Activation of protein kinase C alters voltage dependence of a Na+ channel.

Phorbol esters and purified protein kinase C (PKC) have been shown to down-modulate the voltage-dependent Na+ channels expressed in Xenopus oocytes injected with chick brain RNA. We used the two-electrode voltage-clamp technique to demonstrate that a Na+ channel expressed in oocytes injected with RNA coding for the alpha subunit of the channel alone (VA200, a variant of rat brain type IIA) is also inhibited by PKC activation. The inhibition of Na+ currents, expressed in oocytes injected with either alpha subunit RNA (rat) or total brain RNA (chick), is voltage-dependent, being stronger at negative potentials. It appears to result mainly from a shift in the activation curve to the right and possibly a decrease in the steepness of the voltage dependence of activation. There is little effect on the inactivation process and maximal Na+ conductance. Thus, PKC modulates the Na+ channel by a mechanism involving changes in voltage-dependent properties of its main, channel-forming alpha subunit.     

Kinetic evidence for two Na channels in frog skeletal muscle

The kinetics of voltage-clamped sodium currents were studied in frog skeletal muscle. Sodium currents in frog skeletal muscle activate and inactivate following an initial delay in response to a depolarizing voltage pulse. Inactivation occurs via a double exponential decay exhibiting fast and slow components for virtually all depolarizing pulses used.The deactivation of Na currents exhibits two exponential components, one decaying rapidly, while the other decays slowly in time; the relative amplitude of the two components changes with the duration of the activating pulse. The two deactivation phases remain after pharmacological elimination of inactivation.In individual fibers, the percent amplitude of the slow inactivation component correlates with the percent amplitude of the slow deactivation component.Tetrodotoxin differentially blocks the slow deactivation component.These observations are interpreted as the activation, inactivation and deactivation of two subtypes (fast and slow) of Na channels.Studies of the slow deactivation phase magnitude vs the duration of the eliciting pulse provide a way to determine the kinetics of the slow Na channel in muscle.Ammonium substitution for Na in the Ringer produces a voltage dependent activation and inactivation of current which exhibits only one decay phase, and eliminates the slow decay phase of current, suggesting that adjustments of the ionic environment of the channels can mask the presence of one of the channel subtypes.     

A voltage-gated calcium-selective channel encoded by a sodium channel-like gene

BSC1, which was originally identified by its sequence similarity to voltage-gated Na(+) channels, encodes a functional voltage-gated cation channel whose properties differ significantly from Na(+) channels. BSC1 has slower kinetics of activation and inactivation than Na(+) channels, it is more selective for Ba(2+) than for Na(+), it is blocked by Cd(2+), and Na(+) currents through BSC1 are blocked by low concentrations of Ca(2+). All of these properties are more similar to voltage-gated Ca(2+) channels than to voltage-gated Na(+) channels. The selectivity for Ba(2+) is partially due to the presence of a glutamate in the pore-forming region of domain III, since replacing that residue with lysine (normally present in voltage-gated Na(+) channels) makes the channel more selective for Na(+). BSC1 appears to be the prototype of a novel family of invertebrate voltage-dependent cation channels with a close structural and evolutionary relationship to voltage-gated Na(+) and Ca(2+) channels.     

Efficient expression of rat brain type IIA Na+ channel alpha subunits in a somatic cell line.

Type IIA rat brain Na+ channel alpha subunits were expressed in CHO cells by nuclear microinjection or by transfection using a vector containing both metallothionein and bacteriophage SP6 promoters. Stable cell lines expressing Na+ channels were isolated, and whole-cell Na+ currents of 0.9-14 nA were recorded. The mean level of whole-cell Na+ current (4.5 nA) corresponds to a cell surface density of approximately 2 channels active at the peak of the Na+ current per microns 2, a density comparable to that observed in the cell bodies of central neurons. The expressed Na+ channels had the voltage dependence, rapid activation and inactivation, and rapid recovery from inactivation characteristic of Na+ channels in brain neurons, bound toxins at neurotoxin receptor sites 1 and 3 with normal properties, and were posttranslationally processed to a normal mature size of 260 kd. Expression of Na+ channel cDNA in CHO cells driven by the metallothionein promoter accurately and efficiently reproduces native Na+ channel properties and provides a method for combined biochemical and physiological analysis of Na+ channel structure and function.     

