Two different tetrodotoxin-separable inward sodium currents in the membrane of isolated cardiomyocytes

Isolated ventricular myocytes of 3 to 5 weeks old rats were studied under conditions of intracellular perfusion and voltage clamp. The existence of two inward sodium currents with different TTX-sensitivities and different properties was shown. The fast sodium current was more sensitive to TTX (Kd about 8 X 10(-8) mol/l). The block of the slow sodium current by TTX was less specific (Kd about 7 X 10(-6) mol/l). There was an about four fold difference in the inactivation time constants between these currents. The maximum on the I-V curve of the slow sodium current was shifted along the voltage axis by about 15 mV in the positive direction as compared with that of the fast sodium current. A slow current carried by calcium ions was observed in sodium-free solution. The kinetics and TTX-sensitivity of this current were similar to those of the slow sodium current. The amplitude of this current was 15 to 20 times lower as compared with the slow sodium current observed in Na-containing solution with 10(-6) mol/l TTX (a concentration which completely blocked the fast sodium current). It is suggested that the slow voltage-gated TTX-sensitive channels described are not highly selective and pass both sodium and calcium ions.     

Na(+) current contribution to the diastolic depolarization in newborn rabbit SA node cells

Isolated newborn, but not adult, rabbit sinoatrial node (SAN) cells exhibit spontaneous activity that (unlike adult) are highly sensitive to the Na(+) current (I(Na)) blocker TTX. To investigate this TTX action on automaticity, cells were voltage clamped with ramp depolarizations mimicking the pacemaker phase of spontaneous cells (-60 to -20 mV, 35 mV/s). Ramps elicited a TTX-sensitive current in newborn (peak density 0.89 +/- 0.14 pA/pF, n = 24) but not adult (n = 5) cells. When depolarizing ramps were preceded by steplike depolarizations to mimic action potentials, ramp current decreased 54.6 +/- 8.0% (n = 3) but was not abolished. Additional experiments demonstrated that ramp current amplitude depended on the slope of the ramp and that TTX did not alter steady-state holding current at pacemaker potentials. This excluded a steady-state Na(+) window component and suggested a kinetic basis, which was investigated by measuring TTX-sensitive I(Na) during long step depolarizations. I(Na) exhibited a slow but complete inactivation time course at pacemaker voltages (tau = 33.9 +/- 3.9 ms at -50 mV), consistent with the rate-dependent ramp data. The data indicate that owing to slow inactivation of I(Na) at diastolic potentials, a small TTX-sensitive current flows during the diastolic depolarization in neonatal pacemaker myocytes.     

Sodium currents during differentiation in a human neuroblastoma cell line

The electrophysiological properties of a human neuroblastoma cell line, LA-N-5, were studied with the whole-cell configuration of the patch clamp technique before and after the induction of differentiation by retinoic acid, a vitamin A metabolite. Action potentials could be elicited from current clamped cells before the induction of differentiation, suggesting that some neuroblasts of the developing sympathetic nervous system are excitable. The action potential upstroke was carried by a sodium conductance, which was composed of two types of sodium currents, described by their sensitivity to tetrodotoxin (TTX) as TTX sensitive and TTX resistant. TTX-sensitive and TTX-resistant sodium currents were blocked by nanomolar and micromolar concentrations of TTX, respectively. The voltage sensitivity of activation and inactivation of TTX-resistant sodium current is shifted -10 to -30 mV relative to TTX-sensitive sodium current, suggesting that TTX-resistant sodium current could play a role in the initiation of action potentials. TTX-sensitive current comprised greater than 80% of the total sodium current in undifferentiated LA-N-5 cells. The surface density of total sodium current increased from 24.9 to 57.8 microA/microF after cells were induced to differentiate. The increase in total sodium current density was significant (P less than 0.05). The surface density of TTX-resistant sodium current did not change significantly during differentiation, from which we conclude that an increase in TTX-sensitive sodium current underlies the increase in total current.     

