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-收缩耦合。它们是具有共同进化祖先的相关蛋白质，并且它们是 用于控制危及生命的心脏疾病的 I 类和 IV 类抗心律失常药物 (AAD) 的分子靶标 心律失常。 AAD 作用的结构基础尚不清楚。我们已经确定了祖先的晶体结构 细菌导航通道 (NavAb) 的分辨率为 2.7 Å，揭示了电压传感、孔开放和 关闭、离子选择性和缓慢失活。这种结构还揭示了从脂质横向延伸的开窗 双层进入孔中，并为孔阻塞 AAD 的进入提供通道。我们构建了 Ca 选择性形式 NavAb，称为 CavAb，并使用该构建体揭示了原子分辨率下 Ca 选择性的结构基础。我们 现在重点研究 AAD 依赖于状态的 Nav 和 Cav 通道阻断的结构基础。 CavAb 被阻止 由 IV 类 AAD 的所有三个结构子类以状态依赖的方式与 nM 亲和力。我们发现 苯烷基胺维拉帕米与离子选择性过滤器内端孔内的受体位点结合，并以物理方式 阻止它。相反，氨氯地平和其他二氢吡啶结合在孔的面向脂质的外表面上的位点 模块，位于两个电压传感模块之间的接口处，并以变构方式阻塞孔隙。这些结果揭示 首次研究了 Cav 通道的药物受体复合物，并为完整分析其机制奠定了基础 原子级别的 Nav 和 Cav 通道的状态依赖块。我们提出的实验有三个目标。 1.我们 将建立在强有力的初步数据的基础上，揭示具有重要治疗意义的高分辨率结构 苯并硫氮卓地尔硫卓结合到 CavAb 孔中的受体位点，比较其结合的化学性质 维拉帕米，确定开窗在 CavAb 状态依赖性阻断中的作用，并探索突变的影响 取代 AAD 受体位点中的人类残基。 2. 我们将基于强有力的初步数据来揭示高 1 类 AAD（例如利多卡因和氟卡尼）与 NavAb 结合的解析结构，区分结合 亚类 1A、1B 和 1C AAD 的姿势和受体位点构象，决定了开窗在状态中的作用 NavAb 的依赖性阻断，并探索使 NavAb 药物受体人性化的突变的影响。 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 通道的高分辨率结构，定义了其独特的结构基础 生理特性，并阐明 AAD 阻断 Nav1.5 通道的结构基础。我们的结果将至关重要 了解和改进 AAD 对危及生命的心律失常的治疗。