Abstract

Membrane-impermeant quaternary amine local anesthetics QX314 and QX222 can access their binding site on the cytoplasmic side of the selectivity filter from the outside in native cardiac Na⁺channels. Mutation of domain IV S6 Ile-1760 of rat brain IIA Na⁺ channel or the equivalent (Ile-1575) in the adult rat skeletal muscle isoform (μ1) creates an artificial access path for QX. We examined the characteristics of mutation of μ1-I1575 and the resulting QX path. In addition to allowing external QX222 access, I1575A accelerated decay of Na⁺ current and shifted steady-state availability by −27 mV. I1575A had negligible effects on inorganic or organic cation selectivity and block by tetrodotoxin (TTX), saxitoxin (STX), or μ-conotoxin (μ-CTX). It exposed a site within the protein that binds membrane-permeant methanethiosulfonate ethylammonium (MTSEA), but not membrane-impermeant methanethiosulfonate ethyltrimethylammonium (MTSET) and methanethiosulfonate ethylsulfonate (MTSES). MTSEA binding abolished the QX path created by this mutation, without effects on toxin binding. The μ-CTX derivative R13N, which partially occluded the pore, had no effect on QX access. I1575A exposed two Cys residues because a disulfide bond was formed under oxidative conditions, but the exposed Cys residues are not those in domain IV S6, adjacent to Ile-1575. The Cys mutant I1575C was insensitive to external Cd²⁺ and MTS compounds (MTSEA, MTSET, MTSES), and substitution of Ile with a negatively charged residue (I1575E) did not affect toxin binding. Ile-1575 seems to be buried in the protein, and its mutation disrupts the protein structure to create the QX path without disturbing the outer vestibule and its selectivity function.