Structural and gating changes of the sodium channel induced by mutation ofa residue in the upper third of IVS6, creating an external access path forlocal anesthetics

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 a dult rat skeletal muscle isoform (mu1) creates an artificial access path fo r QX. We examined the characteristics of mutation of mu1-I1575 and the resu lting QX path. In addition to allowing external QX222 access, I1575A accele rated decay of Na+ current and shifted steady-state availability by -27 mV. I1575A had negligible effects on inorganic or organic cation selectivity a nd block by tetrodotoxin (TTX), saxitoxin (STX), or mu -conotoxin (mu -CTX) . It exposed a site within the protein that binds membrane-permeant methane thiosulfonate ethylammonium (MTSEA), but not membrane-impermeant methanethi osulfonate ethyltrimethylammonium (MTSET) and methanethiosulfonate ethylsul fonate (MTSES). MTSEA binding abolished the QX path created by this mutatio n, without effects on toxin binding. The mu -CTX derivative R13N, which par tially occluded the pore, had no effect on QX access. I1575A exposed two Cy s 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 compo unds (MTSEA, MTSET, MTSES), and substitution of Ile with a negatively charg ed residue (I1575E) did not affect toxin binding. Ile-1575 seems to be buri ed in the protein, and its mutation disrupts the protein structure to creat e the QX path without disturbing the outer vestibule and its selectivity fu nction.