycine receptor and crude membrane preparations from the mutant mouse spastic indicated that the BD-induced binding site is not located on the postsynaptic glycine receptor. Competition studies revealed that [3H]-strychnine binding sites in untreated and BD-treated membranes have different structural determinants. The ability to effectively inhibit [3H]-strychnine binding to the BD-induced site by cation channel blockers is in accord with reports that strychnine can interact with various cation channels to open or block them. In addition several compounds that inhibit the BD-induced [3H]-strychnine binding can also modulate the reaction of BD with spinal cord membranes if present during the treatment, suggesting a conformational dependent modification. Upon exposure to ultraviolet light [3H]-strychnine is specifically incorporated into a low molecular weight peptide in BD-treated membranes in addition to the ligand binding subunit of the inhibitory glycine receptor, which is the only peptide photolabelled in untreated membranes. The significance of this biochemical and pharmacological characterization of this previously undescribed strychnine binding site is presently unclear. However, the uneven distribution in the CNS and the interaction with important therapeutic agents; local anaesthetics and anti-arrhythmics, indicate the possible biological importance of the novel strychnine binding site. Ill ACKNOWLEDGEMENTS I would like to thank Jonathan Fry for his scientific supervision and optimism throughout the time I have been at University College. I am grateful to the Department and its members for their encouragement in all aspects of my education. I am particularly indebted to: Christine Williams, for her skilful and patient preparation of plates and drug structures, experimental assistance and advice on parenting. Keith Caddy and my buddy Alex who in combination contrived to enable my figures to be printed, although I am grateful for the assistance of many others with regard to this matter. Mabel Kendrick for laboratory luxuries which includes the steady supply of confectionaries. Ian Martin for supplying the glycine receptor alignments along with discussion on my trips to Cambridge or his to London. Dennis Haylett for discussion and supplying several compounds especially the channel blockers that were initially screened. Also I would like to thank the individuals and companies listed in Chapter Two for the generous supply of drugs. In particular I would like to thank G.A.R. Johnston and Hue Tran for synthesizing 2- aminostrychnine and thereby enabling us to bypass the shortage of commercially available compound. I would like to thank my peers at University College especially Boris Barbour, Margaret "Bridgett" Carey and Gerard "de Pad" Christoffi for scientific discussion and a series of lighter moments that helped make my time so stimulating. I would also like to acknowledge the MRC for providing my stipend. In addition I am grateful to the MRC, The Physiological Society, The Biochemical Society and the Department for providing the funding that enabled me to attend conferences and courses. In particular I am grateful to all those who encouraged and made it possible for me to attend MBL, the scientific paradise at Woods Hole. This thesis is my own account of investigations carried out by myself under the supervision of Jonathan Fry. IV ABBREVIATIONS 2-AS 2-aminostrychnine 5-HT 5-hydroxytryptamine Ach acetylcholine B bound ligand BD 2,3-butanedione Bm« maximal number of binding sites BSA bovine serum albumin CAD cationic amphipathic drug cDNA cloned deoxyribonucleic acid CHAPS 3-[(3-Cholamidopropyl)-dimethylammonio]-l- propanesulphonate CHD 1,2-cyclohexanedione CNS central nervous system cpm counts per minute CQS camphorquinone- 10-sulphate D-600 methoxy verapamil DEP diethylpyrocarbonate dpm disintegrations per minute DSA diasotized sulphanilate DT diazonium tetratzole DTT dithiothreitol EDTA ethylenediaminetetraacetic acid FI flurescein isothiocyanate GABA y-amino-N-butyric acid IAA iodoacetamide IC50 concentration of displacer which inhibits 50% of control binding Ig immunoglobulin Iso-THAZ 5,6,7,8-tetrahydro-4H-isooxazolo[3,4]azepin-3-ol Ki association rate constant k.i dissociation rate constant kd kilodalton Kd equilibrium dissociation constant K j equilibrium inhibition constant KLH keyhole limpet haemocyanin M merthiolate mAb monoclonal antibody mRNA messenger ribonucleic acid NA noradrenaline NBS N-bromosuccinimide NEM N-ethylmaleimide NMDA N-methyl-D-aspartic acid PAGE polyacrylamide gel electrophoresis PBB phosphate buffered borate PBS phosphate buffered saline PCR polymeraze chain reaction PEG polyethylene glycol PEI polyethylenimine V PG phenylglyoxal PLP pyridoxal phosphate PMSF phenylmethane sulphonyl fluoride pPG p-hydroxy-phenylglyoxal PTC phosphatidylcholine SBTI soya bean trypsin inhibitor SDS sodium dodecyl sulphate SEM standard error of the mean spa spastic TBPS t-butylbicyclophosphorothionate TCA trichloroacetic acid TEA tetraethylammonium TEMED NNN *,N-tetramethylethylenediamine TNBS trinitrobenzosulphonic acid TNM tetranitromethane UV ultraviolet Single letter code for amino acids A alanine C cysteine D aspartic acid E glutamic acid F phenylalanine G glycine H histidine I isoleucine K lysine L leucine M methionine N asparagine P proline Q glutamine R arginine S serine T threonine V valine W tryptophan Y tyrosine VI CONTENTS PAGE Title I Dedication II Abstract m Acknowledgements IV Abbreviations V Chapter One Introduction i 1.1 Introduction i 1.2 Actions of strychnine on CNS inhibition. i 1.3 Establishing glycine as an inhibitory transmitter in CNS. 3 1.4 Use of strychnine as a molecular probe to study the inhibitory glycine receptor. 4 1.4.a Distribution of glycine receptors in the CNS. 5 1.4.b Ionic modulation of [3H]-strychnine binding. 8 1.5 Biochemical characterization of the inhibitory glycine receptor. 10 1.5.a Solubilization and affinity purification of the inhibitory glycine receptor. 10 1.5.b Photolabelling the inhibitory glycine receptor. 11 1.5.C Antibodies specific for the inhibitory glycine receptor. 12 1.5.d Subunit associations in the inhibitory glycine receptor. 13 1.5.e Developmental regulation of [3H]-strychnine binding and inhibitory glycine receptor. 16 1.6 Molecular Genetic Analysis