Competitive binding interaction between Zn2+ and saxitoxin in cardiac Na+ channels. Evidence for a sulfhydryl group in the Zn2+/saxitoxin binding site.
Mammalian heart Na+ channels exhibit approximately 100-fold higher affinity for block by external Zn2+ than other Na+ channel subtypes. With batrachotoxin-modified Na+ channels from dog or calf heart, micromolar concentrations of external Zn2+ result in a flickering block to a substate level with a conductance of approximately 12% of the open channel at -50 mV. We examined the hypothesis that, in this blocking mode, Zn2+ binds to a subsite of the saxitoxin (STX) binding site of heart Na+ channels by single-channel analysis of the interaction between Zn2+ and STX and also by chemical modification experiments on single heart Na+ channels incorporated into planar lipid bilayers in the presence of batrachotoxin. We found that external Zn2+ relieved block by STX in a strictly competitive fashion. Kinetic analysis of this phenomenon was consistent with a scheme involving direct binding competition between Zn2+ and STX at a single site with intrinsic equilibrium dissociation constants of 30 nM for STX and 30 microM for Zn2+. Because high-affinity Zn2(+)-binding sites often include sulfhydryl groups as coordinating ligands of this metal ion, we tested the effect of a sulfhydryl-specific alkylating reagent, iodoacetamide (IAA), on Zn2+ and STX block. For six calf heart Na+ channels, we observed that exposure to 5 mM IAA completely abolished Zn2+ block and concomitantly modified STX binding with at least 20-fold reduction in affinity. These results lead us to propose a model in which Zn2+ binds to a subsite within or near the STX binding site of heart Na+ channels. This site is also presumed to contain one or more cysteine sulfhydryl groups.
Schild
L
L
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510.
Moczydlowski
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D055592:Biophysical Phenomena; D001703:Biophysics; D002417:Cattle; D004285:Dogs; D066298:In Vitro Techniques; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism*; D012530:Saxitoxin / Q000378:metabolism; D015222:Sodium Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism
D015222:Sodium Channels; D012530:Saxitoxin; D015032:Zinc; D007460:Iodoacetamide