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

描述(申请人提供)：γ-氨基丁酸A型，GABA(A)受体介导大脑中大多数快速突触抑制，是许多抑制剂的靶标，如苯二氮类药物、巴比妥酸盐和酒精。最普遍的受体亚型包括α、β和伽马亚基。然而，含有Delta亚基的GABA(A)受体，而不是伽马，对乙醇表现出独特的敏感性，并可能在调节生理剂量的酒精的行为效应中发挥关键作用。证据还表明，长期使用酒精可能会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增加和受体药理改变。酗酒和酗酒是严重影响身心健康以及家庭和生活方式的严重问题。1380万美国成年人有饮酒问题，其中810万人酗酒。长期酗酒会对大脑造成重大损害，以及需要广泛医疗护理的其他症状，这些症状每年总共给社会造成数十亿美元的损失。几个研究小组在研究含有GABA(A)的三角洲受体的乙醇敏感性时，发现了相互矛盾的结果，乙醇敏感性可能依赖于存在的亚基异构体。这项拟议的研究将以Alpha4-Beta2-Delta受体为模型，通过确定它们的亚单位化学计量比来阐明并进一步表征含有Delta的受体。将确定受体对低剂量乙醇的反应性，并绘制受体上假定的乙醇结合部位图。药物Ro15-4513目前被临床用于对抗过度饮酒的行为影响，它与含有Delta的GABA(A)受体具有高亲和力，被认为与酒精竞争相同的结合部位。Ro15-4513含有一个可光激活的叠氮基团，它将被用来光标记含三角洲的受体。然后，将识别药物光结合到的特定亚基，并使用一系列蛋白水解酶缩小亚基的光标记区。该项目的成功将使我们更好地了解GABA(A)受体如何在大脑中调节酒精的影响，并将提供有助于开发新的药物疗法来治疗许多慢性酒精滥用症状的见解。需要更好的药物来治疗酒精中毒和酗酒。酒精通过与称为GABA(A)受体的特定蛋白质结合来影响大脑。这项拟议的研究将有助于确定酒精与这些受体的结合位置，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.