Structural Basis for Antiarrhythmic Drug Action

抗心律失常药物作用的结构基础

• 批准号: 10063882
• 负责人: WILLIAM A CATTERALL
• 金额: $ 73.49万
• 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 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10063882
• 关键词: Action Potentials; Affinity; Amino Acids; Amlodipine; Anti-Arrhythmia Agents; Arrhythmia; Atrial Fibrillation; Binding; Biochemical; Calcium; Calcium Channel; Cardiac; Cardiac Myocytes; Cells; Chemistry; Clinical; Complex; Coupling; Cryoelectron Microscopy; Crystallization; Data; Dependence; Development; Dihydropyridines; Diltiazem; Drug Antagonism; Drug Receptors; Drug usage; Flecainide; Goals; Heart; Human; Ions; Lead; Learning; Lidocaine; Life; Lipid Bilayers; Lipids; Membrane Potentials; Methods; Molecular; Molecular Conformation; Molecular Target; Mutagenesis; Mutation; Pathway interactions; Pharmaceutical Preparations; Physiological; Preparation; Process; Property; Proteins; Recovery; Resolution; Rest; Role; Safety; Site; Sodium; Sodium Channel; Specificity; Structure; Surface; Therapeutic; Time; Verapamil; Work; X-Ray Crystallography; base; design; drug action; experimental study; heart rhythm; improved; insight; molecular drug target; mutant; next generation; phenylalkylamine; ranolazine; receptor; reconstitution; sensor; side effect; therapeutic target; voltage

ABSTRACT Voltage-gated sodium (Nav) channels initiate action potentials in the heart, and voltage-gated calcium (Cav) channels initiate excitation-contraction coupling. They are related proteins with a common evolutionary ancestor, and they are molecular targets for Class I and Class IV antiarrhythmic drugs (AADs) used in control of life-threatening cardiac arrhythmias. The structural basis for AAD action is unknown. We have determined the crystal structure of an ancestral bacterial Nav channel (NavAb) at 2.7 Å resolution and revealed the structural basis for voltage sensing, pore opening and closing, ion selectivity, and slow inactivation. This structure also revealed fenestrations that lead laterally from the lipid bilayer into the pore and provide an access pathway for entry of pore-blocking AADs. We constructed a Ca-selective form of NavAb, termed CavAb, and used this construct to reveal the structural basis for Ca selectivity at atomic resolution. We are now focusing on the structural basis for state-dependent block of Nav and Cav channels by AADs. CavAb is blocked by all three structural subclasses of Class IV AADs in a state-dependent manner with nM affinity. We found that the phenylalkylamine verapamil binds to a receptor site in the pore, at the inner end of the ion selectivity filter, and physically blocks it. In contrast, amlodipine and other dihydropyridines bind at a site on the lipid-facing outer surface of the pore module, at the interface between two voltage-sensing modules, and allosterically block the pore. These results reveal drug-receptor complexes of Cav channels for the first time and set the stage for complete analysis of the mechanism of state-dependent block of Nav and Cav channels at the atomic level. Our proposed experiments have three goals. 1. We will build upon strong preliminary data to reveal the high-resolution structure of the therapeutically important benzothiazepine diltiazem bound to its receptor site in the pore of CavAb, compare the chemistry of its binding to verapamil, determine the role of fenestrations in state-dependent block of CavAb, and explore the effects of mutations that substitute human residues in the AAD receptor site. 2. We will build on strong preliminary data to reveal the high- resolution structures of Class 1 AADs such as lidocaine and flecainide bound to NavAb, differentiate among the binding poses and receptor site conformations for Subclass 1A, 1B, and 1C AADs, determine the role of fenestrations in state- dependent block of NavAb, and explore the effects of mutations that humanize the NavAb drug receptor. 3. Based on a new homogeneous biochemical preparation, we will use cryo-electron microscopy and X-ray crystallography to determine the structure of a mammalian cardiac Nav1.5 channel at high resolution, define the structural basis for its unique physiological properties, and elucidate the structural basis for AAD block of Nav1.5 channels. Our results will be crucial for understanding and improving therapy of life-threatening cardiac arrhythmias by AADs.

抽象的 电压门控钠（NAV）通道启动心脏中的动作电位，电压门控钙（CAV）通道 启动兴奋反应耦合。它们是具有共同进化祖先的相关蛋白质，它们是 用于控制威胁生命心脏的I类和IV类抗心律失常药物（AADS）的分子靶标 心律不齐。 AAD行动的结构基础尚不清楚。我们已经确定了祖先的晶体结构 分辨率为2.7Å的细菌NAV通道（NAVAB），并揭示了电压传感，打开孔和的结构基础 关闭，离子选择性和缓慢的失活。这种结构还揭示了壁式从脂质横向引起的。 双层进入孔，并为进入孔隙阻塞AADS的进入途径提供了访问途径。我们构建了一种CA选择形式 纳瓦布（Navab）称为cavab，并使用该构造来揭示原子分辨率下CA选择性的结构基础。我们 现在专注于AADS的NAV和CAV通道的状态依赖性块的结构基础。卡瓦布被阻止 IV类AAD的所有三个结构子类都以NM亲和力为依赖状态的方式。我们发现 苯基烷基胺Verapamil在离子选择性过滤器的内端与孔中的受体位点结合，并在物理上结合 阻止它。相比之下，氨氯地平和其他二氢吡啶在孔的面向脂质外表面的位点结合 模块，在两个电压感应模块之间的接口上，并构成变构阻断孔。这些结果揭示了 首次是CAV通道的药物受体复合物，并为完整分析机理奠定了基础 原子水平的NAV和CAV通道的状态依赖性块。我们提出的实验有三个目标。 1 将基于强大的初步数据，以揭示治疗上重要的高分辨率结构 苯锡锡氮卓丁diltiazem与卡瓦布孔中的接收器位点结合，将其结合的化学反应与 维拉帕米（Verapamil 替代人类保留在AAD受体部位。 2。我们将以强大的初步数据为基础，以揭示高 与Navab结合的1类AAD的分辨率结构，例如Lidocaine和Flescainide，在结合之间有区别 子类1a，1b和1c AADS的姿势和受体位点构象，确定了Fenestrations在状态中的作用 Navab的依赖性块，并探索使Navab药物受体人性化的突变的作用。 3。基于 新的均质生化制剂，我们将使用冷冻电子显微镜和X射线晶体学来确定 哺乳动物心脏NAV1.5在高分辨率下的结构，定义其独特的结构基础 生理特性，并阐明NAV1.5通道的AAD块的结构基础。我们的结果至关重要 为了理解和改善AADS危及生命的心律失常的治疗。