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

Abstract

Voltage-gated sodium channel NaV1.5 is responsible for initiating and propagating cardiac action potentials by selectively conducting Na+ into cardiomyocytes. Class-I antiarrhythmic drugs target NaV1.5 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 NaV1.5 and the structural basis for its interaction with antiarrhythmic drugs by cryo-EM, NaV1.5 protein must be expressed at high levels and purified to homogeneity. In this chapter, we discuss the expression and purification of NaV1.5 in a mammalian expression system. We optimized the construct by deleting unstructured intracellular loops of rat NaV1.5 while retaining core functional regions. The resulting rNaV1.5C 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 NaV1.5 for preparing cryo-EM grids. The resulting cryo-EM structure is briefly described.