Structural Basis for Antiarrhythmic Drug Action

摘要 电压门控钠通道(Nav)启动心脏动作电位，电压门控钙通道(Cav)启动心脏动作电位 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，它们是 用于控制危及生命的心脏的I类和IV类抗心律失常药物的分子靶点 心律不齐。反兴奋剂行动的结构基础尚不清楚。我们已经确定了一位祖先的晶体结构 闭合、离子选择性和缓慢失活。这个结构还显示了从脂质横向引导的窗口。 双层进入孔道，并提供进入孔道阻挡AADS的通路。我们构建了一种钙选择形式 ，命名为CavAb，并利用该结构揭示了原子分辨率下钙选择性的结构基础。我们 CavAb被阻止 我们发现， 苯基烷基胺异搏定结合到孔中的受体位置，在离子选择性过滤器的内端，并物理地 挡住了它。相反，氨氯地平和其他二氢吡啶结合在毛孔面向脂质的外表面上的某个位置 在两个电压感应模块之间的界面上，并变构地堵塞气孔。这些结果揭示了 首次发现了CAV通道的药物受体复合体，并为全面分析其作用机制奠定了基础 在原子级别上依赖于状态的NAV和CAV通道阻塞。我们提出的实验有三个目标。1.我们 将建立在强大的初步数据基础上，以揭示治疗上重要的 苯并硫氮类药物地尔硫卓与其在CavAb孔中的受体结合，比较其与 维拉帕米，确定开窗在CavAb状态依赖阻断中的作用，并探索突变的影响 取代AAD受体位点上的人类残基。2.我们将以强劲的初步数据为基础，揭示- 与NAAB结合的1类AADs的拆分结构，如利多卡因和氟卡胺，区分结合 A亚类、1B亚类和1C亚类AADS的姿势和受体位置构象决定了开窗在状态- 3.基于 新的均相生化制剂，我们将使用低温电子显微镜和X射线结晶学来确定 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，阐明NAV1.5通道AAD阻断的结构基础。我们的结果将至关重要 目的了解和改进AADS对危及生命的心律失常的治疗。

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.