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

• Published in: Biophysical journal Vol. 79; no. 3; pp. 1336 - 1345
• Main Authors: Hisatome, Ichiro, Kurata, Yasutaka, Sasaki, Norihito, Morisaki, Takayuki, Morisaki, Hiroko, Tanaka, Yasunori, Urashima, Tadashi, Yatsuhashi, Toru, Tsuboi, Mariko, Kitamura, Fumiyo, Miake, Junichiro, Takeda, Shin-ichi, Taniguchi, Shin-ichi, Ogino, Kazuhide, Igawa, Osamu, Yoshida, Akio, Sato, Ryoichi, Makita, Naomasa, Shigemasa, Chiaki
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.09.2000; Biophysical Society
• Subjects: 2,2'-Dipyridyl - analogs & derivatives; 2,2'-Dipyridyl - pharmacology; Amino Acid Substitution; Anatomy & physiology; Animals; Cysteine; Disulfides - pharmacology; Dithiothreitol - pharmacology; Dose-Response Relationship, Drug; Humans; Membrane Potentials - drug effects; Membrane Potentials - physiology; Mercuric Chloride - pharmacology; Mercury; Muscle, Skeletal; Mutagenesis, Site-Directed; Patch-Clamp Techniques; Rats; Recombinant Proteins - chemistry; Recombinant Proteins - drug effects; Recombinant Proteins - metabolism; Sodium; Sodium Channels - chemistry; Sodium Channels - genetics; Sodium Channels - physiology; Thimerosal - pharmacology; Transfection
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/S0006-3495(00)76386-7

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.