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特定离子通道的影响以及调节它们的激酶。最近在甘氨酸、GABA、烟碱乙酰胆碱和5-HT3受体超家族的跨膜结构域中描述了醇的特定结合位点。我们的假设是，酒精结合在由这些受体的跨膜片段连接的空腔内。我们的目标是确定那些调节酒精结合和功效的位点的性质。这些结合位点可能为醇类在其他类型离子通道内的结合提供了一个共同的基序。我们将建立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一假设。这些突变将由我们的合作者，dr。R. Adron Harris和S. John Mihic在各自资助的实验中。具体来说：目标1。我们将定义确定长链醇的“截断”长度的特定氨基酸残基。我们之前已经证明，甘氨酸α受体跨膜段2 （TM2）上的S267突变和TM3上的A288突变可以将醇“截断”从庚醇转变为十二醇。我们将开发计算分子模型，使我们能够提出突变，这些突变将决定TM1和TM4中额外的残基，这些残基也可能形成假定的结合腔的“壁”。我们将通过迭代来完善我们的模型，其中我们优化初始模型的结构，使用它来预测突变，测试模型是否与最终的实验数据一致，然后以一种更适合数据的方式修改模型。目标2。我们将通过提供一系列醇类似物共价连接到假定结合腔中位点定向半胱氨酸突变的模型，来确定醇对增强激动剂效力的结构要求。由于我们知道单个醇类似物与假定的位点结合，我们可以区分结合和功效。目标3。我们将通过建立预测适合交联的双位点定向半胱氨酸突变的模型来确定对激动剂醇增强作用重要的氨基酸残基的邻近性。我们将预测可以通过直接二硫键形成或与具有1-5个碳间隔的双功能甲基乙硫磺酸试剂连接的残基对。总之，这些计算研究将提供关于酒精结合和功效的决定因素的新知识。

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 specific ion channels and the kinases that modulate them. Specific binding sites for alcohols have recently been described in the transmembrane domain of the superfamily of glycine, GABA, nicotinic acetylcholine, and 5-HT3 receptors. Our hypothesis is that alcohols bind within cavities that are bounded by transmembrane segments of these receptors. Our goal is to define the properties of those sites that regulate binding and efficacy of alcohols. These binding sites may provide a common motif for binding of alcohols within other classes of ion channels. We will build computational models of binding sites and design specific site-directed mutations to test this hypothesis. These mutations will be expressed and tested by our collaborators, Drs. R. Adron Harris and S. John Mihic, in separately funded experiments. Specifically: Aim 1. We will define specific amino acid residues that determine the "cutoff" length of long-chain alcohols. We have previously shown that mutation of S267 in transmembrane segment 2 (TM2) and A288 in TM3 of the glycine alphal receptor can change the alcohol "cutoff' from heptanol to dodecanol. We will develop computational molecular models that allow us to suggest mutations that will determine additional residues in TM1 and TM4 that may also form "walls" of the putative binding cavities. We will refine our models by iterations in which we optimize the structure of an initial model, use it to predict mutations, test if the model is consistent with the resulting experimental data, and then modify the model in a way that would better fit the data. Aim 2. We will determine the structural requirements of alcohols for potentiation of agonist potency by providing models in which a series of alcohol analogs are covalently linked to site-directed cysteine mutations in the putative binding cavities. Since we will know that a single alcohol analog is bound to the putative site, we can distinguish binding from efficacy. Aim 3. We will define the proximity of amino acid residues important for alcohol potentiation of agonists by building models that predict double site-directed cysteine mutations that are appropriate for cross-linking. We will predict pairs of residues that could be linked by direct disulfide formation or with bi-functional methanethiosulfonate reagents with 1-5 carbon spacers. In summary, these computational studies will provide new knowledge about determinants of alcohol binding and efficacy.