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

基本信息

批准号：
8439940
负责人：
RICHARD W OLSEN
金额：
$ 31.07万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-01 至 2018-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8439940
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 chronic alcohol ingestion combat cost desensitization drinking gamma-Aminobutyric Acid hypnotic interest mutant neurotoxicity novel public health relevance radioligand receptor receptor-mediated signaling sedative social success trafficking

项目摘要

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（由申请人提供）：酗酒是最常见的药物滥用形式，给美国带来巨大的经济负担，每年导致超过 100,000 人死亡，还有无数其他导致医疗问题的例子。如此巨大的成本促使人们深入研究这种药物如何影响大脑功能。然而，尽管做出了这些努力，但对于乙醇如何在神经元水平发挥作用尚未达成共识，并且目前还没有针对酒精滥用障碍的有效医学治疗方法。众所周知，乙醇可增强大脑中 GABAA 受体介导的信号传导功能，而 GABAA 受体 (GABAR) 亚基（最显着的是 δ 亚基）的可塑性变化会导致长期饮酒和滥用酒精导致的依赖所产生的行为改变。先前的证据表明，产生突触外或强直性抑制的 delta GABAR 对乙醇的调节敏感。在人类社交饮酒中达到低毫摩尔浓度。虽然这些发现为几十年来积累的证据提供了解释，即抑制性 GABAR 参与介导 EtOH 效应，但重组表达系统中这些受体的独特酒精敏感性仍存在争议。初步数据表明（首次）含有 δ 亚基的重组受体不仅对 EtOH 和 GABA 具有独特的敏感性，而且 δ 共表达导致受体组分的敏感性显着增加（约 1000 倍）。 GABA 结构类似物 THIP/加波沙朵和蝇蕈醇。假设在重组表达系统中缺少促进 delta-GABAR 有效表达表面表达的蛋白质。因此，旨在利用THIP高敏感性的这一特性来筛选通过使用免疫亲和纯化的受体蛋白的最先进的蛋白质组学/质谱方法鉴定的δ亚基结合蛋白，以便找到潜在的δ亚基伴侣蛋白促进细胞表面并可能以其他有趣的方式调节功能。咪唑并二氮杂 EtOH 拮抗剂 Ro15-4513 中的叠氮基具有光亲和性，这一事实将允许识别 δ GABA 受体上 EtOH/Ro15-4513 结合位点的氨基酸残基。基于详细的结构模型假设，将使用重组表达和诱变来确认和验证形成 delta GABAR 上醇位点的确切氨基酸残基。这项工作将有助于更好地了解酒精和镇静催眠药物如何影响大脑功能。乙醇靶标的分子水平鉴定是开发治疗酒精相关认知障碍和酒精成瘾的合理疗法的先决条件。