Miller, P.S.; (2005) Molecular interactions of zinc with the glycine receptor. Doctoral thesis , University of London.

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Abstract

The ionotropic receptors including nicotinic acetylcholine (nAChR), y-aminobutyric acid type A and C (GABAa/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 al 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+. 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.

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