Cardiac Na currents and the inactivating, reopening, and waiting properties of single cardiac Na channels

Tetrodotoxin (TTX)-sensitive Na currents were examined in single dissociated ventricular myocytes from neonatal rats. Single channel and whole cell currents were measured using the patch-clamp method. The channel density was calculated as 2/micron 2, which agreed with our usual finding of four channels per membrane patch. At 20 degrees C, the single channel conductance was 20 pS. The open time distributions were fit by a single-exponential function with a mean open time of approximately 1.0 ms at membrane potentials from -60 to -40 mV. Averaged single channel and whole cell currents were similar when scaled and showed both fast and slow rates of inactivation. The inactivation and activation gating shifted quickly to hyperpolarized potentials for channels in cell-attached as well as excised patches, whereas a much slower shift occurred in whole cells. Slowly inactivating currents were present in both whole cell and single channel current measurements at potentials as positive as -40 mV. In whole cell measurements, the potential range could be extended, and slow inactivation was present at potentials as positive as -10 mV. The curves relating steady state activation and inactivation to membrane potential had very little overlap, and slow inactivation occurred at potentials that were positive to the overlap. Slow inactivation is in this way distinguishable from the overlap or window current, and the slowly inactivating current may contribute to the plateau of the rat cardiac action potential. On rare occasions, a second set of Na channels having a smaller unit conductance and briefer duration was observed. However, a separate set of threshold channels, as described by Gilly and Armstrong (1984. Nature [Lond.]. 309:448), was not found. For the commonly observed Na channels, the number of openings in some samples far exceeded the number of channels per patch and the latencies to first opening or waiting times were not sufficiently dispersed to account for the slowly inactivating currents: the slow inactivation was produced by channel reopening. A general model was developed to predict the number of openings in each sample. Models in which the number of openings per sample was due to a dispersion of waiting times combined with a rapid transition from an open to an absorbing inactivated state were unsatisfactory and a model that was more consistent with the results was identified.     

Inactivation of batrachotoxin-modified Na+ channels in GH3 cells. Characterization and pharmacological modification

Batrachotoxin (BTX)-modified Na+ currents were characterized in GH3 cells with a reversed Na+ gradient under whole-cell voltage clamp conditions. BTX shifts the threshold of Na+ channel activation by approximately 40 mV in the hyperpolarizing direction and nearly eliminates the declining phase of Na+ currents at all voltages, suggesting that Na+ channel inactivation is removed. Paradoxically, the steady-state inactivation (h infinity) of BTX-modified Na+ channels as determined by a two-pulse protocol shows that inactivation is still present and occurs maximally near -70 mV. About 45% of BTX-modified Na+ channels are inactivated at this voltage. The development of inactivation follows a sum of two exponential functions with tau d(fast) = 10 ms and tau d(slow) = 125 ms at -70 mV. Recovery from inactivation can be achieved after hyperpolarizing the membrane to voltages more negative than -120 mV. The time course of recovery is best described by a sum of two exponentials with tau r(fast) = 6.0 ms and tau r(slow) = 240 ms at -170 mV. After reaching a minimum at -70 mV, the h infinity curve of BTX-modified Na+ channels turns upward to reach a constant plateau value of approximately 0.9 at voltages above 0 mV. Evidently, the inactivated, BTX-modified Na+ channels can be forced open at more positive potentials. The reopening kinetics of the inactivated channels follows a single exponential with a time constant of 160 ms at +50 mV. Both chloramine-T (at 0.5 mM) and alpha-scorpion toxin (at 200 nM) diminish the inactivation of BTX-modified Na+ channels. In contrast, benzocaine at 1 mM drastically enhances the inactivation of BTX-modified Na+ channels. The h infinity curve reaches minimum of less than 0.1 at -70 mV, indicating that benzocaine binds preferentially with inactivated, BTX-modified Na+ channels. Together, these results imply that BTX-modified Na+ channels are governed by an inactivation process.     

