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

描述(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，每年超过10万人死亡，还有无数与医疗问题有关的例子。如此巨大的成本促使人们进行密集的研究，以了解这种药物是如何影响大脑功能的。然而，尽管做出了这些努力，关于乙醇在神经元水平上的作用还没有达成共识，目前也没有有效的酒精滥用障碍的药物治疗方法。众所周知，乙醇增强了大脑中GABAA受体介导的信号传递功能，GABAA受体(GABAR)亚单位(最明显的是Delta亚单位)的可塑性变化导致了长期饮酒和滥用导致依赖的行为改变。此前已有证据表明，引起突触外或紧张性抑制的增量GABA对乙醇的调制非常敏感。 在人类社交饮酒中实现的低毫摩尔浓度。虽然这些发现解释了几十年来积累的证据，即抑制性GABA参与了乙醇效应的调节，但对于这些受体在重组表达系统中对酒精的独特敏感性一直存在争议。初步数据表明(首次)含有Delta亚基的重组受体不仅对乙醇和GABA唯一敏感，而且共表达Delta导致受体组分对GABA结构类似物Tip/gaboxadol和Muscimol的敏感性显著增加(~1000倍)。假设在重组表达系统中，存在促进Delta-GABARs高效表达的蛋白质缺失。因此，利用THIP高灵敏度的这一特性，利用免疫亲和纯化受体蛋白的蛋白质组学/质谱学方法筛选最先进的Delta亚基结合蛋白，以寻找潜在的促进细胞表面和潜在地以其他有趣的方式调节功能的Delta亚基伙伴蛋白。咪唑苯并二氮类乙醇拮抗剂Ro15-4513中的叠氮基团是一个光亲和基团，这一事实将使我们能够识别DeltaGABA受体上Etoh/Ro15-4513结合部位的氨基酸残基。基于详细的结构模型假设，形成Delta GABAR上酒精位点的确切氨基酸残基将通过重组表达和突变得到确认和验证。这项工作将有助于更好地理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识别是开发治疗酒精相关性认知障碍和酒精成瘾的合理疗法的先决条件。

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.