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 delayed rectifier current and repolarization in canine cardiac Purkinje cells

Although cardiac Purkinje cells (PCs) are believed to be the source of early afterdepolarizations generating ventricular tachyarrhythmias in long Q-T syndromes (LQTS), the ionic determinants of PC repolarization are incompletely known. To evaluate the role of the slow delayed rectifier current (I(Ks)) in PC repolarization, we studied PCs from canine ventricular false tendons with whole cell patch clamp (37 degrees C). Typical I(Ks) voltage- and time-dependent properties were noted. Isoproterenol enhanced I(Ks) in a concentration-dependent fashion (EC(50) approximately 30 nM), negatively shifted I(Ks) activation voltage dependence, and accelerated I(Ks) activation. Block of I(Ks) with 293B did not alter PC action potential duration (APD) in the absence of isoproterenol; however, in the presence of isoproterenol, 293B significantly prolonged APD. We conclude that, without beta-adrenergic stimulation, I(Ks) contributes little to PC repolarization; however, beta-adrenergic stimulation increases the contribution of I(Ks) by increasing current amplitude, accelerating I(Ks) activation, and shifting activation voltage toward the PC plateau voltage range. I(Ks) may therefore provide an important "braking" function to limit PC APD prolongation in the presence of beta-adrenergic stimulation.     

Background K+ current in isolated canine cardiac Purkinje myocytes.

The current-voltage (I-V) relation of the background current, IK1, was studied in isolated canine cardiac Purkinje myocytes using the whole-cell, patch-clamp technique. Since Ba2+ and Cs+ block IK1, these cations were used to separate the I-V relation of IK1 from that of the whole cell. The I-V relation of IK1 was measured as the difference between the I-V relations of the cell in normal Tyrode (control solution) and in the presence of either Ba2+ (1 mM) or Cs+ (10 mM). Our results indicate that IK1 is an inwardly rectifying K+ current whose conductance depends on extracellular potassium concentration. In different [K+]0's the I-V relations of IK1 exhibit crossover. In addition the I-V relation of IK1 contains a region of negative slope (even when that of the whole cell does not). We also examined the relationship between the resting potential of the myocyte, Vm, and [K+]0 and found that it exhibits the characteristic anomalous behavior first reported in Purkinje strands (Weidmann, S., 1956, Elektrophysiologie der Herzmuskelfaser, Med. Verlag H. Huber), where lowering [K+]0 below 4 mM results in a depolarization.     

Inactivation of calcium channel current in the calf cardiac Purkinje fiber. Evidence for voltage- and calcium-mediated mechanisms

We have studied the influence of divalent cations on Ca channel current in the calf cardiac Purkinje fiber to determine whether this current inactivates by voltage- or Ca-mediated mechanisms, or by a combination of the two. We measured the reversal (or zero current) potential of the current when Ba, Sr, or Ca were the permeant divalent cations and determined that depletion of charge carrier does not account for time-dependent relaxation of Ca channel current in these preparations. Inactivation of Ca channel current persists when Ba or Sr replaces Ca as the permeant divalent cation, but the voltage dependence of the rate of inactivation is markedly changed. This effect cannot be explained by changes in external surface charge. Instead, we interpret the results as evidence that inactivation is both voltage and Ca dependent. Inactivation of Sr or Ba currents reflects a voltage-dependent process. When Ca is the divalent charge carrier, an additional effect is observed: the rate of inactivation is increased as Ca enters during depolarizing pulses, perhaps because of an additional Ca-dependent mechanism.     

Slow inactivation of a tetrodotoxin-sensitive current in canine cardiac Purkinje fibers.

