Block of sodium channels by divalent mercury: role of specific cysteinyl residues in the P-loop region.
Divalent mercury (Hg(2+)) blocked human skeletal Na(+) channels (hSkM1) in a stable dose-dependent manner (K(d) = 0.96 microM) 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 (mu1) 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 microM). However, Y401C+E758C mu1 after reduction with DTT had a significantly higher sensitivity to Hg(2+) (K(d) = 0.36 microM) than wild-type hSkM1. Mutants C753Amu1 (K(d) = 8.47 microM) or C1521A mu1 (K(d) = 8.63 microM) 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 microM) than was hSkM1. The C373Y hH1 mutant exhibited higher resistance (K(d) = 1.12 microM) 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 microM) 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 microM) 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 microM) to Hg(+), accounting for heart-specific high sensitivity to the divalent mercury.
Hisatome
I
I
First Department of Internal Medicine, Tottori University Faculty of Medicine, Yonago 683, Japan. hisatome@grape.med.tottori-u.ac.jp
Kurata
Y
Y
Sasaki
N
N
Morisaki
T
T
Morisaki
H
H
Tanaka
Y
Y
Urashima
T
T
Yatsuhashi
T
T
Tsuboi
M
M
Kitamura
F
F
Miake
J
J
Takeda
S i
Si
Taniguchi
S i
Si
Ogino
K
K
Igawa
O
O
Yoshida
A
A
Sato
R
R
Makita
N
N
Shigemasa
C
C
D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives / Q000494:pharmacology; D019943:Amino Acid Substitution; D000818:Animals; D003545:Cysteine; D004220:Disulfides / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008627:Mercuric Chloride / Q000494:pharmacology*; D018482:Muscle, Skeletal; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D015222:Sodium Channels / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D013849:Thimerosal / Q000494:pharmacology; D014162:Transfection
D004220:Disulfides; D011994:Recombinant Proteins; D015222:Sodium Channels; C001105:2,2'-dipyridyl disulfide; D013849:Thimerosal; D008627:Mercuric Chloride; D015082:2,2'-Dipyridyl; D003545:Cysteine; D004229:Dithiothreitol