Structure and Function of Pentameric Ligand-Gated Ion Channels

项目摘要/摘要 该提案的总体目标是确定变构机制的结构基础 五聚配体门控离子通道(PLGIC)的门控和调制。PLGIC超级家族掌管着至关重要的 生理过程，如胃肠功能、运动功能和疼痛传递。反常的 通道功能与情绪障碍、成瘾、慢性疼痛和癌症有关。当前使用 治疗策略受累于我们对pLGIC功能的分子细节的有限了解，pLGIC功能的起源 它们的功能多样性，以及下游的信号事件。使用单粒子低温EM，我们最近 静息状态和两种状态下阳离子pLGIC全长5-羟色胺受体(5HT3AR)的可解结构 5-羟色胺激活的构象。建立在这一技术进步和进一步的生化基础上 优化，我们的目标是确定潜在的门控和脂质调节的构象变化在完全- 阳离子5-HT3R和阴离子甘氨酸受体(GlyR)中长度的同、异构体受体 子族。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。具体来说，我们将确定高分辨率 PLGIC在多种功能状态、调节器结合构象下的快照 膜脂组分，以及与细胞内结合蛋白的复合体。这些结构将是 通过膜环境和广泛功能的蛋白质动力学研究来验证和补充 分析。综上所述，我们拟议的工作有望提供该通道的分子蓝图 治疗靶向的生理相关构象及其分子机制的研究 基础渠道功能。这些发现将反过来为设计新的治疗剂铺平道路。 更安全、更有效。

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.