Structural Basis for Antiarrhythmic Drug Action

摘要 电压门控钠（Nav）通道启动心脏中的动作电位，电压门控钙（Cav）通道启动心脏中的动作电位。 启动兴奋-收缩偶联。它们是具有共同进化祖先的相关蛋白质， 用于控制危及生命的心脏病的I类和IV类抗心律失常药物（AAD）的分子靶点 心律不齐AAD作用的结构基础尚不清楚。我们已经确定了一个祖先的晶体结构 细菌Nav通道（NavAb）在2.7 μ m的分辨率，并揭示了电压传感，孔开放和 关闭、离子选择性和缓慢失活。该结构还揭示了从脂质侧向引导的开窗 在一些实施方案中，AAD可以通过双层进入孔中，并为孔阻断AAD的进入提供通路。我们构建了一个钙选择性形式 的NavAb，称为CavAb，并使用该构建体来揭示原子分辨率下Ca选择性的结构基础。我们 目前，AAD对Nav和Cav通道的状态依赖性阻断的结构基础正受到关注。CavAb受阻 通过IV类AAD的所有三个结构亚类以状态依赖性方式以nM亲和力进行。我们发现 苯基烷基胺维拉帕米在离子选择性过滤器的内端结合到孔中的受体位点， 与此相反，二氢吡啶和其他二氢吡啶结合在孔的面向脂质的外表面上的一个位点上， 模块，在两个电压传感模块之间的界面处，并且变构地阻塞孔。这些结果揭示 药物-受体复合物的Cav通道的第一次，并设置了阶段，为完整的分析机制， 在原子水平上的Nav和Cav通道的状态依赖性阻断。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，以揭示高分辨率结构的治疗重要的 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状态依赖性阻滞中的作用，并探索突变的影响， 替代AAD受体位点中的人残基。2.我们将建立在强有力的初步数据，以揭示高- 与NavAb结合的1类AAD（如利多卡因和氟卡尼）的解析结构在结合 亚类1A、1B和1C AAD的位置和受体位点构象决定了开窗在状态中的作用， 依赖性阻断NavAb，并探索人源化NavAb药物受体的突变的影响。3.基于 新的均相生化制剂，我们将使用冷冻电子显微镜和X射线晶体学来确定 高分辨率的哺乳动物心脏Nav1.5通道的结构，定义了其独特的结构基础， 生理特性，并阐明了Nav1.5通道的AAD阻断的结构基础。我们的结果将至关重要 了解和改善AAD治疗危及生命的心律失常。

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.