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.     

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.     

Slow and incomplete inactivations of voltage-gated channels dominate encoding in synthetic neurons.

Electrically excitable channels were expressed in Chinese hamster ovary cells using a vaccinia virus vector system. In cells expressing rat brain IIA Na+ channels only, brief pulses (< 1 ms) of depolarizing current resulted in action potentials with a prolonged (0.5-3 s) depolarizing plateau; this plateau was caused by slow and incomplete Na+ channel inactivation. In cells expressing both Na+ and Drosophila Shaker H4 transient K+ channels, there were neuron-like action potentials. In cells with appropriate Na+/K+ current ratios, maintaining stimulation produced repetitive firing over a 10-fold range of frequencies but eventually led to "lock-up" of the potential at a positive value after several seconds of stimulation. The latter effect was due primarily to slow inactivation of the K+ currents. Numerical simulations of modified Hodgkin-Huxley equations describing these currents, using parameters from voltage-clamp kinetics studied in the same cells, accounted for most features of the voltage trajectories. The present study shows that insights into the mechanisms for generating action potentials and trains of action potentials in real excitable cells can be obtained from the analysis of synthetic excitable cells that express a controlled repertoire of ion channels.     

Sodium-conducting channels in cardiac membranes in low calcium.

With no Ca in the patch electrode, two kinds of channels conduct Na in spontaneously beating embryonic chick heart cells. One channel conducts Na primarily during the upstroke of the action potential and is blocked by tetrodotoxin (TTX). The other channel conducts Na primarily during the late plateau and early repolarization phase of the action potential, but only in Ca concentrations below 10(-6) M. This second channel is TTX-insensitive and has a conductance of 50 to 90 pS, depending upon the interpretation of open-channel flickering. These two Na-conducting channels correspond to the channels that normally carry the fast Na current (INa) and the slow Ca current (Isi).     

Outward sodium current in beating heart cells.

This article is a study of the fast Na current during action potentials. We have investigated the outward Na current (Mazzanti, M., and L.J. DeFelice. 1987. Biophys. J. 52:95-100) in more detail, and we have asked whether it goes through the same channels associated with the rapid depolarization phase of action potentials. We address the question by patch clamping single, spontaneously beating, embryonic chick ventricle cells, using two electrodes to record the action potential and the patch current simultaneously. The chief limitation is the capacitive current, and in this article we describe a new method to subtract it. Varying the potential and the Na concentration in the patch pipette, and fitting the corrected currents to a standard model (Ebihara, L., and E.A. Johnson. 1980. Biophys. J. 32:779-790), provides evidence that the outward current is carried by the same channels that conduct the inward current. We compare the currents in beating cells to currents in nonbeating cells using whole-cell and cell-attached patch clamp recordings. The latter tend to show more positive Na reversal potentials, with the implication that internal Na is higher in beating cells. We propose that the plateau of the action potential, which is partly due to an inward Ca current, exceeds Na action current reversal potentials, and that this driving force gives rise to an outward movement of Na ions. The existence of such a current would imply that the fast repolarization phase after the upstroke of cardiac action potentials is partly due to the Na action current.     

Development of slow Ca2+-Na+ channels during organ culture of young embryonic chick hearts

Young (3-days-old) embryonic chick hearts have slowly-rising spontaneous action potentials, dependent on tetrodotoxin-insensitive slow Na+ channels. When the hearts were placed into organ culture for 5-11 days, action potential duration was markedly increased by 260-370%, and a notch appeared between the initial spike phase and the plateau phase in some hearts. The spike amplitude was mainly dependent on [Na]0, whereas the plateau amplitude was dependent on [Ca]0. Thus, the young embryonic hearts develop slow Ca2+-Na+ channels (while retaining the slow Na+ channels) during organ culture, and the spike phase and the plateau phase of the slow action potentials are mainly dependent on currents through slow Na+ channels and through slow Ca2+-Na+ channels, respectively. The effects of Mn2+ (a specific blocker of slow Ca2+-Na+ channels) and verapamil (a blocker of slow Na+ channels as well as of slow Ca2+-Na+ channels) on the spike phase and the plateau phase were examined. Mn2+ (0.5 mM) and verapamil (5 microM) depressed the plateau duration and overshoot. Verapamil did not decrease the maximum rate of rise (Vmax), but Mn++ produced a small, but significant, decrease. High concentrations (10/30 microM) of verapamil depressed the action potential amplitude and Vmax, and abolished the spontaneous action potentials. These results indicate that slow Ca2+-Na+ channels appear de novo during organ culture of young embryonic hearts.     