Effects of pressure overload-induced hypertrophy on TTX-sensitive inward currents in guinea pig left ventricle

We investigated the effects of pressure overload hypertrophy on inward sodium (I Na) and calcium currents (I Ca) in single left ventricular myocytes to determine whether changes in these current systems could account for the observed prolongation of the action potential. Hypertrophy was induced by pressure overload caused by banding of the abdominal aorta. Whole-cell patch clamp experiments were used to measure tetrodotoxin (TTX)-sensitive inward currents. The main findings were that I Ca density was unchanged whereas I Na density after stepping from -80 to -30 mV was decreased by 30% (-9.0 +/- 1.16 pA pF(-1) in control and -6.31 +/- 0.67 pA pF(-1) in hypertrophy, p < 0.05, n = 6). Steady-state activation/inactivation variables of I Na, determined by using double-pulse protocols, were similar in control and hypertrophied myocytes, whereas the time course of fast inactivation of I Na was slowed (p < 0.05) in hypertrophied myocytes. In addition, action potential clamp experiments were carried out in the absence and presence of TTX under conditions where only Ca2+ was likely to enter the cell via TTX-sensitive channels. We show for the first time that a TTX-sensitive inward current was present during the plateau phase of the action potential in hypertrophied but not control myocytes. The observed decrease in I Na density is likely to abbreviate rather than prolong the action potential. Delayed fast inactivation of Na+ channels was not sustained throughout the voltage pulse and may therefore merely counteract the effect of decreased I Na density so that net Na+ influx remains unaltered. Changes in the fast I Na do not therefore appear to contribute to lengthening of the action potential in this model of hypertrophy. However, the presence of a TTX-sensitive current during the plateau could potentially contribute to the prolongation of the action potential in hypertrophied cardiac muscle.     

The properties of batrachotoxin-modified cardiac Na channels, including state-dependent block by tetrodotoxin

Batrachotoxin (BTX) modification and tetrodotoxin (TTX) block of BTX-modified Na channels were studied in single cardiac cells of neonatal rats using the whole-cell patch-clamp recording technique. The properties of BTX-modified Na channels in heart are qualitatively similar to those in nerve. However, quantitative differences do exist between the modified channels of these two tissues. In the heart, the shift of the conductance-voltage curve for the modified channel was less pronounced, the maximal activation rate constant, (tau m)max, of modified channels was considerably slower, and the slow inactivation of the BTX-modified cardiac Na channels was only partially abolished. TTX blocked BTX-modified mammalian cardiac Na channels and the block decreased over the potential range of -80 to -40 mV. The apparent dissociation constant of TTX changed from 0.23 microM at -50 mV to 0.69 microM at 0 mV. No further reduction of block was observed at potentials greater than -40 mV. This is the potential range over which gating from closed to open states occurred. These results were explained by assuming that TTX has a higher affinity for closed BTX-modified channels than for open modified channels. Hence, the TTX-binding rate constants are considered to be state dependent rather than voltage dependent. This differs from the voltage dependence of TTX block reported for BTX-modified Na channels from membrane vesicles incorporated into lipid bilayers and from amphibian node of Ranvier.     

Voltage clamp of the cardiac sodium current at 37 degrees C in physiologic solutions.

The cardiac sodium current was studied in guinea pig ventricular myocytes using the cell-attached patch voltage clamp at 37 degrees C in the presence of 145 mM external sodium concentration. When using large patch pipettes (access resistance, 1-2 M omega), the capacity current transient duration was typically 70 microseconds for voltage clamp steps up to 150 mV. At 37 degrees C the maximum inward sodium current peaked in approximately 200 microseconds after the onset of a clamp step and at this strong depolarization, less than 10% of the sodium current developed during the capacity transient. The sodium current developed smoothly and the descending limb of the current-voltage relationship usually spanned a range of 40 mV. Moreover, currents reduced by inactivation of sodium channels could be scaled to superimpose on the maximum current. Current tails elicited by deactivation followed a monoexponential time course that was very similar for currents of different sizes. Data obtained over a range of temperatures (15 degrees-35 degrees C) showed that the steady-state inactivation and conductance-voltage curves were shifted to more negative voltages at lower temperatures. These results demonstrate the feasibility of investigating the sodium current of mammalian cardiac cells at 37 degrees C in normal physiological solutions.     

Acute thyroid hormone promotes slow inactivation of sodium current in neonatal cardiac myocytes.