Evidence for a population of sleepy sodium channels in squid axon at low temperature

We have studied the effects of temperature changes on Na currents in squid giant axons. Decreases in temperature in the 15-1 degrees C range decrease peak Na current with a Q10 of 2.2. Steady state currents, which are tetrodotoxin sensitive and have the same reversal potential as peak currents, are almost unaffected by temperature changes. After removal of inactivation by pronase treatment, steady state current amplitude has a Q10 of 2.3. Na currents generated at large positive voltages sometimes exhibit a biphasic activation pattern. The first phase activates rapidly and partially inactivates and is followed by a secondary slow current increase that lasts several milliseconds. Peak Na current amplitude can be increased by delivering large positive prepulses, an effect that is more pronounced at low temperatures. The slow activation phase is eliminated after a positive prepulse. The results are consistent with the hypothesis that there are two forms of the Na channel: (a) rapidly activating channels that completely inactivate, and (b) slowly activating "sleepy" channels that inactivate slowly if at all. Some fast channels are assumed to be converted to sleepy channels by cooling, possibly because of a phase transition in the membrane. The existence of sleepy channels complicates the determination of the Q10 of gating parameters and single-channel conductance.     

Y1767C, a novel SCN5A mutation, induces a persistent Na+ current and potentiates ranolazine inhibition of Nav1.5 channels

Long QT syndrome type 3 (LQT3) has been traced to mutations of the cardiac Na(+) channel (Na(v)1.5) that produce persistent Na(+) currents leading to delayed ventricular repolarization and torsades de pointes. We performed mutational analyses of patients suffering from LQTS and characterized the biophysical properties of the mutations that we uncovered. One LQT3 patient carried a mutation in the SCN5A gene in which the cysteine was substituted for a highly conserved tyrosine (Y1767C) located near the cytoplasmic entrance of the Na(v)1.5 channel pore. The wild-type and mutant channels were transiently expressed in tsA201 cells, and Na(+) currents were recorded using the patch-clamp technique. The Y1767C channel produced a persistent Na(+) current, more rapid inactivation, faster recovery from inactivation, and an increased window current. The persistent Na(+) current of the Y1767C channel was blocked by ranolazine but not by many class I antiarrhythmic drugs. The incomplete inactivation, along with the persistent activation of Na(+) channels caused by an overlap of voltage-dependent activation and inactivation, known as window currents, appeared to contribute to the LQTS phenotype in this patient. The blocking effect of ranolazine on the persistent Na(+) current suggested that ranolazine may be an effective therapeutic treatment for patients with this mutation. Our data also revealed the unique role for the Y1767 residue in inactivating and forming the intracellular pore of the Na(v)1.5 channel.     

Enhancement of HERG K(+) currents by Cd(2+) destabilization of the inactivated state

We have studied the functional effects of extracellular Cd(2+) on human ether-a-go-go-related gene (HERG) encoded K(+) channels. Low concentrations (10-200 &mgr;M) of extracellular Cd(2+) increased outward currents through HERG channels; 200 &mgr;M Cd(2+) more than doubled HERG currents and altered current kinetics. Cd(2+) concentrations up to 200 &mgr;M did not change the voltage dependence of channel activation, but shifted the voltage dependence of inactivation to more depolarized membrane potentials. Cd(2+) concentrations >/=500 &mgr;M shifted the voltage dependence of channel activation to more positive potentials. These results are consistent with a somewhat specific ability of Cd(2+) to destabilize the inactivated state. We tested the hypothesis that channel inactivation is essential for Cd(2+)-induced increases in HERG K(+) currents, using a double point mutation (G628C/S631C) that diminishes HERG inactivation (Smith, P. L., T. Baukrowitz, and G. Yellen. 1996. Nature (Lond.). 379:833-836). This inactivation-removed mutant is insensitive to low concentrations of Cd(2+). Thus, Cd(2+) had two distinct effects on HERG K(+) channels. Low concentrations of Cd(2+) caused relatively selective effects on inactivation, resulting in a reduction of the apparent rectification of the channel and thereby increasing HERG K(+) currents. Higher Cd(2+) concentrations affected activation gating as well, possibly by a surface charge screening mechanism or by association with a lower affinity site.     

