Properties of Specific Alcohol Binding Sites

描述(申请人提供)：酗酒和酗酒是主要的健康问题。这些问题的解决很可能需要了解酒精对神经元离子通道蛋白的影响。在配体门控离子通道(LGIC‘s)超家族的跨膜(TM)结构域中已描述了醇的特定结合部位，该超家族包括甘氨酸(GlyRa1)、GABA、烟碱型乙酰胆碱和5-HT3受体。我们的全球假设是，酒精结合在这些受体的TM片段所限制的空腔内，并优先稳定特定的通道亚态。我们的目标是确定那些调节酒精和酒精拮抗剂在GlyRa1中的结合和有效性的位点的属性。这些结合位点可能为醇在其他类型的离子通道和其他重要蛋白质中的结合提供了一个共同的基序。我们将建立GlyRa1结合位点的计算模型，并设计特定的定点突变来检验这些假设。这些突变将由我们的合作者R.A.哈里斯博士构建和测试。目的1.我们将检验GlyRa1的所有四个跨膜螺旋上的氨基酸残基对酒精的结合部位有贡献的假设。我们将开发计算模型来描绘这些场地的尺寸。我们将使用这些模型来预测烷基甲烷硫磺(MTS)试剂的共价结合将在哪里模拟酒精结合。这些预测将在哈里斯实验室通过在卵母细胞中表达含有半胱氨酸定点替代的GlyRa1来验证。他们将把MTS试剂应用于卵母细胞，并测量其对甘氨酸诱导电流的影响。目的2.我们将检验双位点定向半胱氨酸突变可以阐明GlyRa1的精细三级结构的假设，并将我们的模型与基于鱼雷nAChR的模型区分开来。虽然我们的GlyRa1模型和Unwin的nAChR模型的总体结构是一致的，但在跨膜螺旋和结合可能的酒精结合位点的残基的方向上有重要的差异。哈里斯实验室将通过交联定点定向的二半胱氨酸来检验这些预测。总之，了解GlyRa1上的酒精结合位点将增加我们对酒精在LGIC中作用的理解。这一结果可能揭示酒精影响的其他系统中的一般作用基序，并有助于酒精拮抗剂的设计。

DESCRIPTION (provided by applicant): Alcohol abuse and alcoholism are major health problems. It is likely that a solution to these problems will require an understanding of the effects of alcohol on neuronal ion channel proteins. Specific binding sites for alcohols have been described in the transmembrane (TM) domain of the superfamily of ligand-gated ion channels (LGIC's) that includes glycine (GlyRa1), GABA, nicotinic acetylcholine, and 5-HT3 receptors. Our global hypothesis is that alcohols bind within cavities that are bounded by TM segments of these receptors and preferentially stabilize specific channel substates. Our goal is to define the properties of those sites that mediate binding and efficacy of alcohol and alcohol antagonists in GlyRa1. These binding sites may provide a common motif for binding of alcohols within other classes of ion channels and other important proteins. We will build computational models of binding sites in GlyRa1 and design specific site-directed mutations to test these hypothesis. These mutations will be constructed and tested by our collaborator, Dr. R. A. Harris. Aim 1. We will test the hypothesis that amino acid residues from all four transmembrane helices of GlyRa1 contribute to a binding site for alcohols. We will develop computational models to delineate the dimensions of these sites. We will use these models to predict where covalent binding of alkyl methanethiosulfonate (MTS) reagents would mimic alcohol binding. The predictions will be tested in the Harris laboratory by expressing GlyRa1 containing site-directed cysteine substitutions in oocytes. They will apply MTS reagents to the oocytes and measure the effects on glycine-induced currents. Aim 2. We will test the hypothesis that double site directed cysteine mutations can clarify the refined tertiary structure of the GlyRa1 and distinguish our model from one based on the torpedo nAChR. While the overall structure of our GlyRa1 model and the nAChR model of Unwin are in global agreement, there are important differences in the orientation of transmembrane helices and residues bounding a possible alcohol- binding site. The Harris laboratory will test these predictions by crosslinking site-directed di-cysteines. In summary, knowledge of alcohol binding sites in GlyRa1 will increase our understanding of alcohol action in LGIC's. The results may reveal general motifs for action in other systems affected by alcohol and aid in the design of alcohol antagonists.