Mapping the Putative Ethanol Binding Site on alpha4-beta2-delta GABA(A) Receptors

描述（由申请人提供）：γ-氨基丁酸A型（GABA（A））受体介导大脑中大部分快速突触抑制，是许多抑制剂（如苯二氮平类、巴比妥类和酒精）的靶点。最常见的受体亚型包含α、β和γ亚基。然而，含有δ亚基而不是γ亚基的GABA（A）受体对乙醇表现出独特的敏感性，并且可能在生理相关剂量下介导酒精的行为效应中发挥关键作用。证据还表明，长期使用酒精可能导致大脑中受体亚型水平的永久性重排，最终可能导致物质依赖性增加和受体药理学改变。酗酒和酗酒是严重的问题，对身心健康以及家庭和生活方式产生严重影响。13.8 100万美国成年人有饮酒问题，其中810万是酒鬼。慢性酒精滥用会对大脑造成重大损害，以及其他需要广泛医疗护理的症状，每年总共花费社会数十亿美元。几个研究小组在研究含GABA（A）δ受体的乙醇敏感性时显示了相互矛盾的结果，乙醇敏感性可能取决于存在的亚基亚型。拟议的研究将通过确定其亚基化学计量，使用α 4-β 2-δ受体作为模型来澄清和进一步表征含有δ的受体。将建立受体对低剂量乙醇的反应性，并绘制受体上推定的乙醇结合位点。药物Ro 15 -4513目前在临床上用于拮抗过量饮酒的行为效应，它以高亲和力与含δ的GABA（A）受体结合，并被认为与酒精竞争相同的结合位点。Ro 15 -4513含有可光活化的叠氮基，其将用于光标记含δ受体。然后将确定药物光掺入的特定亚基，并使用一系列蛋白水解酶缩小亚基的光标记区域。该项目的成功将更好地了解GABA（A）受体如何介导酒精在大脑中的作用，并将提供有助于开发用于治疗慢性酒精滥用许多症状的新型药物治疗的见解。需要更好的药物来治疗酒精中毒和酒精滥用。酒精通过与称为GABA（A）受体的特定蛋白质结合来影响大脑。这项拟议中的研究将有助于确定酒精与这些受体结合的位置，从而有助于新药的开发。

DESCRIPTION (provided by applicant): The gamma-aminobutyric acid type A, GABA (A), receptor mediates the majority of fast synaptic inhibition in the brain and is a target of many depressants, such as the benzodiazipines, barbiturates, and alcohol.The most prevalent receptor subtype contains alpha, beta, and gamma subunits. However, GABA (A) receptors containing the delta subunit, instead of gamma, display a unique sensitivity to ethanol and may play a key role in mediating the behavioral effects of alcohol at physiologically relevant doses. Evidence also indicates that chronic use of alcohol may cause a permanent rearrangement of receptor subtype levels in the brain, which may ultimately result in increased substance dependence and altered receptor pharmacology. Alcoholism and alcohol abuse are serious problems that have severe repercussions on physical and mental health as well as family and lifestyle. 13.8 million American adults have problems with drinking, 8.1 million of which are alcoholic. Chronic alcohol abuse causes major damage to the brain as well as other symptoms requiring extensive medical care that in total costs society billions of dollars each year. Several research groups have shown conflicting results when investigating ethanol sensitivity of GABA (A) delta-containing receptors, and ethanol sensitivity may be dependent on the subunit isoforms present. The proposed research will clarify and further characterize delta-containing receptors by determining their subunit stoichiometry, using alpha4-beta2-delta receptors as a model. The responsiveness of the receptors to low doses of ethanol will be established and the putative ethanol binding site on the receptors will be mapped. The drug Ro15-4513, which is currently used clinically to antagonize the behavioral effects of excessive alcohol consumption, binds with high affinity to delta-containing GABA (A) receptors and is thought to compete for the same binding site as alcohol. Ro15-4513 contains a photo-activatable azido group that will be used to photolabel delta-containing receptors. The specific subunit into which the drug photoincorporates will then be identified and the photolabeled region of the subunit will be narrowed down using a series of proteolytic digestions. The success of this project will provide a better understanding of how GABA (A) receptors mediate the effects of alcohol in the brain and will provide insight that will aid in the development of novel pharmacotherapeutics for the treatment of many symptoms of chronic alcohol abuse. Better drugs are needed to treat alcoholism and alcohol abuse. Alcohol affects the brain by binding to specific proteins called GABA (A) receptors. The proposed research will help identify where alcohol is binding to these receptors and thus will aid in the development of new drugs.