Sodium current (INa) inactivation kinetics in neonatal cardiac myocytes were analyzed using whole cell voltage clamp before and after acute treatments with thyroid hormone (3,5,3'-triiodo-L-thyronine, T3). In untreated neonatal myocytes, INa inactivation was predominantly mono-exponential, with 93 +/- 3% (S.D.; n = 9) of the peak amplitude decaying with a time constant, tau h1, of 1.8 +/- 0.5 ms at -30 mV. The remaining 7% of control INa decayed more slowly, with a time constant, tau h2, of 9.3 +/- 3.0 ms at -30 mV. The contribution of slowly-inactivating channels to peak current was increased from 7% to 43 +/- 27% within 5 min of exposure to 5-20 nM T3 (nine cells; P less than 0.005). The time constants for both the fast- and slow-inactivating components of peak current (tau h1 and tau h2) were not significantly changed by acute T3 treatment, nor was steady-state INa inactivation (h infinity) affected. Thyroid hormone action on sodium inactivation was partially reversible by lidocaine. These findings indicate that T3 acts at the neonatal cardiac cell membrane to promote slow inactivation kinetics in sodium channels.     

Subthreshold sodium current from rapidly inactivating sodium channels drives spontaneous firing of tuberomammillary neurons

A role for "persistent," subthreshold, TTX-sensitive sodium current in driving the pacemaking of many central neurons has been proposed, but this has been impossible to test pharmacologically. Using isolated tuberomammillary neurons, we assessed the role of subthreshold sodium current in pacemaking by performing voltage-clamp experiments using a cell's own pacemaking cycle as voltage command. TTX-sensitive sodium current flows throughout the pacemaking cycle, even at voltages as negative as -70 mV, and this current is sufficient to drive spontaneous firing. When sodium channels underlying transient current were driven into slow inactivation by rapid stimulation, persistent current decreased in parallel, suggesting that persistent sodium current originates from subthreshold gating of the same sodium channels that underlie the phasic sodium current. This behavior of sodium channels may endow all neurons with an intrinsic propensity for rhythmic, spontaneous firing.     

Voltage-dependent inactivation of the potassium current of embryonic chick hepatocytes

The whole-cell patch electrode voltage clamp technique was used to study the inactivation properties of the delayed rectifying potassium current of single cultured embryonic chick hepatocytes at 20 degrees C. The potassium current activates maximally within 250-500 ms of membrane depolarization, after which it decays with a monoexponential time course. Both steady-state activation and inactivation are voltage dependent. Steady-state inactivation declines from 100% at -5 mV to 0 near -70 mV. with half inactivation at -41 mV. At the resting potential (EM) of these cells (-21.5 +/- 6.0 mV, n = 36) 6-18% of the IK channels are not inactivated and less than 5% are open. Development and removal of inactivation follow single exponential time courses. The inactivation time constant attains a maximum of around 30 s at -35 mV and is sharply voltage dependent at the EM of these cells. Measurement of EM under current clamp shows random oscillations of 5-10 mV amplitude. We suggest that the voltage- and time-dependent properties of IK, in tandem with a time- and voltage-independent, non-selective current also seen here, would provide the mechanism for a fluctuating EM.     

TTX-sensitive Na(+) and nifedipine-sensitive Ca(2+) channels in rat vas deferens smooth muscle cells.

The inward currents in single smooth muscle cells (SMC) isolated from epididymal part of rat vas deferens have been studied using whole-cell patch-clamp method. Depolarising steps from holding potential -90 mV evoked inward current with fast and slow components. The component with slow activation possessed voltage-dependent and pharmacological properties characteristic for Ca(2+) current carried through L-type calcium channels (I(Ca)). The fast component of inward current was activated at around -40 mV, reached its peak at 0 mV, and disappeared upon removal of Na ions from bath solution. This current was blocked in dose-dependent manner by tetrodotoxin (TTX) with an apparent dissociation constant of 6.7 nM. On the basis of voltage-dependent characteristics, TTX sensitivity of fast component of inward current and its disappearance in Na-free solution it is suggested that this current is TTX-sensitive depolarisation activated sodium current (I(Na)). Cell dialysis with a pipette solution containing no macroergic compounds resulted in significant inhibition of I(Ca) (depression of peak I(Ca) by about 81% was observed by 13 min of dialysis), while I(Na) remained unaffected during 50 min of dialysis. These data draw first evidence for the existence of TTX-sensitive Na(+) current in single SMC isolated from rat vas deferens. These Na(+) channels do not appear to be regulated by a phosphorylation process under resting conditions.     

