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.
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 (mu 1) creates an artificial access path for QX. We examined the characteristics of mutation of mu 1-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 mu-conotoxin (mu-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 mu-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 Cd(2+) 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.
Sunami
A
A
The Cardiac Electrophysiology Laboratories, The University of Chicago, Chicago, Illinois 60637, USA.
Glaaser
I W
IW
Fozzard
H A
HA
D019943:Amino Acid Substitution; D000779:Anesthetics, Local / Q000494:pharmacology*; D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002478:Cells, Cultured; D020916:Conotoxins / Q000494:pharmacology; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects / Q000235:genetics*; D008012:Lidocaine / Q000031:analogs & derivatives* / Q000494:pharmacology; D008698:Mesylates / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D018408:Patch-Clamp Techniques; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012530:Saxitoxin / Q000494:pharmacology; D026941:Sodium Channel Blockers; D015222:Sodium Channels / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013779:Tetrodotoxin / Q000494:pharmacology; D014162:Transfection; D014982:Xenopus laevis
D000779:Anesthetics, Local; D002121:Calcium Channel Blockers; D020916:Conotoxins; D008698:Mesylates; D020033:Protein Isoforms; D026941:Sodium Channel Blockers; D015222:Sodium Channels; C093171:methanethiosulfonate ethylammonium; C109153:(2-sulfonatoethyl)methanethiosulfonate; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; C006072:QX-222; C012647:QX-314; D012530:Saxitoxin; D013779:Tetrodotoxin; D008012:Lidocaine; D005020:Ethyl Methanesulfonate