Structural Basis for Antiarrhythmic Drug Action

抗心律失常药物作用的结构基础

• 批准号: 10364048
• 负责人: WILLIAM A CATTERALL
• 金额: $ 74.39万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-06 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10364048
• 关键词: Action Potentials; Affinity; Anti-Arrhythmia Agents; Arrhythmia; Binding; Brugada syndrome; Calcium; Calcium Channel; Cardiac; Cardiac Myocytes; Cells; Clinical; Complex; Coupling; Cryoelectron Microscopy; Crystallization; Cytosol; Dependence; Dihydropyridines; Dilated Cardiomyopathy; Disulfides; Drug Antagonism; Drug Receptors; Drug usage; Elderly; Electron Microscopy; Functional disorder; Image; Inherited; Ion Channel; Ions; Lead; Learning; Length; Life; Lipid Bilayers; Lipids; Long QT Syndrome; Maps; Membrane Potentials; Methods; Modeling; Molecular; Molecular Conformation; Molecular Target; Mutation; Pathogenicity; Pharmaceutical Preparations; Prevention; Resolution; Rest; Role; Safety; Side; Site; Sodium; Sodium Channel; Specificity; Structure; Surface; Timothy syndrome; Work; X-Ray Crystallography; base; crosslink; cryogenics; design; drug action; drug mechanism; heart rhythm; improved; insight; next generation; phenylalkylamine; prevent; receptor; reconstitution; sensor; voltage

Voltage-gated sodium (Nav1.5) channels initiate action potentials and voltage-gated calcium (Cav1.2) channels initiate excitation/contraction coupling in cardiac myocytes. They are molecular targets for mutations that cause arrhythmias and for antiarrhythmic drugs (AADs) used in control and prevention of life-threatening arrhythmias. We have determined the structures of the bacterial Nav channel NavAb and the model calcium channel CavAb by X-ray crystallography, and we have determined the structure of the primary cardiac Nav channel Nav1.5 at high resolution by cryogenic electron microscopy (cryo-EM). This work gave new insight into the structure of the ion selectivity filter and mechanism of Na and Ca selectivity, the structure of the voltage sensors and mechanisms of voltage-dependent gating, the activation and inactivation gates and their functional interaction, the receptor sites for AADs, and the mechanism of access of AADs to their receptor site through the open activation gate and fenestrations in the sides of the pore. Mutations in Nav1.5 that cause inherited cardiac arrhythmias, including Dilated Cardiomyopathy and Long QT Syndrome, map onto the pore module, voltage sensor, activation gate, and fast inactivation gate of Nav and Cav channels, opening the way to probing the pathophysiology of these mutations at the structural level. Here we will investigate the structural basis for Nav and Cav channel function, the complex pore-blocking mechanisms of AADs, and the mechanisms underlying the pathophysiological effects of arrhythmia mutations. Aim 1. We will determine the structure of Nav1.5 channels in resting, open, and inactivated states and analyze the molecular mechanisms for ion selectivity and conductance in the open state of Nav1.5. Aim 2. We will prepare complexes of Nav1.5 in the closed, open, and inactivated states with AADs bound, and we will resolve their structures at high resolution. We will probe structural differences in the drug- receptor complexes formed by Class IA, IB, and IC AADs in order to understand the structural basis for the differences in drug action that lead to their different clinical uses. We will examine the role of the fenestrations in resting-state block by Class IA, IB, and IC AADs. Aim 3. We will insert mutations that cause Dilated Cardiomyopathy and Long QT Syndrome Type-3 into NavAb and Nav1.5, characterize their pathophysiological effects on Na currents and gating pore currents, and resolve their structures at high resolution by X-ray crystallography and/or cryo-EM. Aim 4. We will insert mutations that cause Timothy Syndrome into CavAb and Cav1.2, determine their pathophysiological effects on Ca and Ba currents, and resolve their structures at high resolution by X-ray crystallography and cryo-EM. Overall, these studies open the exciting possibility of understanding cardiac Nav and Cav channels in atomic detail in native and pathogenic conformations and learning how to manipulate the structures of AADs to make them more specific, more effective, and safer.

电压门控钠 (Nav1.5) 通道启动动作电位和电压门控钙 (Cav1.2) 通道 启动心肌细胞的兴奋/收缩耦合。它们是引起突变的分子靶标 心律失常以及用于控制和预防危及生命的心律失常的抗心律失常药物（AAD）。 我们已经确定了细菌 Nav 通道 NavAb 和模型钙通道 CavAb 的结构 通过X射线晶体学，我们确定了主要心脏Nav通道Nav1.5的结构 低温电子显微镜 (cryo-EM) 的高分辨率。这项工作为我们对结构的理解提供了新的见解。 Na和Ca选择性的离子选择性过滤器和机制，电压传感器的结构和机制 电压依赖性门控、激活和失活门及其功能相互作用、受体 AADs 位点，以及 AADs 通过开放激活门进入其受体位点的机制 毛孔侧面的开窗。 Nav1.5 突变导致遗传性心律失常，包括 扩张型心肌病和长 QT 综合征，映射到孔模块、电压传感器、激活门， 以及 Nav 和 Cav 通道的快速失活门，为探索这些通道的病理生理学开辟了道路 结构水平上的突变。这里我们将研究 Nav 和 Cav 通道功能的结构基础， AAD 复杂的毛孔阻塞机制及其病理生理效应的机制 心律失常突变。目标 1. 我们将确定 Nav1.5 通道的静止、开放和 失活状态并分析开放状态下离子选择性和电导的分子机制 导航1.5。目标 2. 我们将用 AAD 制备关闭、打开和失活状态下的 Nav1.5 复合物 绑定，我们将以高分辨率解析它们的结构。我们将探讨药物的结构差异 由 IA、IB 和 IC 类 AAD 形成的受体复合物，以了解 AAD 的结构基础 药物作用的差异导致其临床用途不同。我们将研究开窗的作用 IA、IB 和 IC 类 AAD 的静息态阻滞。目标 3. 我们将插入导致 Dilated 的突变 将心肌病和长 QT 综合征 3 型转化为 NavAb 和 Nav1.5，表征其病理生理学 对 Na 电流和门控孔电流的影响，并通过 X 射线高分辨率解析它们的结构 晶体学和/或冷冻电镜。目标 4. 我们将在 CavAb 中插入导致蒂莫西综合症的突变，并 Cav1.2，确定它们对Ca和Ba电流的病理生理作用，并解析它们在高电压下的结构 通过 X 射线晶体学和冷冻电镜分辨率。总的来说，这些研究开启了令人兴奋的可能性 了解心脏 Nav 和 Cav 通道的天然和致病构象的原子细节， 学习如何操纵 AAD 的结构，使其更具体、更有效、更安全。