Restoration of Fast Inactivation in an Inactivation-Defective Human Heart Sodium Channel by the Cysteine Modifying Reagent Benzyl-MTS: Analysis of IFM-ICM Mutation

It has been suggested that the region linking domain III and IV of voltage-gated sodium channels forms the inactivation gate. A combination of site-directed mutagenesis, cysteine covalent modification, and electrophysiological recording techniques was used to identify the role of the Phe1486, a conserved phenylalanine residue located in the III-IV linker of Na+ channels. This Phe1486 is part of a hydrophobic amino acid cluster (IFM) that was proposed to play an essential role in the fast inactivation of voltage-gated sodium channels. Expression in tsA201 cells of an altered human heart 1 Na+ channel (hH1/F1486C) in which Phe1486 was replaced by a cysteine is associated with the appearance of a residual current, a loss of voltage-dependence of the time constants of inactivation, a shift of the steady-state inactivation to more depolarized voltages, and a recovery from inactivation that is faster than the wild-type hH1. Exposure of the cytoplasmic surface of mutant F1486C to the methanthiosulfonate reagents, MTSEA, MTSET, and MTSES, further disrupted macroscopic inactivation, but exposure to MTSBN completely restores fast inactivation and the voltage-dependence of fast inactivation. These findings support the formulation that the IFM motif of the III-IV-linker of voltage-gated sodium channels serves as an essential component of the inactivation particle and that the phenyl group of Phe1486 may play a crucial role in inactivation gate closure.