Use of Genetically Encoded Photoactivable Cross-Linking Molecules to Probe NaV Channel Fast Inactivation

Abstract

Fast inactivation in voltage-gated sodium channels requires efficient and rapidly reversible conformational changes in the cytoplasmic linker between domains III and IV (DIII-IV) and their putative receptor in the pore region of the channel. Introduced or acquired mutations in this complex lead to defective channel inactivation, and the resulting excessive sodium conductance can cause a myriad of pathophysiological disorders. We set out to better understand fast-inactivation by expressing voltage-gated sodium channels that carry genetically encoded orthogonal photocrosslinking (PC) amino acid molecules in the inactivation complex with the goal of capturing transient, but physiologically important complexes. PC molecules have widely been used to characterize protein-protein interactions through their ability to covalently bond with nearby molecules upon excitation by UV light. Conventional in-vivo use of PC molecules relies on their covalent attachment to introduced cysteine residues, which must be functionally tolerated and equally solvent accessible, and are limited by non-specific labelling of both target and off target proteins in the cellular environment. We have bypassed these limitations by utilizing the amber stop codon suppression system to genetically incorporate the PC unnatural amino acids (UAA) p-benzoyl-L-phenyl alanine and p-azido-L-phenylalanine into NaV1.5 channels in HEK cells. NaV1.5 channels carrying the UAA molecules showed robust expression at a number of sites in the DIII-IV linker with channel currents that displayed near normal activation, fast and steady state inactivation properties, verifying that the UAAs were well tolerated. Simultaneous patch clamp recording and UV irradiation of channels demonstrated that photo-activating the crosslinking UAA has effects on fast inactivation that are dependent upon the incorporation site and the state of the channel during UV exposure. This technique allows resolution of discrete, transient molecular conformations of the inactivation machinery that support sodium channel fast inactivation.