Two functionally distinct 4-aminopyridine-sensitive outward K+ currents in rat atrial myocytes

In the experiments here, the detailed kinetic properties of the Ca(2+)-independent, depolarization-activated outward currents (Iout) in enzymatically dispersed adult rat atrial myocytes were studied. Although there is only slight attenuation of peak Iout during brief (100 ms) voltage steps, substantial decay is evident during long (10 s) depolarizations. The analyses here reveal that current inactivation is best described by the sum of two exponential components, which we have termed IKf and IKs to denote the fast and slow components, respectively, of Iout decay. At all test potentials, IKf inactivates approximately 20-fold more rapidly than IKs. Neither the decay time constants nor the fraction of Iout remaining at the end of 10-s depolarizations varies over the potential range of 0 to +50 mV, indicating that the rates of inactivation and recovery from inactivation are voltage independent. IKf recovers from inactivation completely, independent of the recovery of IKs, and IKf recovers approximately 20 times faster than IKs. The pharmacological properties of IKf and IKs are similar: both components are sensitive to 4-aminopyridine (1-5 mM) and both are relatively resistant to externally applied tetraethylammonium (50 mM). Taken together, these findings suggest that IKf and IKs correspond to two functionally distinct K+ currents with similar voltage-dependent properties and pharmacologic sensitivities, but with markedly different rates of inactivation and recovery from inactivation. From the experimental data, several gating models were developed in which voltage-independent inactivation is coupled either to channel opening or to the activation of the individual channel subunits. Experimental testing of predictions of these models suggests that voltage-independent inactivation is coupled to activation, and that inactivation of only a single subunit is required to result in functional inactivation of the channels. This model closely approximates the properties of IKf and IKs, as well as the composite outward currents, measured in adult rat atrial myocytes.     

Extracellular sodium interacts with the HERG channel at an outer pore site

Most voltage-gated K(+) currents are relatively insensitive to extracellular Na(+) (Na(+)(o)), but Na(+)(o) potently inhibits outward human ether-a-go-go-related gene (HERG)-encoded K(+) channel current (Numaguchi, H., J.P. Johnson, Jr., C.I. Petersen, and J.R. Balser. 2000. Nat. Neurosci. 3:429-30). We studied wild-type (WT) and mutant HERG currents and used two strategic probes, intracellular Na(+) (Na(+)(i)) and extracellular Ba(2+) (Ba(2+)(o)), to define a site where Na(+)(o) interacts with HERG. Currents were recorded from transfected Chinese hamster ovary (CHO-K1) cells using the whole-cell voltage clamp technique. Inhibition of WT HERG by Na(+)(o) was not strongly dependent on the voltage during activating pulses. Three point mutants in the P-loop region (S624A, S624T, S631A) with intact K(+) selectivity and impaired inactivation each had reduced sensitivity to inhibition by Na(+)(o). Quantitatively similar effects of Na(+)(i) to inhibit HERG current were seen in the WT and S624A channels. As S624A has impaired Na(+)(o) sensitivity, this result suggested that Na(+)(o) and Na(+)(i) act at different sites. Extracellular Ba(2+) (Ba(2+)(o)) blocks K(+) channel pores, and thereby serves as a useful probe of K(+) channel structure. HERG channel inactivation promotes relief of Ba(2+) block (Weerapura, M., S. Nattel, M. Courtemanche, D. Doern, N. Ethier, and T. Hebert. 2000. J. Physiol. 526:265-278). We used this feature of HERG inactivation to distinguish between simple allosteric and pore-occluding models of Na(+)(o) action. A remote allosteric model predicts that Na(+)(o) will speed relief of Ba(2+)(o) block by promoting inactivation. Instead, Na(+)(o) slowed Ba(2+) egress and Ba(2+) relieved Na(+)(o) inhibition, consistent with Na(+)(o) binding to an outer pore site. The apparent affinities of the outer pore for Na(+)(o) and K(+)(o) as measured by slowing of Ba(2+) egress were compatible with competition between the two ions for the channel pore in their physiological concentration ranges. We also examined the role of the HERG closed state in Na(+)(o) inhibition. Na(+)(o) inhibition was inversely related to pulsing frequency in the WT channel, but not in the pore mutant S624A.     

