Department of Pharmacology, University of Washington, Seattle, WA, United States; Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing, China.
Department of Pharmacology, University of Washington, Seattle, WA, United States.
Methods Enzymol. 2021;653:89-101. doi: 10.1016/bs.mie.2021.01.030. Epub 2021 Mar 12.
Voltage-gated sodium channel Na1.5 is responsible for initiating and propagating cardiac action potentials by selectively conducting Na into cardiomyocytes. Class-I antiarrhythmic drugs target Na1.5 for treatment of arrhythmias. During the last few years, cryogenic electron microscopy (cryo-EM) has become a powerful technique to determine the structures of ion channels at atomic level. In order to reveal the structural features of Na1.5 and the structural basis for its interaction with antiarrhythmic drugs by cryo-EM, Na1.5 protein must be expressed at high levels and purified to homogeneity. In this chapter, we discuss the expression and purification of Na1.5 in a mammalian expression system. We optimized the construct by deleting unstructured intracellular loops of rat Na1.5 while retaining core functional regions. The resulting rNa1.5 is fully functional and is blocked by Class-I antiarrhythmic drugs in a state-dependent manner. Protocols are presented for expressing and purifying sufficient sample of Na1.5 for preparing cryo-EM grids. The resulting cryo-EM structure is briefly described.
电压门控钠离子通道 Na1.5 通过选择性地将 Na 导入心肌细胞，负责引发和传播心脏动作电位。I 类抗心律失常药物针对 Na1.5 治疗心律失常。在过去的几年中，低温电子显微镜 (cryo-EM) 已成为确定离子通道在原子水平结构的强大技术。为了通过 cryo-EM 揭示 Na1.5 的结构特征及其与抗心律失常药物相互作用的结构基础，必须以高表达水平和均一性来表达和纯化 Na1.5 蛋白。在本章中，我们讨论了在哺乳动物表达系统中表达和纯化 Na1.5。我们通过删除大鼠 Na1.5 的无结构细胞内环来优化构建体，同时保留核心功能区。所得的 rNa1.5 是完全功能性的，并且以状态依赖性方式被 I 类抗心律失常药物阻断。介绍了用于表达和纯化足够量 Na1.5 样品以制备 cryo-EM 网格的方案。简要描述了所得的 cryo-EM 结构。
