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.

Salicylate enhanced tonic and phasic block of Na+ channels induced by class 1 highly liposoluble antiarrhythmic agents. Based on the modulated receptor hypothesis, it is probable that this enhancement was mediated by an increase in the affinity of Na+ channel blockers with high lipid solubility to the inactivated state channels.

It is unknown whether salicylate enhances the action of antiarrhythmic agents on human Na + channels with state dependency and tissue specificity. We therefore investigated effects of salicylate on quinidine-induced block of human cardiac and skeletal muscle Na + channels. Human cardiac wild-type (hH1), LQT3-related mutant (ΔKPQ), and skeletal muscle (hSkM1) Na + channel α subunits were expressed in COS7 cells. Effects of salicylate on quinidine-induced tonic and usedependent block of Na + channel currents were examined by the whole-cell patch-clamp technique. Salicylate enhanced the quinidine-induced tonic and use-dependent block of both hH1 and hSkM1 currents at a holding potential (HP) of −100 mV but not at −140 mV. Salicylate decreased the IC 50 value for the quinidine-induced tonic block of hH1 at an HP of −100 mV, and produced a negative shift in the steady-state inactivation curve of hH1 in the presence of quinidine. According to the modulated receptor theory, it is probable that salicylate decreases the dissociation constant for quinidine binding to inactivated-state channels. Furthermore, salicylate significantly enhanced the quinidine-induced tonic and use-dependent block of the peak and steady-state ΔKPQ channel currents. The results suggest that salicylate enhances quinidine-induced block of Na + channels via increasing the affinity of quinidine to inactivated state channels.Acetylsalicylic acid, the most widely studied antiplatelet drug, suppresses platelet aggregation by inhibiting cyclo-oxygenase. Salicylic acid (C 6 H 4 (OH)COOH) belongs to the group of aromatic monocarboxylic acids, which by themselves do not have an anesthetic action. However, monocarboxylic acids such as salicylic, benzoic, acetic, propionic and butyric acid have been reported to enhance the action of local anesthetics (7, 13). In particular, monocarboxylic acids containing a lipophilic moiety such as an aliphatic hydrocarbon chain or aromatic ring can enhance the action of local anesthetics in nerves; indeed, salicylate has been reported to enhance the blocking action of procainamide on sodium (Na + ) channels in nerves (7). And Katsuki et al. (13) demonstrated that salicylate decreased the intracellular pH, which resulted in an increase of the proportion of charged molecules of procaine and the enhancement of its local anesthetic action on nerves.As for the effect of salicylate on cardiac myocytes, Tanaka et al. (23) reported that it enhanced the action of Na + channel blockers with higher liposolubility by increasing their affinity for inactivated state channels. Since Na + channels in individual

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