Fast Na+ channels in smooth muscle from pregnant rat uterus.

Smooth muscle cells normally do not possess fast Na+ channels, but inward current is carried through two types of Ca2+ channels: slow (L type) Ca2+ channels and fast (T type) Ca2+ channels. Whole-cell voltage clamp was done on single smooth muscle cells isolated from the longitudinal layer of the 18-day pregnant rat uterus. Depolarizing pulses, applied from a holding potential of -90 mV, evoked two types of inward current, fast and slow. The fast inward current decayed within 30 ms, depended on [Na]o, and was inhibited by tetrodotoxin (TTX) (K0.5 = 27 nM). The slow inward current decayed slowly, was dependent on [Ca]o (or Ba2+), and was inhibited by nifedipine. These results suggest that the fast inward current is a fast Na+ channel current and that the slow inward current is a Ca2+ slow channel current. A fast-inactivating Ca2+ channel current was not evident. We conclude that the ion channels that generate inward currents in pregnant rat uterine cells are TTX-sensitive fast Na+ channels and dihydropyridine-sensitive slow Ca2+ channels. The number of fast Na+ channels increased during gestation. The averaged current density increased from 0 on day 5, to 0.19 on day 9, to 0.56 on day 14, to 0.90 on day 18, and to 0.86 pA/pF on day 21. This almost linear increase occurs because of an increase in the fraction of cells that possess fast Na+ channels. The Ca2+ channel current density was also higher during the latter half of gestation. These results indicate that the fast Na+ channels and Ca2+ slow channels in myometrium become more numerous as term approaches, and we suggest that the fast Na+ current may be involved in spread of excitation. Isoproterenol (beta-agonist) did not affect either ICa(s) or INa(f), whereas Mg2+ (K0.5 = 12 mM) and nifedipine (K0.5 = 3.3 nM) depressed ICa(s). Oxytocin had no effect on INa(f) and actually depressed ICa(s) to a small extent. Therefore, the tocolytic action of beta-agonists cannot be explained by an inhibition of ICa(s), whereas that of Mg2+ can be so explained. The stimulating action of oxytocin on uterine contractions cannot be explained by a stimulation of ICa(s).     

Isolation of myocardial L-type calcium channel gating currents with the spider toxin omega-Aga-IIIA

The peptide omega-agatoxin-IIIA (omega-Aga-IIIA) blocks ionic current through L-type Ca channels in guinea pig atrial cells without affecting the associated gating currents. omega-Aga-IIIA permits the study of L- type Ca channel ionic and gating currents under nearly identical ionic conditions. Under conditions that isolate L-type Ca channel currents, omega-Aga-IIIA blocks all ionic current during a test pulse and after repolarization. This block reveals intramembrane charge movements of equal magnitude and opposite sign at the beginning of the pulse (Q(on)) and after repolarization (Q(off)). Q(on) and Q(off) are suppressed by 1 microM felodipine, saturate with increasing test potential, and are insensitive to Cd. The decay of the transient current associated with Q(on) is composed of fast and slow exponential components. The slow component has a time constant similar to that for activation of L-type Ca channel ionic current, over a broad voltage range. The current associated with Q(off) decays monoexponentially and more slowly than ionic current. Similar charge movements are found in guinea pig tracheal myocytes, which lack Na channels and T-type Ca channels. The kinetic and pharmacological properties of Q(on) and Q(off) indicate that they reflect gating currents associated with L-type Ca channels. omega-Aga-IIIA has no effect on gating currents when ionic current is eliminated by stepping to the reversal potential for Ca or by Cd block. Gating currents constitute a significant component of total current when physiological concentrations of Ca are present and they obscure the activation and deactivation of L-type Ca channels. By using omega- Aga-IIIA, we resolve the entire time course of L-type Ca channel ionic and gating currents. We also show that L- and T-type Ca channel ionic currents can be accurately quantified by tail current analysis once gating currents are taken into account.     

