Glycine receptors (GlyR) are inhibitory ligand gated ion channels that is implicated in mediating ethanol's actions in the CMS and spinal cord. A putative binding site for alcohols and anesthetics has been proposed, but not completely defined. Additionally, how this binding site signals modulation of the glycine-gating signal is unclear. We propose to use oocyte electrophysiology and introduced cysteine point mutations to probe the TM2-TM3 linker region for its involvement in binding ethanol or transducing ethanol's modulatory signal. The overall goal of this project is to determine the role of TM2-TM3 linker region in allosteric modulation of a1 GlyR through identification of residues that have a direct or indirect role in the putative alcohol-binding pocket.
Our first aim i s to determine the accessibility of the TM2-TM3 linker region (R271-D284) to PMTS, an alcohol analog that covalently binds cysteines, in the presence and absence of glycine and the effects on alcohol binding. The hypothesis is that PMTS will bind to cysteines introduced into the TM2-TM3 linker and permanently potentiate glycine currents. The next aim is to find residues in the TM2-TM3 linker that are able to cross-link with S267C or A288C (known residues in the putative alcohol-binding pocket). The hypothesis is that residues that reacted to PMTS in the first aim makes up part of the putative alcohol-binding pocket, then they should be close enough to S267C or A288C to form a disulfide bond. Our ultimate goal is to determine where alcohol binds and how it modulates glycine receptor function in order develop drugs that specifically target alcohol's actions on the glycine receptor. Additionally, because glycine shares homology with other cys-loop ion channels, including GABAA, nicotinic acetylcholine, and serotonin-3 receptors, ? ? ?
| Welsh, Brian T; Todorovic, Jelena; Kirson, Dean et al. (2017) Disruption of a putative intersubunit electrostatic bond enhances agonist efficacy at the human ?1 glycine receptor. Brain Res 1657:148-155 |
