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

TM2-TM3 连接子在乙醇调节甘氨酸受体中的作用

• 批准号: 7328986
• 负责人: MICHELLE D Bayly
• 金额: $ 2.73万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7328986
• 关键词: Acetylcholine; Alcohols; Anesthetics; Binding; Binding Sites; Cysteine; Electrophysiology (science); Ethanol; Gated Ion Channel; Glycine; Glycine Receptors; Goals; Ion Channel; Ligands; Mediating; Oocytes; Pharmaceutical Preparations; Point Mutation; Role; Serotonin Receptors 5-HT-3; Signal Transduction; Spinal Cord; analog; crosslink; disulfide bond; mutant; pyridinium 3-methoxyestra-1,3,5(10)-trien-6-yl sulfate; receptor function

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,

描述（由申请人提供）：甘氨酸受体（GlyR）是一种抑制性配体门控离子通道，涉及介导乙醇在CMS和脊髓中的作用。已经提出了一个假定的醇类和麻醉剂的结合位点，但尚未完全确定。此外，这个结合位点如何调节甘氨酸门控信号尚不清楚。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连接区域是否参与结合乙醇或转导乙醇的调节信号。该项目的总体目标是通过鉴定在假定的醇结合口袋中具有直接或间接作用的残基，确定TM2-TM3连接区域在a1 GlyR变构调节中的作用。我们的第一个目标是确定在存在和不存在甘氨酸的情况下，TM2-TM3连接区域（R271-D284）对PMTS（一种共价结合半胱氨酸的醇类似物）的可及性以及对醇结合的影响。假设PMTS将与引入TM2-TM3连接体的半胱氨酸结合，并永久增强甘氨酸电流。下一个目标是在TM2-TM3连接体中找到能够与S267C或A288C交联的残基（已知残基在假定的醇结合口袋中）。假设是，在第一个目标中与PMTS反应的残基构成了假定的醇结合袋的一部分，那么它们应该足够接近S267C或A288C以形成二硫键。我们的最终目标是确定酒精结合的位置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对甘氨酸受体作用的药物。此外，由于甘氨酸与其他半胱氨酸环离子通道具有同源性，包括GABAA、烟碱乙酰胆碱和血清素-3受体，