Structural and pharmacological dynamics of KCNQ2: A cryoEM and biophysics study

项目摘要/摘要 在大脑中，KCNQ2-KCNQ5共同组装形成M-通道，该通道调节神经元的兴奋性，并 几种精神障碍的高效治疗靶点，包括严重抑郁障碍， 精神分裂症、双相情感障碍和注意力缺陷多动障碍。从生理上讲，M通道是 负责静息膜电位的动态控制。胆碱能信号传导途径的研究 受体导致质膜PIP2的耗竭，进而导致M通道的关闭。一个 需要直接的PIP2-通道相互作用来打开KCNQ通道的孔域，然而 这种相互作用的结构基础尚不清楚。这项工作将确定人类的激活状态结构 KCNQ2在脂质环境中与钙调蛋白(CaM)和PIP2结合，用低温电子显微镜观察。在一个 从一般意义上讲，这种结构将扩大对离子通道激活机制的理解。在特定的情况下 因此，激活的KCNQ2-CaM的结构将是结构导向开发的宝贵财富 KCNQ2激动剂，针对上述精神障碍。除了结构研究，这一点 该提案试图描述KCNQ2的药理特性，阐明依赖状态的热力学 以及已知的KCNQ2小分子活化剂的协同效应。这个 测量的相互作用热和熵将报告这些小分子激活剂如何使用它们的 目标，KCNQ2，并可以为未来的药物开发努力提供信息，以提高这些化合物的效力。 最后，该项目将开发和验证KCNQ2激活剂和KCNQ2的直接、体外、高通量检测方法。 抑制剂。这些分析将基于标准的脂质体流动荧光分析(LFFA)，该方法适用于 研究不含PIP2和含PIP2的KCNQ2，以分别测试激活剂和抑制剂。这些化验结果将是 已知的KCNQ2激活剂和抑制剂验证，并将显著扩大目前的武器库 KCNQ2药物筛选技术。为了支持这些拟议的研究，强健的生物化学制剂 开发了KCNQ2-CaM和KCNQ1-CaM，并在KCNQ1-CaM上获得了初步的低温电子显微镜数据 都被收集起来了。此外，申请人还在从事以下工作的同时发展了低温EM方面的技术专长 对古生物钾通道的研究，并对人类质子通道进行了LFFA。这个 这里提出的结构、功能和药理学研究将对分子产生深刻的影响。 神经功能的机制，以及直接为未来的药物开发努力提供信息。

PROJECT SUMMARY / ABSTRACT In the brain, KCNQ2 – KCNQ5 co-assemble to form the M-channel, which regulates neuronal excitability and is a high-impact therapeutic target in several mental disorders, including Major Depressive Disorder, Schizophrenia, Bipolar Disorder and Attention-Deficit Hyperactivity Disorder. Physiologically, the M-channel is responsible for dynamic control of the resting membrane potential. Cholinergic signaling through muscarinic receptors results in depletion of plasma membrane PIP2, which in turn results in closure of the M-channel. A direct PIP2 – channel interaction is required to open the pore domain of KCNQ channels, however the structural basis of this interaction is unknown. This work will determine the activated-state structure of human KCNQ2 bound to calmodulin (CaM) and PIP2 in a lipid environment, using cryo electron microscopy. In a general sense, this structure will expand the understanding of ion channel activation mechanism. In a specific sense, the structure of activated KCNQ2-CaM will be a valuable asset to structure-guided development of KCNQ2 activators, targeting the above-mentioned mental disorders. In addition to structural studies, this proposal seeks to characterize the pharmacology of KCNQ2, elucidating the state-dependent thermodynamics and cooperativity of known KCNQ2 small molecule activators using isothermal titration calorimetry. The interaction enthalpies and entropies measured will report on how these small molecule activators engage their target, KCNQ2, and can inform future drug development efforts to improve the potency of these compounds. Finally, this project will develop and validate direct, in vitro, high-throughput assays for KCNQ2 activators and inhibitors. The assays will be based on a standard liposome flux fluorescence assay (LFFA) that be adapted to study KCNQ2 without and with PIP2 in order to test activators and inhibitors, respectively. These assays will be validated with known KCNQ2 activators and inhibitors, and will significantly expand the current arsenal of KCNQ2 drug screening techniques. In support of these proposed studies, robust biochemical preparations of KCNQ2-CaM and KCNQ1-CaM have been developed, and preliminary cryoEM data on KCNQ1-CaM have been collected. Additionally, the applicant has developed technical expertise in cryoEM while engaged in studies of an archaeal potassium channel, and has conducted LFFAs for a human proton channel. The structural, functional and pharmacological studies proposed here will yield insights into the molecular mechanisms of neuronal function, as well as directly inform future drug development efforts.