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Molecular Interactions of Zinc with the Glycine Receptor

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Molecular Interactions of Zinc with the Glycine Receptor Molecular Interactions of Zinc with the Glycine Receptor A dissertation submitted to the University of London for the degree of Doctor of Philosophy By Paul Steven Miller, B.Sc.(Hons) Department of Pharmacology Medical Sciences Building University College London Gower Street London WC1E6BT October 2004 UMI Number: U592189 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 complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U592189 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 In memory of my Grandfather Abstract The ionotropic receptors including nicotinic acetylcholine (nAChR), y-aminobutyric acid type A and C (GABA a /c ), serotonin (5HT-3) and glycine receptors (GlyR) all comprise the cys-loop receptor family. A pentameric configuration is adopted by these receptors featuring a large extra cellular domain, four transmembrane (M) spanning domains and an intracellular compartment primarily contributed from the M3-4 loop. The recent advent of an X-ray crystal structure of the related ACh binding protein from snail, Lymnaea stagnalis, and an electron micrograph nearing atomic resolution of the transmembrane domains for the Torpedo nAChR provide the opportunity to homology model the structures of all cys-loop ligand-gated receptors and investigate critical elements of receptor function with unprecedented accuracy. By using homology modelling to guide site-directed mutagenesis in combination with whole-cell patch clamp electrophysiology, molecular elements that mediate the biphasic modulation of glycine receptors by Zn2* were investigated. The structure of a previously identified low sensitivity inhibitory Zn2+ site was clarified revealing critical determinants of GlyR subtype selectivity for Zn2* mediated inhibition. Intriguingly these experiments also revealed a novel functional asymmetry at this site suggesting that one specific side of the site directs downstream transduction of the inhibitory Zn2^ effect. This enabled the elucidation of a hydrophobic pathway leading directly through the core of the GlyR from the inhibitory Zn2+ site to the agonist binding site suggesting a mechanism for Zn2* mediated inhibition. This study also investigated the structural basis for Zn2* enhancement of GlyR function. A comparative study of GlyR a l and a2 followed by a directed mutagenesis regional scan led to the identification of a complete and novel site for Zn2+ potentiation. Further studies also elucidated a potential mechanism of action for this site by identifying an important gating component of the GlyR, which exhibited a strong interaction with this novel potentiation site for Zn2+. 1 These studies demonstrate the power of a homology modelling strategy to resolve key elements of cys-loop ligand-gated receptor function. Identifying and understanding receptor modulation by allosteric effectors is a key requisite to understanding receptor function. This work describes the key structural determinants involved in the biphasic Zn2+ modulation of GlyRs both in terms of actual binding sites and downstream effector mechanisms. 2 Contents Abstract l Contents 3 List of Tables 9 List of Figures 10 Chapter 1 - Introduction Inhibitory neurotransmission: The glycine receptor 13 The physiology of glycine receptors Distribution 16 The role of the glycine receptor 17 Glycine receptor synaptic signalling partners 18 Intracellular signalling interactions with the glycine receptor 20 Glycine receptor subtypes: Their respective distributions, properties and functions Isolation of glycine receptor subtypes 20 Glycine receptor a 1 - The rapid responder 23 Glycine receptor a2 - The developmental secretary 25 Glycine receptor a3 - The bearer of great pain 26 Glycine receptor a4 - The enigma 27 Gephyrin and the glycine receptor P-subunit 28 Kinetics and desensitisation Kinetics 28 Desensitisation 33 Structure-Function studies General topology 34 The agonist binding site 36 3 The transduction process 42 The ion channel and its opening mechanism 49 The intracellular domain 53 Pharmacology Alcohols and anaesthetics 55 Picrotoxin (PTX) and cyanotriphenylborate (CTB) 56 Zinc The molecular basis of Zn2^ modulation of glycine receptors 57 Zn2+ as a potential endogenous modulator of glycine receptors 60 Aims of the present study 64 Chapter 2 - Materials and Methods Reagents 66 Molecular biology: Glycine receptor cDNAs 66 Site-directed mutagenesis 67 Cell culture and transfection 70 Neuronal spinal cord cell