Modeling of single noninactivating Na+ channels: evidence for two open and several fast inactivated states

Voltage-gated Na(+) channels play a fundamental role in the excitability of nerve and muscle cells. Defects in fast Na(+) channel inactivation can cause hereditary muscle diseases with hyper- or hypoexcitability of the sarcolemma. To explore the kinetics and gating mechanisms of noninactivating muscle Na(+) channels on a molecular level, we analyzed single channel currents from wild-type and five mutant Na(+) channels. The mutations were localized in different protein regions which have been previously shown to be important for fast inactivation (D3-D4-linker, D3/S4-S5, D4/S4-S5, D4/S6) and exhibited distinct grades of defective fast inactivation with varying levels of persistent Na(+) currents caused by late channel reopenings. Different gating schemes were fitted to the data using hidden Markov models with a correction for time interval omission and compared statistically. For all investigated channels including the wild-type, two open states were necessary to describe our data. Whereas one inactivated state was sufficient to fit the single channel behavior of wild-type channels, modeling the mutants with impaired fast inactivation revealed evidence for several inactivated states. We propose a single gating scheme with two open and three inactivated states to describe the behavior of all five examined mutants. This scheme provides a biological interpretation of the collected data, based on previous investigations in voltage-gated Na(+) and K(+) channels.     

Removal of sodium inactivation and block of sodium channels by chloramine-T in crayfish and squid giant axons.

Modification of sodium channels by chloramine-T was examined in voltage clamped internally perfused crayfish and squid giant axons using the double sucrose gap and axial wire technique, respectively. Freshly prepared chloramine-T solution exerted two major actions on sodium channels: (a) an irreversible removal of the fast Na inactivation, and (b) a reversible block of the Na current. Both effects were observed when chloramine-T was applied internally or externally (5-10 mM) to axons. The first effect was studied in crayfish axons. We found that the removal of the fast Na inactivation did not depend on the states of the channel since the channel could be modified by chloramine-T at holding potential (from -80 to -100 mV) or at depolarized potential of -30 mV. After removal of fast Na inactivation, the slow inactivation mechanism was still present, and more channels could undergo slow inactivation. This result indicates that in crayfish axons the transition through the fast inactivated state is not a prerequisite for the slow inactivation to occur. During chloramine-T treatment, a distinct blocking phase occurred, which recovered upon washing out the drug. This second effect of chloramine-T was studied in detail in squid axons. After 24 h, chloramine-T solution lost its ability to remove fast inactivation but retained its blocking action. After removal of the fast Na inactivation, both fresh and aged chloramine-T solutions blocked the Na currents with a similar potency and in a voltage-dependent manner, being more pronounced at lower depolarizing potentials.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Allosteric Effects of Permeating Cations on Gating Currents during K+ Channel Deactivation

