Unique pharmacology of ligand sites on delta subunit-containing GABA-A receptors

含 δ 亚基的 GABA-A 受体上配体位点的独特药理学

• 批准号: 8901845
• 负责人: RICHARD W OLSEN
• 金额: $ 29.92万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8901845
• 关键词: Acute; Address; Affect; Affinity; Alcohol abuse; Alcohol consumption; Alcohol dependence; Alcoholism; Alcohols; Amino Acids; Antibodies; Anxiety; Behavioral; Benzodiazepines; Binding; Binding Proteins; Binding Sites; Brain; Cattle; Cell Line; Cell surface; Cerebellum; Cessation of life; Chimera organism; Chronic; Consensus; Coupling; Cysteine; Data; Dependence; Development; Disease; Economic Burden; Electrophysiology (science); Epilepsy; Ethanol; Failure; GABA Agonists; GABA Receptor; GABA-A Receptor; Grant; Human; Immune; Impaired cognition; Label; Lead; Ligand Binding; Ligands; Mammalian Cell; Mass Spectrum Analysis; Mediating; Medical; Methods; Molecular; Muscimol; Mutagenesis; Mutation; Neurons; Neurotransmitter Receptor; Oocytes; Pharmaceutical Preparations; Pharmacology; Photoaffinity Labels; Plastics; Point Mutation; Property; Proteins; Proteomics; Rattus; Recombinants; Research; Site; Sleeplessness; Structural Models; Structure; Substance abuse problem; Sulfhydryl Reagents; Surface; Synapses; Synaptic Receptors; System; Testing; Therapeutic; Time; United States; Validation; Work; alcohol abuse therapy; alcohol exposure; alcohol sensitivity; analog; base; brain circuitry; chronic alcohol ingestion; combat; cost; desensitization; drinking; gamma-Aminobutyric Acid; hypnotic; interest; mutant; neurotoxicity; novel; radioligand; receptor; receptor-mediated signaling; sedative; social; success; trafficking

DESCRIPTION (provided by applicant): Alcoholism is the most common form of substance abuse and has enormous economic burden on the United States and more than 100,000 deaths per year, with countless additional examples of contributory medical problems. Such tremendous costs have motivated intensive research efforts to understand how this drug affects brain function. Yet despite these efforts, there is no consensus as to how ethanol acts at a neuronal level, and no effective medical therapeutic treatment for alcohol abuse disorders is currently available. It is known that ethanol enhances the function of GABAA receptor-mediated signaling in brain and that plastic changes in GABAA receptor (GABARs) subunits (most notably the delta subunit) contribute to the behavioral alterations produced by chronic alcohol use and abuse leading to dependence. Previously evidence has been provided that delta GABARs, which give rise to the extrasynaptic or tonic inhibition, are sensitive to modulation by ethanol at low millimolar concentrations achieved in human social drinking. While these findings provide an explanation for decades of accumulated evidence that inhibitory GABARs are involved in mediating EtOH effects, there has been controversy over the unique of alcohol-sensitivity of these receptors in recombinant expression systems. Preliminary data show (for the first time) that recombinant receptors containing delta subunits are not only uniquely sensitive to EtOH and GABA but also that delta co-expression leads to receptors fractions with a rather dramatic (~1000-fold) increase in sensitivity for the GABA structural analogs THIP/gaboxadol and muscimol. The hypothesis is that in recombinant expression systems there are proteins missing that promote efficient expression surface expression of delta-GABARs. Therefore it is intended to use this property of high THIP sensitivity to screen for delta subunit-binding proteins identifid by a state of the art proteomic/mass spectroscopy approach using immune-affinity purified receptors protein, in order to find potential delta subunit partner proteins that promote cell surface and potentially modulate the function in other interesting ways. The fact that the azido group in the imidazobenzodiazepine EtOH antagonist Ro15-4513 is a photoaffinity will allow the identification of amino acid residues in the EtOH/Ro15-4513 binding site on delta GABA receptors. Based on a detailed structural model hypothesis, the exact amino acid residues that form the alcohol site on delta GABARs will be confirmed and verified using recombinant expression and mutagenesis. This work will help lead to a better understanding of how alcohol and sedative-hypnotic drugs affect brain function. The molecular level identification of ethanol targets is a prerequisite for the development of rational therapies to treat alcohol- related cognitive impairment and alcohol addiction.

描述（申请人提供）：酒精中毒是滥用药物的最常见形式，对美国的经济负担很大，每年有100,000多人死亡，还有无数其他有关医疗问题的例子。如此巨大的成本激发了深入的研究工作，以了解该药物如何影响大脑功能。然而，尽管做出了这些努力，但目前尚无关于乙醇在神经元水平上的作用，目前尚无有效的医学治疗治疗方法的共识。众所周知，乙醇增强了脑中GABAA受体介导的信号传导的功能，并且GABAA受体（GABARS）亚基中的塑性变化（最值得注意的是Delta亚基）有助于慢性酒精使用和滥用导致依赖性的慢性酒精使用和滥用。以前已经证明，导致外交或补品抑制的三角洲Gabar对乙醇的调节敏感 人类社交饮酒中达到的低摩尔浓度。尽管这些发现提供了数十年的积累证据，表明抑制性GABAR参与了介导ETOH效应，但关于这些受体在重组表达系统中这些受体的独特性的独特性一直存在争议。初步数据表明（首次）表明，包含三角亚基的重组受体不仅对EtOH和GABA唯一敏感，而且还会导致Delta共表达导致受体分数具有相当大的（〜1000倍）的GABA结构类似物Thip/Gababoxadol和Muscimcimolcimolcimolcimolcimological and Muscimcimolcimolcimolcimologations signal（〜1000倍）。假设是，在重组表达系统中，缺少蛋白质可促进三角洲-Gabar的有效表达表达。因此，使用免疫 - 亲和力纯化的受体蛋白的TAR蛋白质组学/质谱法鉴定Delta亚基结合蛋白的这种特性筛选了Delta亚基结合蛋白，以发现潜在的Delta亚基伙伴蛋白质，以找到潜在的Delta亚基伙伴蛋白，从而促进细胞表面并在其他有趣的方法中调节功能。 Imidazobenzodiazepine eTOH拮抗剂RO15-4513中的氮杂组是光接度，这一事实将允许在Delta GABA受体上鉴定ETOH/RO15-4513结合位点中的氨基酸残基。基于一个详细的结构模型假设，将使用重组表达和诱变来确认并验证构成Delta Gabars酒精位点的精确氨基酸残基。这项工作将有助于更好地了解酒精和镇静催眠药如何影响大脑功能。乙醇靶标的分子水平鉴定是发展与酒精相关的认知障碍和酒精成瘾的理性疗法的先决条件。