Role of L1462 in Nav1.5 Channel DIII-S6 in Voltage-dependent Gating and Antiarrhythmic Block

Abstract

Mutations of domain III S6 (DIII-S6) Leu-1465 of rat brain IIA Na+ channel (Nav1.2) or the equivalent (L1280) in the adult rat skeletal muscle isoform (Nav1.4) affect local anesthetic and anticonvulsant block (Yarov-Yarovoy et al., 2001; Nau et al., 2003). We examined the role of the equivalent position (L1462) of the human heart isoform (Nav1.5) in voltage-dependent gating and antiarrhythmic block. Whole-cell Na+ currents were measured in HEK293 cells transiently expressing the recombinant wild type (WT) or mutant channels and the beta-1 subunit. Compared to WT, all the mutants of L1462 (L1462A, L1462C, L1462F) accelerated the current decay and shifted the voltage dependence of activation towards more positive direction. L1462 mutants had no effects on the voltage dependence of fast inactivation and the recovery from fast inactivation, but increased the fraction of intermediate inactivation component. Internal charged methanethiosulfonate, MTSES blocked L1462C/C373F with a high frequency of stimulation (20 Hz), where C373 is known to face the outer pore and be the critical residue for isoform differences in tetrodotoxin block. L1462F had no obvious effects on tonic block, but increased use-dependent block by the class Ic antiarrhythmic drug, flecainide, which binds preferentially to activated channels. L1462F increased affinity of activated channels for flecainide by 4-fold, while L1462F had little effects on affinity of inactivated channels. For internal membrane-impermeant QX314, L1462F increased use-dependent block and affinity for QX314 binding to activated channels by 5-fold. These results suggest that L1462 faces the pore in the open state and is involved in activated channel block by antiarrhythmic drugs as well as inactivated channel block by local anesthetics.