Effects of benzocaine on the kinetics of normal and batrachotoxin-modified Na channels in frog node of Ranvier.

The effects of benzocaine (0.5-1 mM) on normal Na currents, and on Na current and gating charge movement (Q) of batrachotoxin (BTX)-modified Na channels were analyzed in voltage-clamped frog node of Ranvier. Without BTX treatment the decay of Na current during pulses to between -40 and 0 mV could be decomposed into two exponential components both in the absence and in the presence of benzocaine. Benzocaine did not significantly alter the inactivation time constant of either component, but reduced both their amplitudes. The amplitude of the slow inactivating component was more decreased by benzocaine than the amplitude of the fast one, leading to an apparently faster decline of the overall Na current. After removal of Na inactivation and charge movement immobilization by BTX, benzocaine decreased the amplitude of INa with no change in time course. INa, QON, and QOFF were all reduced by the same factor. The results suggest that the rate of reaction of benzocaine with its receptor is slow compared to the rates of channel activation and inactivation. The differential effects of benzocaine on the two components of Na current inactivation in normal channels can be explained assuming two types of channel with different rates of inactivation and different affinities for the drug.     

Permeation and gating in CaV3.1 (alpha1G) T-type calcium channels effects of Ca2+, Ba2+, Mg2+, and Na+

We examined the concentration dependence of currents through Ca(V)3.1 T-type calcium channels, varying Ca(2+) and Ba(2+) over a wide concentration range (100 nM to 110 mM) while recording whole-cell currents over a wide voltage range from channels stably expressed in HEK 293 cells. To isolate effects on permeation, instantaneous current-voltage relationships (IIV) were obtained following strong, brief depolarizations to activate channels with minimal inactivation. Reversal potentials were described by P(Ca)/P(Na) = 87 and P(Ca)/P(Ba) = 2, based on Goldman-Hodgkin-Katz theory. However, analysis of chord conductances found that apparent K(d) values were similar for Ca(2+) and Ba(2+), both for block of currents carried by Na(+) (3 muM for Ca(2+) vs. 4 muM for Ba(2+), at -30 mV; weaker at more positive or negative voltages) and for permeation (3.3 mM for Ca(2+) vs. 2.5 mM for Ba(2+); nearly voltage independent). Block by 3-10 muM Ca(2+) was time dependent, described by bimolecular kinetics with binding at approximately 3 x 10(8) M(-1)s(-1) and voltage-dependent exit. Ca(2+)(o), Ba(2+)(o), and Mg(2+)(o) also affected channel gating, primarily by shifting channel activation, consistent with screening a surface charge of 1 e(-) per 98 A(2) from Gouy-Chapman theory. Additionally, inward currents inactivated approximately 35% faster in Ba(2+)(o) (vs. Ca(2+)(o) or Na(+)(o)). The accelerated inactivation in Ba(2+)(o) correlated with the transition from Na(+) to Ba(2+) permeation, suggesting that Ba(2+)(o) speeds inactivation by occupying the pore. We conclude that the selectivity of the "surface charge" among divalent cations differs between calcium channel families, implying that the surface charge is channel specific. Voltage strongly affects the concentration dependence of block, but not of permeation, for Ca(2+) or Ba(2+).     

