Structure and Function of Pentameric Ligand-Gated Ion Channels

五聚体配体门控离子通道的结构和功能

• 批准号: 10317065
• 负责人: Sudha Chakrapani
• 金额: $ 61.99万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-21 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10317065
• 关键词: Binding Proteins; Biochemical; Cations; Cell physiology; Complement; Complex; Cryoelectron Microscopy; Development; Drug Targeting; Electrophysiology (science); Environment; Event; Functional disorder; Gastrointestinal Diseases; Glycine Receptors; Goals; HTR3A gene; Ion Channel Gating; Knowledge; Length; Ligands; Lipids; Malignant Neoplasms; Mediating; Membrane; Membrane Lipids; Mental disorders; Molecular; Molecular Conformation; Mood Disorders; Motor; Pain; Peripheral Nervous System; Physiologic pulse; Physiological; Physiological Processes; Process; Proteins; Resolution; Rest; Role; Serotonin; Signal Transduction; Structure; Synaptic Transmission; Techniques; Therapeutic; Therapeutic Agents; Therapeutic Uses; Work; addiction; chronic pain; design; gastrointestinal function; multidisciplinary; nervous system disorder; novel therapeutics; pain perception; particle; receptor; serotonin receptor; therapeutic target; transmission process

Project Summary/Abstract The overarching goal of the proposal is to determine the structural basis for allosteric mechanisms governing gating and modulation in pentameric ligand-gated ion channels (pLGIC). The pLGIC superfamily governs crucial physiological processes such as gastrointestinal functions, motor functions, and pain transmission. Aberrant channel functions are implicated in mood disorders, addiction, chronic pain, and cancer. Currently used therapeutic strategies suffer from our limited knowledge of the molecular details of pLGIC function, the origin of their functional diversity, and the downstream signaling events. Using single-particle cryo-EM, we recently solved structures of the full-length serotonin receptor (5HT3AR), a cationic pLGIC, in the resting state and two serotonin-activated conformations. Building on this technical advancement and further biochemical optimization, we aim to determine the conformational changes underlying gating and lipid modulation in the full- length homomeric and heteromeric receptors within the cationic 5-HT3R and anionic glycine receptor (GlyR) subfamilies. To achieve these goals we will use an approach that combines multidisciplinary techniques, including cryo-EM, pulsed-EPR, and electrophysiology. Specifically, we will determine high-resolution snapshots of pLGIC in multiple functional states, in modulator-bound conformations, in the presence of membrane lipid constituents, and in complex with intracellular-binding proteins. These structures will be validated and complemented with protein dynamic studies in a membrane environment and extensive functional analysis. Taken together, our proposed work is expected to provide molecular blueprints of the channel in physiologically relevant conformations for therapeutic targeting and unravel the molecular mechanisms underlying channel function. These findings will, in turn, pave the way for design of novel therapeutic agents that are safer and more effective.

项目摘要/摘要 该提案的总体目标是确定控制变构机制的结构基础 五聚合配体门控离子通道（PLGIC）中的门控和调节。 PLGIC超家族控制着至关重要的 生理过程，例如胃肠功能，运动功能和疼痛传播。异常 通道功能与情绪障碍，成瘾，慢性疼痛和癌症有关。当前使用 治疗策略遭受了我们对PLGIC功能分子细节的有限了解，起源于 它们的功能多样性以及下游信号事件。使用单粒子冷冻EM，我们最近 全长5-羟色胺受体（5HT3AR）的结构，一种阳离子PLGIC，在静止状态和两个 5-羟色胺激活的构象。以这种技术进步和进一步的生化为基础 优化，我们旨在确定全面门控和脂质调制的构象变化 阳离子5-HT3R和阴离子甘氨酸受体（GLYR）内的长度同源物和杂体受体 亚家族。为了实现这些目标，我们将使用一种结合多学科技术的方法， 包括冷冻EM，脉冲 - EPR和电生理学。具体而言，我们将确定高分辨率 PLGIC在多个功能状态中的快照，在调制器结合的构象中，在存在下 膜脂质成分，并具有细胞内结合蛋白的复杂性。这些结构将是 经过验证并补充膜环境中的蛋白质动态研究和广泛的功能 分析。综上 生理上相关的治疗靶向和分子机制的构象 基础通道功能。这些发现反过来将为设计新型治疗剂的设计铺平道路 更安全，更有效。