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The principal voltage-sensitive sodium channel from human heart has been cloned, sequenced, and functionally expressed. The cDNA, designated hH1, encodes a 2016-amino acid protein that is homologous to other members of the sodium channel multigene family and bears >90% identity to the tetrodotoxin-insensitive sodium channel characteristic of rat heart and of immature and denervated rat skeletal muscle. Northern blot analysis demonstrates an =9.0-kilobase transcript expressed in human atrial and ventricular cardiac muscle but not in adult skeletal muscle, brain, myometrium, liver, or spleen. When expressed in Xenopus oocytes, hHl exhibits rapid activation and inactivation kinetics similar to native cardiac sodium channels. The single channel conductance of hHl to sodium ions is about twice that of the homologous rat channel and hHl is more resistant to block by tetrodotoxin (ICso = 5.7 pM). hHl is also resistant to Iu-conotoxin but sensitive to block by therapeutic concentrations of lidocaine in a use-dependent manner.

Sodium channels have four homologous domains (D1-D4) each with six putative transmembrane segments (S1-$6). The highly charged $4 segments in each domain are postulated voltage sensors for gating. We made 15 charge-neutralizing or -reversing substitutions in the first or third basic residues (arginine or lysine) by replacement with histidine, glutamine, or glutamate in $4 segments of each domain of the human heart Na ÷ channel. Nine of the mutations cause shifts in the conductance-voltage (G-V) midpoints, and all but two significantly decrease the voltage dependence of peak Na + current, consistent with a role of $4 segments in activation. The decreases in voltage dependence of activation were equivalent to a decrease in apparent gating charge of 0.5-2.1 elementary charges (%) per channel for single charge-neutralizing mutations. Three charge-reversing mutations gave decreases of 1.2-1.9 eo per channel in voltage dependence of activation. The steady-state inactivation (h~) curves were fit by single-component Boltzmann functions and show significant decreases in slope for 9 of the 15 mutants and shifts of midpoints in 9 mutants. The voltage dependence of inactivation time constants is markedly decreased by mutations only in $4D4, providing further evidence that this segment plays a unique role in activation-inactivation coupling.

Site-3 toxins have been shown to inhibit a component of gating charge (33% of maximum gating charge, Q(max)) in native cardiac Na channels that has been identified with the open-to-inactivated state kinetic transition. To investigate the role of the three outermost arginine amino acid residues in segment 4 domain IV (R1, R2, R3) in gating charge inhibited by site-3 toxins, we recorded ionic and gating currents from human heart Na channels with mutations of the outermost arginines (R1C, R1Q, R2C, and R3C) expressed in fused, mammalian tsA201 cells. All four mutations had ionic currents that activated over the same voltage range with slope factors of their peak conductance-voltage (G-V) relationships similar to those of wild-type channels, although decay of I(Na) was slowest for R1C and R1Q mutant channels and fastest for R3C mutant channels. After Na channel modification by Ap-A toxin, decays of I(Na) were slowed to similar values for all four channel mutants. Toxin modification produced a graded effect on gating charge (Q) of mutant channels, reducing Q(max) by 12% for the R1C and R1Q mutants, by 22% for the R2C mutant, and by 27% for the R3C mutant, only slightly less than the 31% reduction seen for wild-type currents. Consistent with these findings, the relationship of Q(max) to G(max) was significantly shallower for R1 mutants than for R2C and R3C mutant Na channels. These data suggest that site-3 toxins primarily inhibit gating charge associated with movement of the S4 in domain IV, and that the outermost arginine contributes the largest amount to channel gating, with other arginines contributing less.