K⁺ channel gating currents are usually measured in the absence of permeating ions, when a common feature of channel closing is a rising phase of off-gating current and slow subsequent decay. Current models of gating invoke a concerted rearrangement of subunits just before the open state to explain this very slow charge return from opening potentials. We have measured gating currents from the voltage-gated K⁺ channel, Kv1.5, highly overexpressed in human embryonic kidney cells. In the presence of permeating K⁺ or Cs⁺, we show, by comparison with data obtained in the absence of permeant ions, that there is a rapid return of charge after depolarizations. Measurement of off-gating currents on repolarization before and after K⁺ dialysis from cells allowed a comparison of off-gating current amplitudes and time course in the same cells. Parallel experiments utilizing the low permeability of Cs⁺ through Kv1.5 revealed similar rapid charge return during measurements of off-gating currents at E_Cs. Such effects could not be reproduced in a nonconducting mutant (W472F) of Kv1.5, in which, by definition, ion permeation was macroscopically absent. This preservation of a fast kinetic structure of off-gating currents on return from potentials at which channels open suggests an allosteric modulation by permeant cations. This may arise from a direct action on a slow step late in the activation pathway, or via a retardation in the rate of C-type inactivation. The activation energy barrier for K⁺ channel closing is reduced, which may be important during repetitive action potential spiking where ion channels characteristically undergo continuous cyclical activation and deactivation.     

Na+ permeation and block of hERG potassium channels

The inactivation gating of hERG channels is important for the channel function and drug-channel interaction. Whereas hERG channels are highly selective for K+, we have found that inactivated hERG channels allow Na+ to permeate in the absence of K+. This provides a new way to directly monitor and investigate hERG inactivation. By using whole cell patch clamp method with an internal solution containing 135 mM Na+ and an external solution containing 135 mM NMG+, we recorded a robust Na+ current through hERG channels expressed in HEK 293 cells. Kinetic analyses of the hERG Na+ and K+ currents indicate that the channel experiences at least two states during the inactivation process, an initial fast, less stable state followed by a slow, more stable state. The Na+ current reflects Na+ ions permeating through the fast inactivated state but not through the slow inactivated state or open state. Thus the hERG Na+ current displayed a slow inactivation as the channels travel from the less stable, fast inactivated state into the more stable, slow inactivated state. Removal of fast inactivation by the S631A mutation abolished the Na+ current. Moreover, acceleration of fast inactivation by mutations T623A, F627Y, and S641A did not affect the hERG Na+ current, but greatly diminished the hERG K+ current. We also found that external Na+ potently blocked the hERG outward Na+ current with an IC50 of 3.5 mM. Mutations in the channel pore and S6 regions, such as S624A, F627Y, and S641A, abolished the inhibitory effects of external Na+ on the hERG Na+ current. Na+ permeation and blockade of hERG channels provide novel ways to extend our understanding of the hERG gating mechanisms.     

Gating of cardiac Na+ channels in excised membrane patches after modification by alpha-chymotrypsin.

Single cardiac Na+ channels were investigated after intracellular proteolysis to remove the fast inactivation process in an attempt to elucidate the mechanisms of channel gating and the role of slow inactivation. Na+ channels were studied in inside-out patches excised from guinea-pig ventricular myocytes both before and after very brief exposure (2-4 min) to the endopeptidase, alpha-chymotrypsin. Enzyme exposure times were chosen to maximize removal of fast inactivation and to minimize potential nonspecific damage to the channel. After proteolysis, the single channel current-voltage relationship was approximately linear with a slope conductance of 18 +/- 2.5 pS. Na+ channel reversal potentials measured before and after proteolysis by alpha-chymotrypsin were not changed. The unitary current amplitude was not altered after channel modification suggesting little or no effect on channel conductance. Channel open times were increased after removal of fast inactivation and were voltage-dependent, ranging between 0.7 (-70 mV) and 3.2 (-10 mV) ms. Open times increased with membrane potential reaching a maximum at -10 mV; at more positive membrane potentials, open times decreased again. Fast inactivation appeared to be completely removed by alpha-chymotrypsin and slow inactivation became more apparent suggesting that fast and slow inactivation normally compete, and that fast inactivation dominates in unmodified channels. This finding is not consistent with a slow inactivated state that can only be entered through the fast inactivated state, since removal of fast inactivation does not eliminate slow inactivation. The data indicate that cardiac Na+ channels can enter the slow inactivated state by a pathway that bypasses the fast inactivated state and that the likelihood of entering the slow inactivated state increases after removal of fast inactivation.     

