Structural analysis of alcohol-dependent activation of GIRKs

 描述（由申请人提供）：酒精（乙醇）是美国和世界范围内的主要滥用药物，每年因酒精相关病例死亡的人数超过10万。迫切需要治疗酒精使用障碍的新药物疗法。反复饮酒通过乙醇直接调节不同类型的离子通道来改变大脑中的神经活动。乙醇的药理作用在毫摩尔浓度下发生。为了了解乙醇是如何改变大脑回路的，有必要阐明乙醇对大脑蛋白质低亲和力调节的分子和化学机制。对GIRK通道的研究提供了乙醇调节的最佳实例之一。GIRK通道被乙醇直接激活，并已明确涉及乙醇饮用行为。缺乏GIRK2亚基的小鼠比野生型小鼠自我施用更多的乙醇，而缺乏GIRK3亚基的小鼠增加了酗酒。这些研究表明，GIRK2和GIRK3亚基对乙醇的敏感性可能不同。GIRK通道拥有一个酒精口袋，已经在GIRK2通道的原子分辨率下描述过。然而，对含有GIRK3的异聚体通道的结构知之甚少。据推测，含GIRK3的异聚体通道中的醇口袋比GIRK2通道更好地协调乙醇，导致对乙醇的更高敏感性。这项资助将确定GIRK通道亚基的酒精敏感性的化学和结构差异，使用纯化的同源和异源四聚体GIRK通道与胆固醇重组成定义的脂质，并解决酒精口袋的原子结构。还将确定酒精对纯化的GIRK通道的微观门控特性的影响，以检验以下假设：与G蛋白稳定开放状态的机制相反，酒精通过增加开放频率来增加开放通道概率。最后，GIRK醇袋的原子分辨率结构将用于使用计算机筛选来识别新型乙醇调节剂。候选药物将使用高通量测定法用纯化的GIRK通道进行测试，然后用在腹侧被盖区多巴胺神经元中表达的天然GIRK通道进行离体研究。定义乙醇的物理口袋对于理解乙醇的低亲和力结合如何改变通道活性和影响大脑功能至关重要。这笔拨款的结果可以开创酒精滥用和依赖新型治疗方法的开发。

 DESCRIPTION (provided by applicant): Alcohol (ethanol) is a major drug of abuse in the U.S. and worldwide, with deaths from alcohol-related cases numbering over a hundred thousand per year. New pharmacotherapies for treating alcohol use disorders are urgently needed. Repeated alcohol consumption alters the neural activity in the brain through ethanol's direct modulation of different types of ion channels. The pharmacological effects of ethanol occur with milliMolar concentrations. In order to understand how ethanol alters brain circuits it is essential to elucidate the molecular and chemical mechanisms underlying ethanol's low affinity modulation of brain proteins. Studies of GIRK channels have provided one of the best examples of modulation by ethanol. GIRK channels are activated directly by ethanol and have been clearly implicated in ethanol-drinking behaviors. Mice lacking GIRK2 subunits self-administer more ethanol than wild-type mice while mice lacking GIRK3 subunits increase binge- drinking of alcohol. These studies suggest that GIRK2 and GIRK3 subunits may differ in their sensitivity to ethanol. GIRK channels possess a pocket for alcohol that has been described at the atomic resolution for GIRK2 channels. However, little is known about the structure of heteromeric GIRK3-containing channels. It is hypothesized that the alcohol pocket in heteromeric GIRK3-containing channels coordinates ethanol better than GIRK2 channels, resulting in a higher sensitivity to alcohol. This grant will determine the chemical and structural differences in alcoho-sensitive properties of GIRK channel subunits using purified homo- and hetero- tetrameric GIRK channels reconstituted into defined lipids with cholesterol, and solving atomic structures of alcohol pockets. The effect of alcohol on microscopic gating properties of purified GIRK channels will also be determined to test the hypothesis that alcohol increases the open channel probability via an increase in the frequency of opening, in contrast to the mechanism of G proteins that stabilize an open state. Lastly, the atomic resolution structure of the GIRK alcohol pocket will be used to identify novel ethanol modulators, using in silico screening. Candidate drugs will be tested using a high-throughput flux assay with purified GIRK channels and then with native GIRK channels expressed in ventral tegmental area dopamine neurons for ex vivo studies. Defining the physical pocket for ethanol is critical for understanding how the low affinit binding of ethanol changes channel activity and affects brain function. Results from this grant could pioneer the development of novel treatments for alcohol abuse and dependence.