Voltage-dependent regulation of modal gating in the rat SkM1 sodium channel expressed in Xenopus oocytes

The TTX-sensitive rat skeletal muscle sodium channel (rSkM1) exhibits two modes of inactivation (fast vs slow) when the alpha subunit is expressed alone in Xenopus oocytes. In this study, two components are found in the voltage dependence of normalized current inactivation, one having a V1/2 in the expected voltage range (approximately -50 mV, I(N)) and the other with a more hyperpolarized V1/2 (approximately -130 mV, IH) at a holding potential of -90 mV. The I(N) component is associated with the gating mode having rapid inactivation and recovery from inactivation of the macroscopic current (N-mode), while IH corresponds to the slow inactivation and recovery mode (H-mode). These two components are interconvertible and their relative contribution to the total current varies with the holding potential: I(N) is favored by hyperpolarization. The interconversion between the two modes is voltage dependent and is well fit to a first-order two-state model with a voltage dependence of e-fold/8.6 mV and a V1/2 of -62 mV. When the rat sodium channel beta 1-subunit is coinjected with rSkM1, IH is essentially eliminated and the inactivation kinetics of macroscopic current becomes rapid. These two current components and their associated gating modes may represent two conformations of the alpha subunit, one of which can be stabilized either by hyperpolarization or by binding of the beta 1 subunit.     

Characterization of Na(+) currents in isolated dorsal unpaired median neurons of Locusta migratoria and effect of the alpha-like scorpion toxin BmK M1

A primary cell culture was developed for efferent dorsal unpaired median (DUM) neurons of the locust. The isolated somata were able to generate Tetrodotoxin (TTX)-sensitive action potentials in vitro. The alpha-like scorpion toxin BmK M1, from the Asian scorpion Buthus martensi Karsch, prolonged the duration of the action potential up to 50 times. To investigate the mechanism of action of BmK M1, the TTX-sensitive voltage gated Na(+) currents were studied in detail using the whole cell patch clamp technique. BmK M1 slowed down and partially inhibited the inactivation of the TTX-sensitive Na(+) current in a dose dependent manner (EC50=326.8+/-34.5 nM). Voltage and time dependence of the Na(+) current were described in terms of the Hodgkin-Huxley model and compared in control conditions and in the presence of 500 nM BmK M1. The BmK M1 shifted steady state inactivation by 10.8 mV to less negative potentials. The steady state activation was shifted by 5.5 mV to more negative potentials, making the activation window larger. Moreover, BmK M1 increased the fast time constant of inactivation, leaving the activation time constant unchanged. In summary, BmK M1 primarily affected the inactivation parameters of the voltage gated Na(+) current in isolated locust DUM neurons.     