culture preparation 71 Solutions 72 Electrophysiology 73 Data acquisition and analysis 74 Structural modelling 77 Statistics 78 4 Chapter 3 - Molecular determinants of glycine receptor aP subunit sensitivities to Zn2+ inhibition Introduction 80 Results Differential Zn2* sensitivity between GlyR a l and GlyR a2 subunits 81 Histidine 107 determines the differential Zn2+ sensitivity between GlyR a subunits 84 GlyR a3 exhibits low sensitivity to inhibition by Zn2+ 84 Mechanism for Zn2+ inhibition on GlyR a l and GlyR a2 subtypes 86 Rate of onset of Zn2+ inhibition at GlyR a l and a2 86 Differential sensitivity to Zn2+ inhibition is unaffected by the GlyR p subunit 90 GlyRs from cultured spinal cord neurones show a low non-al-like sensitivity to Zn2+ inhibition 90 Identification of the structural elements required for Zn2+ inhibition 95 Using the GlyR p subunit to investigate functional asymmetry at the inhibitory Zn2+ binding site 98 Discussion a l H I07 is responsible for high sensitivity GlyR mediated Zn2+ inhibition 101 Zn2^ inhibition affects the apparent affinity of the GlyR 101 The mechanism of Zn2^ inhibition 102 Native GlyRs retain the sensitivity to Zn2+ inhibition of their recombinant counterparts 104 The properties of the p subunit support an asymmetric role for the inhibitory Zn2^ site 105 A proposed molecular basis for the action of inhibitory Zn2+ 106 Comparisons with the GABAaR family of inhibitory Zn2+ binding sites 107 Conclusion 108 5 Chapter 4 - Evidence for an allosteric transduction pathway across the extracellular domain of the glycine receptor Introduction 111 Results Disruption of the GlyR a l ‘-4 face of the Zn2+ inhibition site generates a family of low potency zinc-activated GlyRs (ZAGs) 114 Disruption of the GlyR a l ‘+‘ face of the Zn2+ inhibition site does not induce ZAG activity 119 A putative hydrophobic transduction pathway between the Zn2^ inhibition and the agonist-binding sites 121 Disruption of the hydrophobic pathway generates spontaneously open GlyRs that are dependent on background Zn2+ and protons 122 Disruption of the hydrophobic pathway ablates Zn2+ mediated inhibition 126 Disruption of the hydrophobic pathway generates a family of high sensitivity ZAG receptors 126 ZAG receptors require binding components of the Zn2+ inhibitory site 131 The formation of high sensitivity ZAG receptors is specific to residues of the hydrophobic transduction apparatus 136 Both low and high sensitivity ZAG receptors exhibit a slower rate of macroscopic current activation 136 Discussion Evidence favouring an asymmetric function of the Zn2+ inhibition site 141 A simple hypothesis to explain ZAG formation 142 Evidence supporting the hydrophobic pathway as a transduction pathway 144 Reliance of Zn2^ binding residues at the high and low potency ZAGs and the original Zn2+ inhibition site 145 Consequences for the hydrophobic transduction pathway in determining macroscopic rates of receptor activation 146 The significance of related studies on ZAC formation 147 The rate of progress: As measured by the NMD A receptor 147 Conclusion 149 6 Chapter 5 - Identification of a Zn2+ potentiation site in glycine receptors and a mechanism of action involving the cys-loop gating domain Introduction 152 Results GlyR a l and a2 exhibit distinct sensitivities to Zn2+ mediated potentiation 154 GlyR a l E l92, D194 and H215 are all essential for high sensitivity to potentiating Zn2r 156 Asymmetry of function at the putative Zn2+ potentiation site 159 Comparative influence of GlyR a l El 92 and D 194 on p-strand 9 with H215 of p-strand 10 162 GlyR a l T151 is a critical control element for Zn2+ potentiation 166 The 5-HT3AR Zn2^ potentiation site does not operate through its GlyR T151 homologue 167 GlyR a 1 T151 influences apparent agonist gating 169 GlyR a l T151 and residues in the proposed Zn2^ potentiation site region can interact in mutated receptors 171 Discussion Evidence for a discrete Zn2+ potentiation site in GlyR a l 176 Comparison of the GlyR a l inhibition and potentiation sites 176 Transduction roles for residues at the GlyR a l Zn2+ potentiation site 177 A link between the GlyR a l Zn2^ potentiation site and the channel gating domain 178 Implications for Zn2+ potentiation at other GlyR subtypes 179 Implications for Zn2+ enhancement of 5-HT3ARs and nAChRs 180 Physiological significance of GlyR Zn2+ potentiation 182 Conclusion 182 7 Chapter 6 - Summary and Final Thoughts Acknowledgements Reference List List of Tables Chapter 1 1.1. The functions associated with residues in the GlyR transmembrane domains 52 Chapter 3 3.1. GlyR sensitivities to glycine
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