Structural Basis for Antiarrhythmic Drug Action

电压门控钠（Nav1.5）通道启动动作电位，电压门控钙（Cav1.2）通道启动动作电位 启动心肌细胞兴奋/收缩偶联。它们是突变的分子靶点， 心律失常和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NavAb和模型钙通道CavAb的结构 通过X射线晶体学，我们已经确定了主要心脏Nav通道Nav1.5的结构， 通过低温电子显微镜（cryo-EM）获得高分辨率。这项工作使人们对宇宙的结构有了新的认识。 离子选择性过滤器和Na、Ca选择性机理，电压传感器的结构和机理 电压依赖性门控，激活和失活门及其功能相互作用，受体 AAD的受体位点，以及AAD通过开放的激活门进入其受体位点的机制， 孔的侧面有开窗。导致遗传性心律失常的Nav1.5突变，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，为探索这些疾病的病理生理学开辟了道路。 结构层面的突变。在这里，我们将研究Nav和Cav通道功能的结构基础， AAD的复杂孔阻塞机制，以及病理生理学效应的潜在机制 心律不齐突变。目标1。我们将确定Nav1.5通道的结构，在休息，开放， 失活状态，并分析了开放状态下离子选择性和电导的分子机制 Nav1.5的目标二。我们将制备Nav1.5与AAD的封闭、开放和失活状态的复合物 我们将以高分辨率解析它们的结构。我们将探索药物的结构差异- 受体复合物形成的IA类，IB类，和IC AAD，以了解的结构基础， 药物作用的差异导致其不同的临床用途。我们将研究开窗的作用 IA类、IB类和IC类AAD的静息状态阻滞。目标3。我们将插入导致扩张的突变 将心肌病和长QT综合征3型分为NavAb和Nav1.5，表征其病理生理学 对钠电流和门控孔电流的影响，并通过X射线以高分辨率解析它们的结构 晶体学和/或冷冻EM。目标4。我们将在CavAb中插入导致蒂莫西综合征的突变， Cav1.2，确定其对Ca和Ba电流的病理生理作用，并在高电压下解析其结构 通过X射线晶体学和cryo-EM的分辨率。总的来说，这些研究开启了令人兴奋的可能性， 了解天然和致病构象中心脏Nav和Cav通道的原子细节， 学习如何操纵抗艾滋病药物的结构，使它们更具体，更有效，更安全。

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.