The pacemaker current in cardiac Purkinje myocytes

It is generally assumed that in cardiac Purkinje fibers the hyperpolarization activated inward current i(f) underlies the pacemaker potential. Because some findings are at odds with this interpretation, we used the whole cell patch clamp method to study the currents in the voltage range of diastolic depolarization in single canine Purkinje myocytes, a preparation where many confounding limitations can be avoided. In Tyrode solution ([K+]o = 5.4 mM), hyperpolarizing steps from Vh = -50 mV resulted in a time-dependent inwardly increasing current in the voltage range of diastolic depolarization. This time- dependent current (iKdd) appeared around -60 mV and reversed near EK. Small superimposed hyperpolarizing steps (5 mV) applied during the voltage clamp step showed that the slope conductance decreases during the development of this time-dependent current. Decreasing [K+]o from 5.4 to 2.7 mM shifted the reversal potential to a more negative value, near the corresponding EK. Increasing [K+]o to 10.8 mM almost abolished iKdd. Cs+ (2 mM) markedly reduced or blocked the time-dependent current at potentials positive and negative to EK. Ba2+ (4 mM) abolished the time-dependent current in its usual range of potentials and unmasked another time-dependent current (presumably i(f)) with a threshold of approximately -90 mV (> 20 mV negative to that of the time-dependent current in Tyrode solution). During more negative steps, i(f) increased in size and did not reverse. During i(f) the slope conductance measured with small (8-10 mV) superimposed clamp steps increased. High [K+]o (10.8 mM) markedly increased and Cs+ (2 mM) blocked i(f). We conclude that: (a) in the absence of Ba2+, a time-dependent current does reverse near EK and its reversal is unrelated to K+ depletion; (b) the slope conductance of that time-dependent current decreases in the absence of K+ depletion at potentials positive to EK where inactivation of iK1 is unlikely to occur. (c) Ba2+ blocks this time-dependent current and unmasks another time-dependent current (i(f)) with a more negative (> 20 mV) threshold and no reversal at more negative values; (d) Cs+ blocks both time-dependent currents recorded in the absence and presence of Ba2+. The data suggest that in the diastolic range of potentials in Purkinje myocytes there is a voltage- and time-dependent K+ current (iKdd) that can be separated from the hyperpolarization- activated inward current i(f).     

I(Ca(TTX)) channels are distinct from those generating the classical cardiac Na(+) current

The Na(+) current component I(Ca(TTX)) is functionally distinct from the main body of Na(+) current, I(Na). It was proposed that I(Ca(TTX)) channels are I(Na) channels that were altered by bathing media containing Ca(2+), but no, or very little, Na(+). It is known that Na(+)-free conditions are not required to demonstrate I(Ca(TTX).) We show here that Ca(2+) is also not required. Whole-cell, tetrodotoxin-blockable currents from fresh adult rat ventricular cells in 65 mm Cs(+) and no Ca(2+) were compared to those in 3 mM Ca(2+) and no Cs(+) (i.e., I(Ca(TTX))). I(Ca(TTX)) parameters were shifted to more positive voltages than those for Cs(+). The Cs(+) conductance-voltage curve slope factor (mean, -4.68 mV; range, -3.63 to -5.72 mV, eight cells) is indistinguishable from that reported for I(Ca(TTX)) (mean, -4.49 mV; range, -3.95 to -5.49 mV). Cs(+) current and I(Ca(TTX)) time courses were superimposable after accounting for the voltage shift. Inactivation time constants as functions of potential for the Cs(+) current and I(Ca(TTX)) also superimposed after voltage shifting, as did the inactivation curves. Neither of the proposed conditions for conversion of I(Na) into I(Ca(TTX)) channels is required to demonstrate I(Ca(TTX)). Moreover, we find that cardiac Na(+) (H1) channels expressed heterologously in HEK 293 cells are not converted to I(Ca(TTX)) channels by Na(+)-free, Ca(2+)-containing bathing media. The gating properties of the Na(+) current through H1 and those of Ca(2+) current through H1 are identical. All observations are consistent with two non-interconvertable Na(+) channel populations: a larger that expresses little Ca(2+) permeability and a smaller that is appreciably Ca(2+)-permeable.     

Saxitoxin and tetrodotoxin. Electrostatic effects on sodium channel gating current in crayfish axons.

The effects of extracellular saxitoxin (STX) and tetrodotoxin (TTX) on gating current (IgON) were studied in voltage clamped crayfish giant axons. At a holding potential (VH) of -90 mV, integrated gating charge (QON) was found to be 56% suppressed when 200 nM STX was added to the external solution, and 75% suppressed following the addition of 200 nM TTX. These concentrations of toxin are sufficiently high to block greater than 99% of sodium channels. A smaller suppression of IgON was observed when 1 nM STX was used (KD = 1-2 nM STX). The suppression of IgON by external toxin was found to be hold potential dependent, with only minimal suppression observed at the most hyperpolarized hold potentials, -140 to -120 mV. The maximal effect of these toxins on IgON was observed at hold potentials where the QON vs. VH plot was found to be steepest, -100 to -80 mV. The suppression of IgON induced by TTX is partially relieved following the removal of fast inactivation by intracellular treatment with N-bromoacetamide (NBA). The effect of STX and TTX on IgON is equivalent to a hyperpolarizing shift in the steady state inactivation curve, with 200 nM STX and 200 nM TTX inducing shifts of 4.9 +/- 1.7 mV and 10.0 +/- 2.1 mV, respectively. Our results are consistent with a model where the binding of toxin displaces a divalent cation from a negatively charged site near the external opening of the sodium channel, thereby producing a voltage offset sensed by the channel gating apparatus.     

