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
- The Cardiac Electrophysiology Laboratories, Departments of Neurobiology, Pharmacology & Physiology, and Medicine, The University of Chicago, Chicago, Illinois
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.
Footnotes
-
Send reprint requests to: Harry A. Fozzard, M.D., Cardiac Electrophysiology Laboratories (MC6094), University of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637. E-mail:foz{at}hearts.bsd.uchicago.edu
-
This work was supported by National Institutes of Health Grant P01-HL20592.
-
Preliminary reports of this work have been published in abstract form [Sunami A, Dudley SC, Lipkind G, Fozzard HA (1998) A mutation of the sodium channel suggesting that domain IV S6 contributes to the outer vestibule. Biophys J74:A398 and Sunami A, Lipkind G, Glaaser IW, Fozzard HA (1999) Characterizing structural rearrangement of the sodium channel outer vestibule induced by S6 mutants. Biophys J76:A81].
- Abbreviations:
- TTX
- tetrodotoxin
- μ1
- adult rat skeletal muscle Na+ channel α-subunit
- MTSEA
- methanethiosulfonate ethylammonium
- MTSET
- methanethiosulfonate ethyltrimethylammonium
- MTSES
- methanethiosulfonate ethylsulfonate
- CTX
- conotoxin
- STX
- saxitoxin
- I-V
- current-voltage
- DTT
- dithiothreitol
- Cu(phe)3
- Cu(II)(1, 10-phenanthroline)3
- WT
- wild-type
-
- Received June 8, 2000.
- Accepted December 19, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
