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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.

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The structure and function of many cysteine-containing proteins critically depend on the oxidation state of the sulfhydryl groups. In such proteins, selective modification of sulfhydryl groups can be used to probe the relation between structure and function. We examined the effects of sulfhydryloxidizing and -reducing agents on the function of the heterologously expressed pore-forming subunits of the cloned rabbit smooth muscle L-type Ca2+ channel and the human cardiac tetrodotoxin-insensitive Na+ channel. The known sequences of the channels suggest the presence of three or four cysteine residues within the putative pores of Ca2+ or Na+ channels, respectively, as well as multiple other cysteines in regions of unknown function. We determined the effects of sulfhydryl modification on Ca2+ and Na+ channel gating and permeation by using the whole-cell and single-channel variants of the patch-clamp technique. Within 10 minutes of exposure to 2,2'-dithiodipyridine (DTDP, a specific lipophilic oxidizer of sulfhydryl groups), Ca2+ current was reduced compared with the control value, with no significant change in the kinetics and no shift in the current-voltage relations. The effect could be readily reversed by 1,4-dithiothreitol (an agent that reduces disulfide bonds). Similar results were obtained by using the hydrophilic sulfhydryl-oxidizing agent thimerosal. The effects were Ca(2+)-channel specific: DTDP induced no changes in expressed human cardiac Na+ current. Single-channel Ba2+ current recordings revealed a reduction in open probability and mean open time by DTDP but no change in single-channel conductance, implying that the reduction of macroscopic Ca2+ current reflects changes in gating and not permeation. In summary, the pore-forming (alpha 1) subunit of the L-type Ca2+ channel contains functionally important free sulfhydryl groups that modulate gating. These free sulfhydryl groups are accessible from the extracellular side by an aqueous pathway.

A pair of conserved methionine residues, located on the cytoplasmic linker between segments $4 and $5 in the fourth domain of human heart Na channels (hill), plays a role in the kinetics and voltage dependence of inactivation. Substitution of these residues by either glutamine (M1551M1652/QQ) or alanine (MM/AA) increases the inactivation time constant (Th) at depolarized voltages, shifts steady-state inactivation (h~) in a depolarized direction, and decreases the time constant for recovery from inactivation. The data indicate that the mutations affect the rate constants for both binding and unbinding of a hypothetical inactivation particle from its binding site. Cytoplasmic application of the pentapeptide KIFMK in Na channels mutated to remove inactivation produces current decays resembling inactivation (Eaholtz, G., T. Scheuer, and W. A. Catterall. 1994. Neuron. 12: 1041-1048. KIFMK produces a concentration-dependent, voltage-independent increase in the decay rate of MM/QQ and MM/AA currents at positive membrane potentials (/~ ~30 I.tM), while producing only a small increase in the decay rate of wild-type currents at a concentration of 200 I~M. Although MM/QQ inactivates N2.5-fold faster than MM/AA in the absence of peptide, the estimated rate constants for peptide block and unblock do not differ in these mutants. External Na § ions antagonize the block by cytoplasmic KIFMK of MM/AA channels, but not the inactivation kinetics of this mutant in the absence of peptide. The effect of external [Na +] is interpreted as a voltage-dependent knock-off mechanism. The data provide evidence that KIFMK can only block channels when they are open and that peptide block does not mimic the inactivation process. Key words: cardiac muscle 9 complementary DNA 9 expression 9 mutagenesis INTRODUCTION