Two modes of gating during late Na+ channel currents in frog sartorius muscle

Na+ currents were measured during 0.4-s depolarizing pulses using the cell-attached variation of the patch-clamp technique. Patches on Cs-dialyzed segments of sartorius muscle of Rana pipiens contained an estimated 25-500 Na+ channels. Three distinct types of current were observed after the pulse onset: a large initial surge of inward current that decayed within 10 ms (early currents), a steady "drizzle" of isolated, brief, inward unitary currents (background currents), and occasional "cloudbursts" of tens to hundreds of sequential unitary inward currents (bursts). Average late currents (background plus bursts) were 0.12% of peak early current amplitude at -20 mV. 85% of the late currents were carried by bursting channels. The unit current amplitude was the same for all three types of current, with a conductance of 10.5 pS and a reversal potential of +74 mV. The magnitudes of the three current components were correlated from patch to patch, and all were eliminated by slow inactivation. We conclude that all three components were due to Na+ channel activity. The mean open time of the background currents was approximately 0.25 ms, and the channels averaged 1.2 openings for each event. Neither the open time nor the number of openings of background currents was strongly sensitive to membrane potential. We estimated that background openings occurred at a rate of 0.25 Hz for each channel. Bursts occurred once each 2,000 pulses for each channel (assuming identical channels). The open time during bursts increased with depolarization to 1-2 ms at -20 mV, whereas the closed time decreased to less than 20 ms. The fractional open time during bursts was fitted with m infinity 3 using standard Na+ channel models. We conclude that background currents are caused by a return of normal Na+ channels from inactivation, while bursts are instances where the channel's inactivation gate spontaneously loses its function for prolonged periods.     

Transfer of twelve charges is needed to open skeletal muscle Na+ channels

Voltage-dependent Na+ channels are thought to sense membrane potential with fixed charges located within the membrane's electrical field. Measurement of open probability (Po) as a function of membrane potential gives a quantitative indication of the number of such charges that move through the field in opening the channel. We have used single- channel recording to measure skeletal muscle Na+ channel open probability at its most negative extreme, where channels may open as seldom as once per minute. To prevent fast inactivation from masking the voltage dependence of Po, we have generated a clone of the rat skeletal muscle Na+ channel that is lacking in fast inactivation (IFM1303QQQ). Using this mutant channel expressed in Xenopus oocytes, and the extra resolution afforded by single-channel analysis, we have extended the resolution of the hyperpolarized tail of the Po curve by four orders of magnitude. We show that previous measurements, which indicated a minimum of six effective gating charges, may have been made in a range of Po values that had not yet arrived at its limiting slope. In our preparation, a minimum of 12 charges must function in the activation gating of the channel. Our results will require reevaluation of kinetic models based on six charges, and they have major implications for the interpretation of S4 mutagenesis studies and structure/function models of the Na+ channel.     

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.     

Kinetic analysis of the action of Leiurus scorpion alpha-toxin on ionic currents in myelinated nerve

