Properties of Specific Alcohol Binding Sites

描述（由申请人提供）：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）超家族的跨膜（TM）结构域中已经描述了醇的特异性结合位点，所述配体门控离子通道（LGIC）超家族包括甘氨酸（GlyRa 1）、GABA、烟碱乙酰胆碱和5-HT 3受体。我们的全球性假设是，醇类结合在这些受体的TM片段所界定的空腔内，并优先稳定特定的通道substates。我们的目标是确定这些网站的属性，介导的结合和疗效的酒精和酒精拮抗剂GlyRa 1。这些结合位点可以提供用于在其他类型的离子通道和其他重要蛋白质内结合醇的共同基序。我们将建立GlyRa 1结合位点的计算模型，并设计特定的定点突变来验证这些假设。这些突变将由我们的合作者R博士构建和测试。A.哈里斯目标1.我们将测试的假设，从所有四个跨膜螺旋的GlyRa 1的氨基酸残基有助于醇的结合位点。我们将开发计算模型来描绘这些网站的尺寸。我们将使用这些模型来预测烷基甲硫基磺酸盐（MTS）试剂的共价结合将模拟醇结合。将在Harris实验室通过在卵母细胞中表达含有定点半胱氨酸取代的GlyRa 1来测试预测。他们将MTS试剂应用于卵母细胞，并测量对甘氨酸诱导电流的影响。目标二。我们将测试的假设，双位点定向半胱氨酸突变可以澄清精细的三级结构的GlyRa 1和区分我们的模型从一个基于鱼雷nAChR。虽然我们的GlyRa 1模型和Unwin的nAChR模型的整体结构是全局一致的，但跨膜螺旋和限制可能的醇结合位点的残基的方向存在重要差异。哈里斯实验室将通过交联定点双半胱氨酸来测试这些预测。总之，GlyRa 1中的酒精结合位点的知识将增加我们对LGIC中酒精作用的理解。这些结果可能揭示了在其他受酒精影响的系统中作用的一般模式，并有助于设计酒精拮抗剂。

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.