Defining Alcohol Binding Sites in Ligand-Gated Ion Channels

描述(申请人提供)：酗酒和酗酒是严重的健康问题，影响着1700多万人，每年造成近2000亿美元的损失。这些问题的解决很可能源于对酒精对神经元离子通道和调节它们的蛋白质的影响的更好理解。这项拟议研究的目标将是在原子水平上了解酒精结合位点的性质的重要一步。我们相信，这些知识将对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。具体地说，我们将研究配体门控离子通道(LGIC)中的酒精结合部位，包括GABAaRs和GlyRs。我们的同源建模和实验方法将提供GABAAR的三维可视化，以增加我们对酒精作用的理解。这一创新方法将尖端计算和神经科学技术与分子生物学相结合。我们预计我们的结果将对神经系统其他重要受体中更广泛类别的酒精结合部位产生重大影响。我们的方法通过三个具体目标集中在酒精结合位点的三个方面：酒精结合位点在哪里；亚基内和亚基间 LGICs中的亚基(目标1)，LGICs中的哪些特定残基或片段介导这些位点上的酒精结合效应(目标1和2)，以及这些位点如何调节配体结合(目标2和3)。在目标1中，特鲁德尔和贝尔塔奇尼将建立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这些模型。根据德克萨斯大学奥斯汀分校的分包合同，哈里斯和霍华德将测试这些突变受体的功能。我们将反复改进Trudell团队将使用这些模型来预测突变影响的模型；Harris团队将测试这些模型是否一致 然后，特鲁德尔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：加巴尔最重要的酒精效应场所是什么？是亚基内还是亚基间？他们还将测试GABAAR TM3螺旋在激活过程中必须旋转的假设，以便纳入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中哪些残基是乙醇结合的特异性残基并将这些残基突变到它们在GLIC中的相应同源位置，来从同源但天然对乙醇不敏感的离子通道GLIC中的GABAaRs重建酒精结合位点。在目标3中，特鲁德尔和贝尔塔奇尼将使用三个对接程序来研究酒精类似物的结合。我们的研究人员已被证明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。我们的建议响应了NIAAA在计算神经科学方面的倡议和NIH路线图：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，这些拮抗剂可能会彻底改变酒精滥用和依赖的治疗方法。

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.