The effects of a neurotoxin, purified from the venom of the scorpion Leiurus quinquestriatus, on the ionic currents of toad single myelinated fibers were studied under voltage-clamp conditions. Unlike previous investigations using crude scorpion venom, purified Leiurus toxin II alpha at high concentrations (200-400 nM) did not affect the K currents, nor did it reduce the peak Na current in the early stages of treatment. The activation of the Na channel was unaffected by the toxin, the activation time course remained unchanged, and the peak Na current vs. voltage relationship was not altered. In contrast, Na channel inactivation was considerably slowed and became incomplete. As a result, a steady state Na current was maintained during prolonged depolarizations of several seconds. These steady state Na currents had a different voltage dependence from peak Na currents and appeared to result from the opening of previously inactivated Na channels. The opening kinetics of the steady state current were exponential and had rates approximately 100-fold slower than the normal activation processes described for transitions from the resting state to the open state. In addition, the dependence of the peak Na current on the potential of preceding conditioning pulses was also dramatically altered by toxin treatment; this parameter reached a minimal value near a membrane potential of -50 mV and then increased continuously to a "plateau" value at potentials greater than +50 mV. The amplitude of this plateau was dependent on toxin concentration, reaching a maximum value equal to approximately 50% of the peak current; voltage-dependent reversal of the toxin's action limits the amplitude of the plateauing effect. The measured plateau effect was half-maximum at a toxin concentration of 12 nM, a value quite similar to the concentration producing half of the maximum slowing of Na channel inactivation. The results of Hill plots for these actions suggest that one toxin molecule binds to one Na channel. Thus, the binding of a single toxin molecule probably both produces the steady state currents and slows the Na channel inactivation. We propose that Leiurus toxin inhibits the conversion of the open state to inactivated states in a voltage-dependent manner, and thereby permits a fraction of the total Na permeability to remain at membrane potentials where inactivation is normally complete.     

Modifications of human cardiac sodium channel gating by UVA light

Voltage-gated Na(+) channels are membrane proteins responsible for the generation of action potentials. In this report we demonstrate that UVA light elicits gating changes of human cardiac Na+ channels. First, UVA irradiation hampers the fast inactivation of cardiac Nav1.5 Na(+) channels expressed in HEK293t cells. A maintained current becomes conspicuous during depolarization and reaches its maximal quasi steady-state level within 5-7 min. Second, the activation time course is slowed by UVA light; modification of the activation gating by UVA irradiation continues for 20 min without reaching steady state. Third, along with the slowed activation time course, the peak current is reduced progressively. Most Na(+) currents are eliminated during 20 min of UVA irradiation. Fourth, UVA light increases the holding current nonlinearly; this phenomenon is slow at first but abruptly fast after 20 min. Other skeletal muscle Nav1.4 isoforms and native neuronal Na(+) channels in rat GH(3) cells are likewise sensitive to UVA irradiation. Interestingly, a reactive oxygen metabolite (hydrogen peroxide at 1.5%) and an oxidant (chloramine-T at 0.5 mM) affect Na(+) channel gating similarly, but not identically, to UVA. These results together suggest that UVA modification of Na(+) channel gating is likely mediated via multiple reactive oxygen metabolites. The potential link between oxidative stress and the impaired Na(+) channel gating may provide valuable clues for ischemia/reperfusion injury in heart and in CNS.     

A study of the molecular sources of nonideal osmotic pressure of bovine serum albumin solutions as a function of pH.

Two types of the late Na channels, burst and background, were studied in Purkinje and ventricular cells. In the whole-cell configuration, steady-state Na currents were recorded at potentials (-70 to -80 mV) close to the normal cell resting potential. The question of the contribution of late Na channels to this background Na conductance was investigated. During depolarization, burst Na channels were active for periods (up to approximately 5 s), which exceeded the action potential duration. However, they eventually closed without reopening, indicating the presence of slow and complete inactivation. When, at the moment of burst channel opening, the potential was switched to -80 mV, the channel closed quickly without reopening. We conclude that the burst Na channels cannot contribute significantly to the background Na conductance. Background Na channels undergo incomplete inactivation. After a step depolarization, their activity decreased in time, approaching a steady-state level. Background Na channel openings could be recorded at constant potentials in the range from -120 to 0 mV. After step depolarizations to potentials near -70 mV and more negative, a significant fraction of Na current was carried by the background Na channels. Analysis of the background channel behavior revealed that their gating properties are qualitatively different from those of the early Na channels. We suggest that background Na channels represent a special type of Na channel that can play an important role in the initiation of cardiac action potential and in the TTX-sensitive background Na conductance.