Membrane current following activity in canine cardiac Purkinje fibers

Membrane current following prolonged periods of rapid stimulation was examined in short (less than 1.5 mm) canine cardiac Purkinje fibers of radius less than 0.15 mm. The Purkinje fibers were repetitively stimulated by delivering trains of depolarizing voltage clamp pulses at rapid frequencies. The slowly decaying outward current following repetitive stimulation ("post-drive" current) is eliminated by the addition of 10(-5) M dihydro-ouabain. The post-drive current is attributed to enhanced Na/K exchange caused by Na loading during the overdrive. Depolarizing voltage clamp pulses initiated from negative (- 80 mV) or depolarized (-50 mV) holding potentials can give rise to post- drive current because of activation of tetrodotoxin-sensitive or D600- sensitive channels. The magnitude of the post-drive current depends on the frequency of voltage clamp pulses, the duration of each pulse, and the duration of the repetitive stimulation. The time constant of decay of the post-drive current depends on extracellular [K] in accordance with Michaelis-Menten kinetics. The Km is 1.2 mM bulk [K], [K]B. The mean time constant in 4 mM [K]B is 83 s. Epinephrine (10(-5) M) decreases the time constant by 20%. The time constant is increased by lowering [Ca]o between 4 and 1 mM. Lowering [Ca]o further, to 0.1 mM, eliminates post-drive current following repetitive stimulation initiated from depolarized potentials. The latter result suggests that slow inward Ca2+ current may increase [Na]i via Na/Ca exchange.     

Biophysical properties and slow voltage-dependent inactivation of a sustained sodium current in entorhinal cortex layer-II principal neurons: a whole-cell and single-channel study.

The functional and biophysical properties of a sustained, or "persistent," Na(+) current (I(NaP)) responsible for the generation of subthreshold oscillatory activity in entorhinal cortex layer-II principal neurons (the "stellate cells") were investigated with whole-cell, patch-clamp experiments. Both acutely dissociated cells and slices derived from adult rat entorhinal cortex were used. I(NaP), activated by either slow voltage ramps or long-lasting depolarizing pulses, was prominent in both isolated and, especially, in situ neurons. The analysis of the gating properties of the transient Na(+) current (I(NaT)) in the same neurons revealed that the resulting time-independent "window" current (I(NaTW)) had both amplitude and voltage dependence not compatible with those of the observed I(NaP), thus implying the existence of an alternative mechanism of persistent Na(+)-current generation. The tetrodotoxin-sensitive Na(+) currents evoked by slow voltage ramps decreased in amplitude with decreasing ramp slopes, thus suggesting that a time-dependent inactivation was taking place during ramp depolarizations. When ramps were preceded by increasingly positive, long-lasting voltage prepulses, I(NaP) was progressively, and eventually completely, inactivated. The V(1/2) of I(NaP) steady state inactivation was approximately -49 mV. The time dependence of the development of the inactivation was also studied by varying the duration of the inactivating prepulse: time constants ranging from approximately 6.8 to approximately 2.6 s, depending on the voltage level, were revealed. Moreover, the activation and inactivation properties of I(NaP) were such as to generate, within a relatively broad membrane-voltage range, a really persistent window current (I(NaPW)). Significantly, I(NaPW) was maximal at about the same voltage level at which subthreshold oscillations are expressed by the stellate cells. Indeed, at -50 mV, the I(NaPW) was shown to contribute to >80% of the persistent Na(+) current that sustains the subthreshold oscillations, whereas only the remaining part can be attributed to a classical Hodgkin-Huxley I(NaTW). Finally, the single-channel bases of I(NaP) slow inactivation and I(NaPW) generation were investigated in cell-attached experiments. Both phenomena were found to be underlain by repetitive, relatively prolonged late channel openings that appeared to undergo inactivation in a nearly irreversible manner at high depolarization levels (-10 mV), but not at more negative potentials (-40 mV).     

