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

酒精中毒是最常见的药物滥用形式，对美国造成巨大的经济负担，每年有超过10万人死亡，还有无数其他的医疗问题。如此巨大的成本促使人们进行深入的研究，以了解这种药物如何影响大脑功能。然而，尽管有这些努力，乙醇如何在神经元水平上发挥作用还没有达成共识，目前还没有有效的药物治疗酒精滥用障碍。已知乙醇增强脑中GABAA受体介导的信号传导的功能，并且GABAA受体（GABAR）亚基（最显著的是δ亚基）的可塑性变化有助于由慢性酒精使用和滥用产生的行为改变，从而导致依赖。先前的证据已经提供，引起突触外或强直性抑制的δ GABAR对乙醇的调节敏感， 在人类社交饮酒中达到的低毫摩尔浓度。虽然这些发现为数十年来积累的证据提供了解释，即抑制性GABARs参与介导EtOH效应，但对重组表达系统中这些受体的独特酒精敏感性存在争议。初步数据显示（首次），含有δ亚基的重组受体不仅对EtOH和GABA独特敏感，而且δ共表达导致受体组分对GABA结构类似物THIP/加波沙朵和蝇蕈醇的敏感性显著增加（约1000倍）。假设是在重组表达系统中缺失促进δ-GABAR的有效表达表面表达的蛋白质。因此，旨在使用高THIP敏感性的这种特性来筛选通过现有技术的蛋白质组学/质谱方法使用免疫亲和纯化的受体蛋白鉴定的δ亚基结合蛋白，以发现促进细胞表面并以其他感兴趣的方式潜在地调节功能的潜在δ亚基伴侣蛋白。咪唑并苯并二氮杂卓EtOH拮抗剂Ro 15 -4513中的叠氮基具有光亲和性的事实将允许鉴定δ GABA受体上EtOH/Ro 15 -4513结合位点中的氨基酸残基。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δ 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。酒精靶点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前提。

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.