Structural investigation of the gating and regulatory mechanism of voltage-gated Ca2+ channels

电压门控Ca2通道的门控和调节机制的结构研究

• 批准号: 10059258
• 负责人: Nieng Yan
• 金额: $ 32.94万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-01 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10059258
• 关键词: 3-Dimensional; Acute; Adaptor Signaling Protein; Algorithms; Arrhythmia; Basic Science; Binding; Biochemical; Biological Assay; Calcium; Calcium Channel; Calcium ion; Cardiovascular Diseases; Cell Death; Cell membrane; Cells; Chemicals; Clinical Data; Complex; Coupling; Cryoelectron Microscopy; Cysteine; Disease; Dissection; Docking; Drug Industry; Drug Targeting; Electron Microscope; Electrophysiology (science); Epilepsy; Event; FDA approved; Future; Genetic Transcription; Goals; Homology Modeling; Hypertension; Hypokalemic periodic paralysis; Image; Investigation; Ions; Isradipine; Ligands; Malignant hyperpyrexia due to anesthesia; Mediating; Membrane; Membrane Potentials; Methods; Molecular; Molecular Conformation; Mutagenesis; Mutation; Mutation Analysis; Myopathy; Nimodipine; Oryctolagus cuniculus; Peptides; Pharmaceutical Preparations; Pharmacotherapy; Phase; Physiological; Physiological Processes; Play; Preparation; Production; Proteins; Regulation; Reporting; Research; Resolution; Role; RyR1; Ryanodine Receptor Calcium Release Channel; Sampling; Side; Signal Transduction; Site; Skeletal Muscle; Specificity; Structure; Structure-Activity Relationship; Titan; Toxin; Verapamil; Work; base; crosslink; density; design; drug development; drug discovery; electron energy; gabapentin; improved; interest; molecular dynamics; nervous system disorder; neurotransmission; particle; pregabalin; prevent; reconstruction; response; tool; voltage

Calcium ions (Ca2+) play a critical role in diverse physiological processes such as contraction, secretion, neurotransmission, gene transcription, and cell death. The voltage-gated calcium (Cav) channels open upon membrane depolarization, converting the membrane electrical signals to intracellular Ca2+-mediated events. Malfunction or dysregulation of Cav channels is associated with a broad spectrum of neurological, cardiovascular, and muscular disorders. Despite the physiological and pathophysiological significance of Cav channels, further progress has been acutely limited by the dearth of structural information. Indeed, the only available structure of any eukaryotic Cav channel is that of the Cav1.1 channel complex, which my group determined using single-particle electron cryo-microscopy (cryo-EM). Cav channels are targeted by multiple FDA-approved drugs for the treatment of neurological and cardiovascular disorders, and their activity is modulated by various peptide toxins. These ligands could be used to stabilize the Cav channels in various functional states, facilitating the dissection of the gating mechanism. In turn, structural elucidation of Cav channels in complex with the drugs and toxins will elucidate the molecular basis for their modes of action. These structures will guide mutagenesis for functional and mechanistic characterizations, serve as an important framework for homology modeling, ligand docking, and molecular dynamics simulation analyses, and eventually facilitate potential drug discovery. The overarching goal of this proposal is to achieve an improved mechanistic understanding of Cav channels through high-resolution structural determination of Cav1.1 in complex with various modulatory ligands using single-particle cryo-EM. In Aim 1, we will further improve the resolution of the Cav1.1 channel to beyond 3 Å by optimizing cryo-sample preparation and hardware configuration. Improved resolution will afford a more accurate structural template for molecular dynamics simulation analysis. In Aim 2, we will biochemically recapitulate the interactions between the purified Cav1.1 channel and various drugs and toxins, and elucidate the structures of Cav1.1 in complex with well-defined ligands. These structures will guide the design of mutations for functional characterizations and mechanistic investigations in cell-based electrophysiological assays. In Aim 3, we will investigate the structural basis for the modulation of Cav1.1 by the adaptor protein Stac3. This study will encompass crosslinking, mass spectrometric analysis, and new algorithms for cryo-EM to unravel the recognition between Stac3 and Cav1.1. Completion of the proposed research will advance our understanding of the function and disease-causing mechanisms of Cav channels as well as facilitate future drug discovery.

钙离子（Ca2+）在潜水员物理过程中起关键作用，例如收缩，分泌， 神经传递，基因转录和细胞死亡。电压门控钙（CAV）通道打开 膜去极化，将膜电信号转换为细胞内Ca2+介导的事件。 CAV通道的故障或失调与广泛的神经系统有关 心血管和肌肉疾病。尽管CAV具有物理和病理生理学意义 渠道，由于结构信息的死亡，进一步的进步受到了严重限制。确实，唯一的 任何真核CAV通道的可用结构都是Cav1.1通道复合物的结构，我的小组 使用单粒子电子冷冻微镜（Cryo-EM）确定。 CAV通道是由多个针对的 FDA批准的药物用于治疗神经系统和心血管疾病，其活性是 由各种肽毒素调节。这些配体可用于稳定各种骑士通道 功能状态，支持门控机制的解剖。反过来，CAV的结构阐明 与药物和毒素复杂的通道将阐明其作用模式的分子基础。 这些结构将指导功能和机械特征的诱变，并作为 同源建模，配体对接和分子动力学仿真分析的重要框架，以及 最终有利于潜在的药物发现。该提议的总体目标是实现改进 通过高分辨率的CAV1.1的机械理解对CAV通道的理解 使用单粒子冷冻EM与各种调节配体的复合物。在AIM 1中，我们将进一步改善 通过优化冷冻样本制备和硬件，将CAV1.1通道分辨率超过3Å 配置。改进的分辨率将为分子动力学提供更准确的结构模板 仿真分析。在AIM 2中，我们将在生化上概括纯化的Cav1.1之间的相互作用 通道和各种药物和毒素，并阐明了Cav1.1的结构，并定义明确 配体。这些结构将指导功能特征和机械性的突变设计 基于细胞的电生理测定法。在AIM 3中，我们将研究 衔接蛋白STAC3对CAV1.1的调节。这项研究将涵盖交联，质谱法 分析和冷冻EM的新算法，以阐明STAC3和CAV1.1之间的识别。完成 拟议的研究将提高我们对CAV功能和致病机制的理解 渠道以及促进未来的药物发现。