Expression and purification of the cardiac sodium channel NaV1.5 for cryo-EM structure determination

Abstract

Voltage-gated sodium channel Na_V1.5 is responsible for initiating and propagating cardiac action potentials by selectively conducting Na⁺ into cardiomyocytes. Class-I antiarrhythmic drugs target Na_V1.5 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 Na_V1.5 and the structural basis for its interaction with antiarrhythmic drugs by cryo-EM, Na_V1.5 protein must be expressed at high levels and purified to homogeneity. In this chapter, we discuss the expression and purification of Na_V1.5 in a mammalian expression system. We optimized the construct by deleting unstructured intracellular loops of rat Na_V1.5 while retaining core functional regions. The resulting rNa_V1.5_C 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 Na_V1.5 for preparing cryo-EM grids. The resulting cryo-EM structure is briefly described.

Keywords: Affinity chromatography; Antiarrhythmic drug; Cryogenic electron microscopy; FLAG epitope; Fluorescent size exclusion chromatography; Heart; Ion selectivity filter; Pore; Sodium channel; Voltage sensor.