Block of tetrodotoxin-resistant Na+ channel pore by multivalent cations: gating modification and Na+ flow dependence

Tetrodotoxin-resistant (TTX-R) Na(+) channels are much less susceptible to external TTX but more susceptible to external Cd(2+) block than tetrodotoxin-sensitive (TTX-S) Na(+) channels. Both TTX and Cd(2+) seem to block the channel near the "DEKA" ring, which is probably part of a multi-ion single-file region adjacent to the external pore mouth and is involved in the selectivity filter of the channel. In this study we demonstrate that other multivalent transitional metal ions such as La(3+), Zn(2+), Ni(2+), Co(2+), and Mn(2+) also block the TTX-R channels in dorsal root ganglion neurons. Just like Cd(2+), the blocking effect has little intrinsic voltage dependence, but is profoundly influenced by Na(+) flow. The apparent dissociation constants of the blocking ions are always significantly smaller in inward Na(+) currents than those in outward Na(+) current, signaling exit of the blocker along with the Na(+) flow and a high internal energy barrier for "permeation" of these multivalent blocking ions through the pore. Most interestingly, the activation and especially the inactivation kinetics are slowed by the blocking ions. Moreover, the gating changes induced by the same concentration of a blocking ion are evidently different in different directions of Na(+) current flow, but can always be correlated with the extent of pore block. Further quantitative analyses indicate that the apparent slowing of channel activation is chiefly ascribable to Na(+) flow-dependent unblocking of the bound La(3+) from the open Na(+) channel, whereas channel inactivation cannot happen with any discernible speed in the La(3+)-blocked channel. Thus, the selectivity filter of Na(+) channel is probably contiguous to a single-file multi-ion region at the external pore mouth, a region itself being nonselective in terms of significant binding of different multivalent cations. This region is "open" to the external solution even if the channel is "closed" ("deactivated"), but undergoes imperative conformational changes during the gating (especially the inactivation) process of the channel.     

Slow currents through single sodium channels of the adult rat heart

The currents through single Na+ channels from the sarcolemma of ventricular cells dissociated from adult rat hearts were studied using the patch-clamp technique. All patches had several Na+ channels; most had 5-10, while some had up to 50 channels. At 10 degrees C, the conductance of the channel was 9.8 pS. The mean current for sets of many identical pulses inactivated exponentially with a time constant of 1.7 +/- 0.6 ms at -40 mV. Careful examination of the mean currents revealed a small, slow component of inactivation at pulse potentials ranging from -60 to -30 mV. The time constant of the slow component was between 8 and 14 ms. The channels that caused the slow component had the same conductance and reversal potential as the fast Na+ currents and were blocked by tetrodotoxin. The slow currents appear to have been caused by repeated openings of one or more channels. The holding potential influenced the frequency with which such channel reopening occurred. The slow component was prominent during pulses from a holding potential of -100 mV, while it was very small during pulses from -140 mV. Ultraslow currents through the Na+ channel were observed occasionally in patches that had large numbers of channels. They consisted of bursts of 10 or more sequential openings of a single channel and lasted for up to 150 ms. We conclude that the single channel data cannot be explained by standard models, even those that have two inactivated states or two open states of the channel. Our results suggest that Na+ channels can function in several different "modes," each with a different inactivation rate.     

