Defining Alcohol Binding Sites in Ligand-Gated Ion Channels

酒精滥用和酒精中毒是严重的健康问题，影响超过1700万人，每年花费近2000亿美元。更好地理解酒精对神经元离子通道和调节它们的蛋白质的影响，可能会解决这些问题。这项研究的目标将是在原子水平上理解酒精结合位点特性的重要一步。我们相信，这些知识对于未来设计和选择可以减少酒精引起的渴望或成瘾的药物至关重要。具体来说，我们将研究乙醇在配体门控离子通道（LGIC）中的结合位点，包括GABAaR和GlyR。我们的同源建模和实验方法将提供三维可视化的GABAaRs，以增加我们对酒精的行动的理解。这种创新的方法将尖端的计算和神经科学技术与分子生物学相结合。我们期望我们的结果将对神经系统其他重要受体中更广泛的酒精结合位点产生重大影响。我们的方法通过三个特定的目的关注酒精结合位点的三个方面：酒精结合位点在哪里;亚基内与亚基间 LGIC中哪些特定残基或片段介导这些位点的醇结合效应（目的1和2），以及这些位点如何调节配体结合（目的2和3）。 在目标1中，Trudell和Bertaccini将建立GABA受体中酒精结合位点的计算模型，并设计定点突变来测试模型。哈里斯和霍华德将在德克萨斯大学奥斯汀分校的一项研究中测试这些突变受体的功能。我们将反复完善模型，Trudell小组将使用模型来预测突变的影响; Harris小组将测试模型是否一致 然后Trudell小组将修改模型以适应新的数据。他们将解决这个有争议的问题：GABAaR中最重要的酒精作用位点是什么？是亚单位内还是亚单位间？他们还将测试GABAaR TM 3螺旋在激活过程中必须旋转的假设，以结合所有最近的实验数据。在目标2中，Trudell和Harris实验室将通过确定GABAR中哪些残基对EtOH结合具有特异性并将其突变到GLIC中相应的同源位置，在同源但天然的EtOH不敏感离子通道GLIC中从GABAaR中重建醇结合位点。在目标3中，Trudell和Bertaccini将使用三个对接程序来研究酒精类似物的结合。我们的研究人员在酒精研究方面取得了成就，并拥有实现我们目标所需的资源。我们的建议是响应NIAAA在计算神经科学和NIH路线图倡议：生物信息学和计算生物学。这些重要的研究将提供设计酒精结合拮抗剂所需的基本知识，这可能会彻底改变酒精滥用和依赖的治疗。

DESCRIPTION (provided by applicant): Alcohol abuse and alcoholism are significant health problems that affect over 17 million people and cost nearly $200 billion annually. It is likely tha a solution to these problems will result from a better understanding of alcohol effects on neuronal ion channels and the proteins that modulate them. The goal of the proposed research will be a significant step towards understanding the properties of alcohol-binding sites at an atomic level. We believe this knowledge will be essential for future design and selection of drugs that could reduce craving or addiction induced by alcohol. Specifically, we will study the sites fo alcohol binding in ligand- gated ion channels (LGICs), which include GABAaRs and GlyRs. Our homology modeling and experimental methods will provide 3-dimensional visualization of GABAaRs to increase our understanding of alcohol's action. This innovative approach combines cutting-edge computational and neuroscience techniques with molecular biology. We expect our results will have a significant impact on the broader class of alcohol- binding sites in other important receptors of the nervous system. Our Approach focuses on three aspects of alcohol-binding sites via three Specific Aims: Where are alcohol-binding sites; intra-subunit versus inter- subunit in LGICs (Aim 1), which specific residues or segments in LGICs mediate the effect of alcohol binding at these sites (Aims 1 and 2), and how do these sites modulate ligand binding (Aims 2 and 3). In Aim 1, Trudell and Bertaccini will build computational models of alcohol-binding sites in GABA receptors and design site-directed mutations to test the models. Harris and Howard, under a subcontract to the University of Texas, Austin, will test the function of these mutated receptors. We will iteratively refine the models the Trudell group will use the models to predict the effects of mutations; the Harris group will test if the models are consistent with experimental data; and the Trudell group will then modify the models to fit the new data. They will address this controversial question: What is the most important alcohol effect site in GABAaR? Is it Intra-subunit or Inter-subunit? They will also test the hypothesis that the GABAaR TM3 helix must rotate during activation in order to incorporate all recent experimental data. In Aim 2, the Trudell and Harris laboratories will recreate the alcohol-binding site from GABAaRs in the homologous but natively EtOH insensitive ion channel, GLIC, by determining which residues are specific to EtOH binding in GABAR and mutating these into their corresponding homologous positions within GLIC. In Aim 3, Trudell and Bertaccini will use three docking programs to investigate binding of alcohol analogs. Our investigators have proven accomplishment in alcohol research and possess the resources necessary to accomplish our Aims. Our proposal is responsive to both the NIAAA initiative in computational neuroscience and the NIH Roadmap: Bioinformatics and Computational Biology. These significant studies will provide essential knowledge needed to design alcohol-binding antagonists which could revolutionize treatment for alcohol abuse and dependence.