Single ion occupancy and steady-state gating of Na channels in squid giant axon

The properties of the small fraction of tetrodotoxin (TTX)-sensitive Na channels that remain open in the steady state were studied in internally dialyzed voltage clamped squid giant axons. The observed Ussing flux ratio exponent (n') of 0.97 plus minus 0.03 (calculated from simultaneous measurements of TTX-sensitive current and (22)Na efflux) and nonindependent behavior of Na current at high internal [Na] are explained by a one-site ("1s") permeation model characterized by a single effective binding site within the channel pore in equilibrium with internal Na ions (apparent equilibrium dissociation constant K(Nai)(0) = 0.61 +/- 0.08 M). Steady-state open probability of the TTX-sensitive channels can be modeled by the product p(a)p(infinity), where p(a) represents voltage-dependent activation described by a Boltzmann distribution with midpoint V(a) = -7 mV and effective valence z(a) = 3.2 (Vandenberg, C.A., and F. Bezanilla. 1991. BIOPHYS: J. 60:1499--1510) coupled to voltage-independent inactivation by an equilibrium constant (Bezanilla, F., and C.M. Armstrong. 1977. J. Gen. Physiol. 70:549--566) K(eq) = 770. The factor p(infinity) represents voltage-dependent inactivation with empirical midpoint V(infinity)= -83 plus minus 5 mV and effective valence z(infinity) = 0.55 plus minus 0.03. The composite p(a)p(infinity)1s model describes the steady-state voltage dependence of the persistent TTX-sensitive current well.     

Sodium current in voltage clamped internally perfused canine cardiac Purkinje cells.

Study of the excitatory sodium current (INa) intact heart muscle has been hampered by the limitations of voltage clamp methods in multicellular preparations that result from the presence of large series resistance and from extracellular ion accumulation and depletion. To minimize these problems we voltage clamped and internally perfused freshly isolated canine cardiac Purkinje cells using a large bore (25-microns diam) double-barreled flow-through glass suction pipette. Control of [Na+]i was demonstrated by the agreement of measured INa reversal potentials with the predictions of the Nernst relation. Series resistance measured by an independent microelectrode was comparable to values obtained in voltage clamp studies of squid axons (less than 3.0 omega-cm2). The rapid capacity transient decays (tau c less than 15 microseconds) and small deviations of membrane potential (less than 4 mV at peak INa) achieved in these experiments represent good conditions for the study of INa. We studied INa in 26 cells (temperature range 13 degrees-24 degrees C) with 120 or 45 mM [Na+]o and 15 mM [Na+]i. Time to peak INa at 18 degrees C ranged from 1.0 ms (-40 mV) to less than 250 microseconds (+ 40 mV), and INa decayed with a time course best described by two time constants in the voltage range -60 to -10 mV. Normalized peak INa in eight cells at 18 degrees C was 2.0 +/- 0.2 mA/cm2 with [Na+]o 45 mM and 4.1 +/- 0.6 mA/cm2 with [Na+]o 120 mM. These large peak current measurements require a high density of Na+ channels. It is estimated that 67 +/- 6 channels/micron 2 are open at peak INa, and from integrated INa as many as 260 Na+ channels/micron2 are available for opening in canine cardiac Purkinje cells.     

Slow inactivation of L-type calcium current distorts the measurement of L- and T-type calcium current in Purkinje myocytes

We have examined slow inactivation of L-type calcium current in canine Purkinje myocytes with the whole cell patch clamp technique. Slow inactivation is voltage dependent. It is negligible at -50 mV but can inactivate more than half of available iCaL at -10 mV. There are two major consequences of this slow inactivation. First, standard protocols for the measurement of T-type current can dramatically overestimate its contribution to total calcium current, and second, the position and steepness of the inactivation versus voltage curve for iCaL will depend on the method of measurement. Given the widespread attempts to identify calcium current components and characterize them biophysically, an important first step should be to determine the extent of slow inactivation of calcium current in each preparation.