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特定离子通道的影响以及调节它们的激酶。最近已经描述了醇的特异性结合位点在甘氨酸、GABA、烟碱乙酰胆碱和5-HT 3受体超家族的跨膜结构域中。我们的假设是，醇类结合在这些受体的跨膜片段所包围的空腔内。我们的目标是确定这些网站的属性，调节酒精的结合和功效。这些结合位点可以提供用于在其他种类的离子通道内结合醇的共同基序。我们将建立结合位点的计算模型，并设计特定的定点突变来验证这一假设。这些突变将由我们的合作者R. Adron Harris和S. John Mihic，在独立资助的实验中。具体而言：目标1。我们将定义特定的氨基酸残基，确定长链醇的“截止”长度。我们以前已经表明，突变的跨膜段2（TM 2）和A288的跨膜段3的甘氨酸受体可以改变醇的"截止"从庚醇到十二醇。我们将开发计算分子模型，使我们能够提出突变，这将决定在TM1和TM4中的其他残基，也可能形成假定的结合腔的“墙”。我们将通过迭代来完善我们的模型，在迭代中，我们优化初始模型的结构，使用它来预测突变，测试模型是否与所得的实验数据一致，然后以更好地拟合数据的方式修改模型。目标二。我们将通过提供模型，其中一系列的醇类似物共价连接到推定的结合腔中的定点半胱氨酸突变，来确定醇的激动剂效力增强的结构要求。由于我们知道一个单一的醇类似物结合到推定的位点，我们可以区分结合和功效。目标3：我们将通过构建模型来确定对酒精增强激动剂重要的氨基酸残基的接近度，该模型预测适合于交联的双位点定向半胱氨酸突变。我们将预测可以通过直接二硫键形成或与具有1 - 5个碳间隔的双官能甲硫基磺酸酯试剂连接的残基对。总之，这些计算研究将提供有关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.