%0 Journal Article %A Akihiko Sunami %A Ian W. Glaaser %A Harry A. Fozzard %T Structural and Gating Changes of the Sodium Channel Induced by Mutation of a Residue in the Upper Third of IVS6, Creating an External Access Path for Local Anesthetics %D 2001 %R 10.1124/mol.59.4.684 %J Molecular Pharmacology %P 684-691 %V 59 %N 4 %X 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. %U https://molpharm.aspetjournals.org/content/molpharm/59/4/684.full.pdf