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。要解决这些问题，很可能需要了解酒精对神经元离子通道蛋白的影响。在包括甘氨酸（GlyRa1）、GABA、烟碱乙酰胆碱和5-HT3受体的配体门控离子通道（LGIC）超家族的跨膜（TM）结构域中，已经描述了醇的特定结合位点。我们的总体假设是，酒精结合在由这些受体的TM片段结合的空腔内，并优先稳定特定的通道亚态。我们的目标是确定介导GlyRa1中酒精和酒精拮抗剂结合的位点的特性和功效。这些结合位点可能为醇类在其他种类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，并设计特定的位点导向突变来验证这些假设。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。我们将验证GlyRa1所有四个跨膜螺旋的氨基酸残基都有助于醇的结合位点的假设。我们将开发计算模型来描绘这些地点的尺寸。我们将使用这些模型来预测烷基甲乙硫磺酸（MTS）试剂的共价结合将模拟醇的结合。这些预测将在Harris实验室通过在卵母细胞中表达含有位点定向半胱氨酸取代的GlyRa1来进行测试。他们将把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2。我们将验证双位点定向半胱氨酸突变可以澄清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模型的整体结构是一致的，但在跨膜螺旋和残基结合可能的醇结合位点的方向上存在重要差异。哈里斯实验室将通过交联定向二半胱氨酸来验证这些预测。总之，对GlyRa1中醇结合位点的了解将增加我们对醇在LGIC中的作用的理解。该结果可能揭示受酒精影响的其他系统中作用的一般原理，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.