Block of sodium channels by divalent mercury: Role of specific cysteinyl residues in the P-loop region

Divalent mercury (Hg2+) blocked human skeletal Na+ channels (hSkN1) in a st able dose-dependent manner (K-d = 0.96 mu M) in the absence of reducing age nt. Dithiothreitol (DTT) significantly prevented Hg2+ block Of hSkM1, and H g2+ block was also readily reversed by DTT. Both thimerosal and 2,2'-dithio dipyridine had little effect on hSkM1; however, pretreatment with thimerosa l attenuated Hg2+ block of hSkM1. Y401C+E758C rat skeletal muscle Na+ chann els (mu 1) that form a disulfide bond spontaneously between two cysteines a t the 401 and 758 positions showed a significantly lower sensitivity to Hg2 + (K-d = 18 mu M). However, Y401C+E758C mu 1 after reduction with DTT had a significantly higher sensitivity to Hg2+ (K-d = 0.36 mu M) than wild-type hSkM1. Mutants C753A mu 1 (K-d = 8.47 mu M) or C1521A mu 1 (K-d = 8.63 mu M ) exhibited significantly lower sensitivity to Hg2+ than did wild-type hSkM 1, suggesting that these two conserved cysteinyl residues of the P-loop reg ion may play an important role in the Hg2+ block of the hSkM1 isoform. The heart Na+ channel (hH1) was significantly more sensitive to low-dose Hg2+ ( K-d = 0.43 mu M) than was hSkM1. The C373Y hH1 mutant exhibited higher resi stance (K-d = 1.12 mu M) to Hg2+ than did wild-type hH1. In summary, Hg2+ p robably inhibits the muscle Na+ channels at more than one cysteinyl residue in the Na+ channel P-loop region. Hg2+ exhibits a lower K-d value (<1.23 m u M) for inhibition by terming a sulfur-Hg-sulfur bridge, as compared to re action at a single cysteinyl residue with a higher K-d value (>8.47 mu M) b y forming sulfur-Hg+ covalently. The heart Na+ channel isoform with more th an two cysteinyl residues in the P-loop region exhibits an extremely high s ensitivity (K-d < 0.43 mu M) to Hg+, accounting for heart-specific high sen sitivity to the divalent mercury.