Biophys J, September 2000, p. 1336-1345, Vol. 79, No. 3

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

Ichiro Hisatome,^* Yasutaka Kurata, Norihito Sasaki,^* Takayuki Morisaki, Hiroko Morisaki, Yasunori Tanaka,^* Tadashi Urashima,^* Toru Yatsuhashi,^* Mariko Tsuboi,^* Fumiyo Kitamura,^* Junichiro Miake,^* Shin-ichi Takeda,^* Shin-ichi Taniguchi,^* Kazuhide Ogino,^* Osamu Igawa,^* Akio Yoshida,^* Ryoichi Sato,^§ Naomasa Makita,^¶ and Chiaki Shigemasa^*

 ^*First Department of Internal Medicine, Tottori University Faculty of Medicine, Yonago 683, Japan;  Department of Physiology, Kanazawa Medical University, Ishikawa, Japan;  Department of Bioscience, National Institute of Cardiovascular Center, Osaka, Japan;  ^§Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois, USA; and  ^¶Department of Cardiovascular Medicine, Hokkaido University School of Medicine, Sapporo, Japan

Divalent mercury (Hg²⁺) 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 Hg²⁺ block of hSkM1, and Hg²⁺ block was also readily reversed by DTT. Both thimerosal and 2,2'-dithiodipyridine had little effect on hSkM1; however, pretreatment with thimerosal attenuated Hg²⁺ 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 Hg²⁺ (K_d = 18 µM). However, Y401C+E758C µ1 after reduction with DTT had a significantly higher sensitivity to Hg²⁺ (K_d = 0.36 µM) than wild-type hSkM1. Mutants C753Aµ1 (K_d = 8.47 µM) or C1521A µ1 (K_d = 8.63 µM) exhibited significantly lower sensitivity to Hg²⁺ than did wild-type hSkM1, suggesting that these two conserved cysteinyl residues of the P-loop region 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 µM) than was hSkM1. The C373Y hH1 mutant exhibited higher resistance (K_d = 1.12 µM) to Hg²⁺ than did wild-type hH1. In summary, Hg²⁺ probably 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 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.

Biophys J, September 2000, p. 1336-1345, Vol. 79, No. 3
© 2000 by the Biophysical Society   0006-3495/00/09/1336/10  $2.00

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