Molecular Mechanisms of Desensitization and Drug Modulation in Ligand-Gated Ion Channels

描述（由申请人提供）：本提案的长期目标是确定五聚体配体门控离子通道（LGIC）中变构机制的结构基础。在神经递质结合后，LGIC迅速从关闭到开放到脱敏通道构象。尽管对脱敏的生理和病理生理作用有了实质性的了解，但关于这种状态的结构信息很少。本研究的重点是利用细菌LGIC同源物GLIC和ELIC来鉴定脱敏机制，并确定药物如何调节封闭、开放和脱敏状态之间的转换。我们建议将光谱技术应用于膜环境中GLIC和ELIC的功能定义状态。我们的研究将涉及多学科方法，包括光谱技术（EPR和荧光）、重组脂蛋白体的膜片钳测量以及使用等温滴定量热法的热力学测量。我们将1）测量脱敏构象下的构象转变。2)确定脂质-水-蛋白质界面在门控转换中的作用。3)确定全身麻醉药和酒精对别构调节的结构基础。这些实验将测试的核心假设，膜脂质发挥关键作用，在保持构象的完整性，在驱动门控转换，并在管理药物调制。预期所提出的研究是重要的，因为它们将提供生理相关膜环境中LGIC门控的动态视图。这些发现将建立一个结构框架，以增强我们在分子水平上对LGIC功能的理解，从而改进治疗策略和药物设计。

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to determine the structural basis for allosteric mechanisms in pentameric ligand gated ion channels (LGIC). Upon neurotransmitter binding, LGIC rapidly switch from closed to open to desensitized channel conformations. Despite a substantial understanding of the physiological and pathophysiological role of desensitization, there is very little structural information available on this state. The fcus of this study is to use bacterial LGIC homologues, GLIC and ELIC, to identify the mechanism of desensitization and to determine how drugs modulate transitions between the closed, open and desensitized states. We propose to apply spectroscopic techniques to functionally defined states of GLIC and ELIC in a membrane environment. Our studies will involve a multidisciplinary approach that includes spectroscopic techniques (EPR and fluorescence), patch-clamp measurements in reconstituted proteoliposomes, and thermodynamic measurements using isothermal titration calorimetry. We will 1) Measure the conformational transitions underlying the desensitized conformation. 2) Determine the role of lipid-water-protein interface in gating transitions. 3) Identify the structural basis for allosteric modulation by general anesthetics and alcohols. These experiments will test the central hypothesis that membrane lipids play a key role in maintaining the conformational integrity, in driving gating transitions, and in governing drug modulation. The proposed studies are expected to be significant in that they will provide a dynamic view of LGIC gating in a physiologically- relevant membrane environment. These findings will establish a structural framework to enhance our understanding of LGIC function at the molecular level and thereby improve therapeutic strategies and drug design.