The role of the TM2-TM3 linker in ethanol modulation of the Glycine Receptor

描述(申请人提供)：甘氨酸受体(GlyR)是一种抑制配体门控离子通道，与乙醇在CMS和脊髓中的作用有关。已经提出了酒精和麻醉剂的假定结合部位，但还没有完全确定。此外，该结合位点如何发出甘氨酸门控信号的调制信号尚不清楚。我们建议利用卵母细胞电生理学和引入半胱氨酸点突变来探测TM2-TM3连接区参与乙醇结合或转导乙醇的调制信号。本项目的总体目标是通过鉴定在推测的酒精结合口袋中具有直接或间接作用的残基来确定TM2-TM3连接区在A1 GlyR变构调节中的作用。我们的第一个目标是确定TM2-TM3连接区(R271-D284)对PMTS的可及性，PMTS是一种与半胱氨酸共价结合的酒精类似物，在甘氨酸存在和不存在的情况下，以及对酒精结合的影响。假设PMTS将与引入TM2-TM3连接子的半胱氨酸结合，并永久增强甘氨酸电流。下一个目标是在TM2-TM3连接子中找到能够与S267C或A288C(假定的酒精结合口袋中的已知残基)交联的残基。假设在第一个目的中与PMTS反应的残基构成了推测的酒精结合口袋的一部分，然后它们应该足够接近S267C或A288C以形成二硫键。我们的最终目标是确定酒精在哪里结合，以及它如何调节甘氨酸受体的功能，以便开发专门针对酒精在甘氨酸受体上的作用的药物。此外，由于甘氨酸与其他环状离子通道，包括GABAA、烟碱型乙酰胆碱和5-羟色胺-3受体具有同源性，

DESCRIPTION (provided by applicant): 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 is 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,