Vol. 59, Issue 4, 684-691, April 2001

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

Akihiko Sunami, Ian W. Glaaser, and Harry A. Fozzard

The Cardiac Electrophysiology Laboratories, Departments of Neurobiology, Pharmacology & Physiology, and Medicine, The University of Chicago, Chicago, Illinois

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.