Properties of Specific Alcohol Binding Sites

特定醇结合位点的特性

基本信息

批准号：
7458033
负责人：
JAMES Robert TRUDELL
金额：
$ 30.5万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-07-01 至 2010-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7458033
关键词：
3-Dimensional Acetylcholine Affect Agonist Agreement Alcohols Amino Acids Arts Binding Binding Sites Class Computer Simulation Cysteine Data Dependence Dimensions Disulfides Dodecanol Engineering Gated Ion Channel Glycine Goals Health Helix (Snails)Heptanol In Vitro Intoxication Ion Channel Ion Channel Protein Knowledge Laboratories Ligands Measures Mediating Membrane Methods Modeling Modification Molecular Mutation Neurons Numbers Oocytes Property Proteins Rate Reagent Research Personnel Rotation Series Serotonin Receptors 5-HT-3 Site Solutions Structure Sulfhydryl Compounds System Testing Torpedo Transmembrane Domain alcohol effect alcoholism/alcohol abuse alpha helix analog base computer generated crosslink design ear helix gamma-Aminobutyric Acid in vivo interfacial methanethiosulfonate molecular modeling programs propanethiol receptor

项目摘要

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.

描述（由申请人提供）：酗酒和酗酒是主要的健康问题。解决这些问题可能需要了解酒精对神经元离子通道蛋白的影响。醇的特异性结合位点已在配体门控离子通道 (LGIC) 超家族的跨膜 (TM) 结构域中得到描述，该超家族包括甘氨酸 (GlyRa1)、GABA、烟碱乙酰胆碱和 5-HT3 受体。我们的总体假设是，醇类结合在以这些受体的 TM 片段为界的空腔内，并优先稳定特定的通道亚状态。我们的目标是确定 GlyRa1 中介导酒精和酒精拮抗剂的结合和功效的位点的特性。这些结合位点可以为其他类别的离子通道和其他重要蛋白质内的醇的结合提供共同的基序。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. Harris 博士构建和测试。目标 1. 我们将检验以下假设：GlyRa1 的所有四个跨膜螺旋的氨基酸残基均有助于醇的结合位点。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硫代磺酸盐 (MTS) 试剂的共价结合将在何处模拟酒精结合。 Harris 实验室将通过在卵母细胞中表达含有定点半胱氨酸取代的 GlyRa1 来测试这些预测。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 2. 我们将测试以下假设：双位点定向半胱氨酸突变可以澄清 GlyRa1 的精细三级结构，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 模型的整体结构在全球范围内一致，但跨膜螺旋的方向和限制可能的醇结合位点的残基存在重要差异。哈里斯实验室将通过交联定点二半胱氨酸来测试这些预测。总之，对 GlyRa1 中酒精结合位点的了解将增加我们对 LGIC 中酒精作用的理解。结果可能揭示受酒精影响的其他系统中作用的一般基序，并有助于设计酒精拮抗剂。