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 vol tage-gated sodium channels forms the inactivation gate. A combination of site-directed mutagenesis, cysteine covalent modification, and elec trophysiological recording techniques was used to identify the role of the Phe(1486), a conserved phenylalanine residue located in the III-I V linker of Na+ channels, This Phe(1486) is part of a hydrophobic amin o acid cluster (IFM) that was proposed to play an essential role in th e fast inactivation of voltage-gated sodium channels. Expression in ts A201 cells of an altered human heart 1 Na+ channel (hHI/F14SGC) in whi ch Phe(1486) was replaced by a cysteine is associated with the appeara nce of a residual current, a loss of voltage-dependence of the time co nstants of inactivation, a shift of the steady-state inactivation to m ore depolarized voltages, and a recovery from inactivation that is fas ter than the wild-type hill. Exposure of the cytoplasmic surface of mu tant F1486C to the methanthiosulfonate reagents, MTSEA, MTSET, and MTS ES, further disrupted macroscopic inactivation, but exposure to MTSBN completely restores fast inactivation and the voltage-dependence of fa st inactivation. These findings support the formulation that the IFM m otif of the III-IV-linker of voltage-gated sodium channels serves as a n essential component of the inactivation particle and that the phenyl group of Phe(1486) may play a crucial role in inactivation gate closu re. (C) 1997 Academic Press.