Block of Sodium Channels by Divalent Mercury: Role of Specific Cysteinyl Residues in the P-Loop Region

Abstract

Divalent mercury (Hg 2+) blocked human skeletal Na + channels (hSkM1) in a stable dose-dependent manner ( K d = 0.96 μM) in the absence of reducing agent. Dithiothreitol (DTT) significantly prevented Hg 2+ block of hSkM1, and Hg 2+ block was also readily reversed by DTT. Both thimerosal and 2,2′-dithiodipyridine had little effect on hSkM1; however, pretreatment with thimerosal attenuated Hg 2+ block of hSkM1. Y401C+E758C rat skeletal muscle Na + channels ( μ1) that form a disulfide bond spontaneously between two cysteines at the 401 and 758 positions showed a significantly lower sensitivity to Hg 2+ ( K d = 18 μM). However, Y401C+E758C μ1 after reduction with DTT had a significantly higher sensitivity to Hg 2+ ( K d = 0.36 μM) than wild-type hSkM1. Mutants C753A μ1 ( K d = 8.47 μM) or C1521A μ1 ( K d = 8.63 μM) exhibited significantly lower sensitivity to Hg 2+ than did wild-type hSkM1, suggesting that these two conserved cysteinyl residues of the P-loop region may play an important role in the Hg 2+ block of the hSkM1 isoform. The heart Na + channel (hH1) was significantly more sensitive to low-dose Hg 2+ ( K d = 0.43 μM) than was hSkM1. The C373Y hH1 mutant exhibited higher resistance ( K d = 1.12 μM) to Hg 2+ than did wild-type hH1. In summary, Hg 2+ probably inhibits the muscle Na + channels at more than one cysteinyl residue in the Na + channel P-loop region. Hg 2+ exhibits a lower K d value (<1.23 μM) for inhibition by forming a sulfur-Hg-sulfur bridge, as compared to reaction at a single cysteinyl residue with a higher K d value (>8.47 μM) by forming sulfur-Hg + covalently. The heart Na + channel isoform with more than two cysteinyl residues in the P-loop region exhibits an extremely high sensitivity ( K d < 0.43 μM) to Hg +, accounting for heart-specific high sensitivity to the divalent mercury.