Rate of opening of a population of Na channels in frog skeletal muscle fibers

Linear Systems convolution analysis of muscle sodium currents was used to predict the opening rate of sodium channels as a function of time during voltage clamp pulses. If open sodium channel lifetimes are exponentially distributed, the channel opening rate corresponding to a sodium current obtained at any particular voltage, can be analytically obtained using a simple equation, given single channel information about the mean open-channel lifetime and current.Predictions of channel opening rate during voltage clamp pulses show that sodium channel inactivation arises coincident with a decline in channel opening rate.Sodium currents pharmacologically modified with Chloramine-T treatment so that they do not inactivate, show a predicted sustained channel opening rate.Large depolarizing voltage clamp pulses produce channel opening rate functions that resemble gating currents.The predicted channel opening rate functions are best described by kinetic models for Na channels which confer most of the charge movement to transitions between closed states.Comparisons of channel opening rate functions with gating currents suggests that there may be subtypes of Na channel with some contributing more charge movement per channel opening than others.Na channels open on average, only once during the transient period of Na activation and inactivation.After transiently opening during the activation period and then closing by entering the inactivated state, Na channels reopen if the voltage pulse is long enough and contribute to steady-state currents.The convolution model overestimates the opening rate of channels contributing to the steady-state currents that remain after the transient early Na current has subsided.     

Contributions of charged residues in a cytoplasmic linking region to Na channel gating

Na channels inactivate quickly after opening, and the very highly positively charged cytoplasmic linking region between homologous domains III and IV of the channel molecule acts as the inactivation gate. To test the hypothesis that the charged residues in the domain III to domain IV linker have a role in channel function, we measured currents through wild-type and two mutant skeletal muscle Na channels expressed in Xenopus oocytes, each lacking two or three charged residues in the inactivation gate. Microscopic current measures showed that removing charges hastened activation and inactivation. Macroscopic current measures showed that removing charges altered the voltage dependence of inactivation, suggesting less coupling of the inactivation and activation processes. Reduced intracellular ionic strength shifted the midpoint of equilibrium activation gating to a greater extent, and shifted the midpoint of equilibrium inactivation gating to a lesser extent in the mutant channels. The results allow the possibility that an electrostatic mechanism contributes to the role of charged residues in Na channel inactivation gating.     

Characterization of the isoform-specific differences in the gating of neuronal and muscle sodium channels

Human heart (hH1), human skeletal muscle (hSkM1), and rat brain (rIIA) Na channels were expressed in cultured cells and the activation and inactivation of the whole-cell Na currents measured using the patch clamp technique. hH1 Na channels were found to activate and inactivate at more hyperpolarized voltages than hSkM1 and rIIA. The conductance versus voltage and steady state inactivation relationships have midpoints of -48 and -92 mV (hH1), -28 and -72 mV (hSkM1), and -22 and -61 mV (rIIA). At depolarized voltages, where Na channels predominately inactivate from the open state, the inactivation of hH1 is 2-fold slower than that of hSkM1 and rIIA. The recovery from fast inactivation of all three isoforms is well described by a single rapid component with time constants at -100 mV of 44 ms (hH1), 4.7 ms (hSkM1), and 7.6 ms (rIIA). After accounting for differences in voltage dependence, the kinetics of activation, inactivation, and recovery of hH1 were found to be generally slower than those of hSkM1 and rIIA. Modeling of Na channel gating at hyperpolarized voltages where the channel does not open suggests that the slow rate of recovery from inactivation of hH1 accounts for most of the differences in the steady-state inactivation of these Na channels.     

The characteristics of the sodium currents across EGTA-modified calcium channels in the membrane of cultured rat cardiomyocytes]

In isolated, cultured neonatal rat ventricular myocytes sodium currents through calcium channels induced by lowering of extracellular calcium concentration 100 nmol/l have been investigated by whole-cell patch clamp technique. Such Na(+)-carried currents are modulated by classic Ca2+ agonists and antagonists. The potential-dependent characteristics of Na+ current are shifted at 20 mV in hyperpolarizing direction as compared to initial Ca(2+)-carried current. The inactivation decay of Na+ current through Ca2+ channels has the monoexponential behaviour. The possible action of extracellular Ca2+ lowering on Ca2+ channel selective filter and gating mechanisms is suggested.