We used the two-microelectrode voltage clamp technique and tetrodotoxin (TTX) to investigate the possible occurrence of slow inactivation of sodium channels in canine cardiac Purkinje fibers under physiologic conditions. The increase in net outward current during prolonged (5-20 s) step depolarizations (range -70 to +5 mV) following the application of TTX is time dependent, being maximal immediately following depolarization, and declining thereafter towards a steady value. To eliminate the possibility that this time-dependent current was due to inadequate voltage control of these multicellular preparations early during square clamp pulses, we also used slowly depolarizing voltage clamp ramps (range 5-100 mV/s) to ensure control of membrane potential. TTX-sensitive current also was observed with these voltage ramps; the time dependence of this current was demonstrated by the reduction of the peak current magnitude as the ramp speed was reduced. Reducing the holding potential within the voltage range of sodium channel inactivation also decreased the TTX-sensitive current observed with identical speed ramps. These results suggest that the TTX-sensitive time-dependent current is a direct measure of slow inactivation of canine cardiac sodium channels. This current may play an important role in modulating the action potential duration.     

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.     

Low conductance sodium channels in canine cardiac Purkinje cells.

Low conductance sodium (Na) channels have been observed in nerve, skeletal muscle, and cardiac cells. In cardiac tissues the higher amplitude, more commonly observed Na channel was first investigated in detail by Cachelin et al. (Cachelin, A.B., J.E. de Peyer, S. Kokubun, and H. Reuter, 1983, J. Physiol. (Lond.), 340:389-402). They also reported low amplitude Na channel events. We have studied this low conductance Na channel in single canine cardiac Purkinje cells using cell-attached patches. Patch pipette solutions contained either 140 or 280 mM NaCl, and cells were bathed in a solution of 150 mM KCl to bring their resting potential close to zero. In 140 mM Na+, during steps to -50 mV, the lower and higher openings had amplitudes of 0.57 +/- 0.2 and 1.2 +/- 0.2 pA (means +/- SD of Gaussian fits). In 280 mM Na+ at -50 mV, amplitudes were 0.72 +/- 0.2 and 1.55 +/- 0.2 pA. Over a substantial voltage range, the lower events had amplitudes of about one-third that of the higher events. The frequency of the low conductance openings varied in different patches from zero to 22% of total openings. Histograms of open durations and latencies at several voltages suggested no difference in kinetics between the two channel events. The behavior of the low conductance channels was more consistent with a second population of channels rather than a second open state.     

Expression of T-type calcium current precedes neurite extension in neuroblastoma cells.

1. N1E-115 mouse neuroblastoma cells morphologically differentiate by extending neurites in a period of seven days after addition of 2% DMSO to the culture medium. We used the whole-cell patch clamp technique to measure calcium currents in these cells under conditions where voltage clamp of the whole membrane was assured. 2. Current densities of both T and L type calcium currents were identical in cells included to differentiate with dibutyryl cyclic AMP and cells induced to differentiate with dimethylsulphoxide (DMSO). Cells differentiated with DMSO were used for all subsequent experiments. 3. All morphologically differentiated cells showed a T type calcium current. In contrast, a minority of morphologically undifferentiated cells did not show a T current. 4. Once expressed, both T and L currents did not change either in current density or in behaviour over a period of five days. 5. These data demonstrate that expression of a T current always precedes neurite extension, and suggest a role for calcium currents in triggering morphological differentiation.     

Simulated calcium current can both cause calcium loading in and trigger calcium release from the sarcoplasmic reticulum of a skinned canine cardiac Purkinje cell

Skinned canine cardiac Purkinje cells were stimulated by regularly repeated microinjection-aspiration sequences that were programmed to simulate the fast initial component of the transsarcolemmal Ca2+ current and the subsequent slow component corresponding to noninactivating Ca2+ channels. The simulated fast component triggered a tension transient through Ca2+-induced release of Ca2+ from the sarcoplasmic reticulum (SR). The simulated slow component did not affect the tension transient during which it was first introduced but it potentiated the subsequent transients. The potentiation was not observed when the SR function had been destroyed by detergent. The potentiation decreased progressively when the slow component was separated by an increasing time interval from the fast component. The potentiation was progressive over several beats under conditions that decreased the rate of Ca2+ accumulation into the SR (deletion of calmodulin from the solutions; a decrease of the temperature from 22 to 12 degrees C). In the presence of a slow component, an increase of frequency caused a positive staircase, and the introduction of an extrasystole caused a postextrasystolic potentiation. There was a negative staircase and no postextrasystolic potentiation in the absence of a slow component. These results can be explained by a time- and Ca2+-dependent functional separation of the release and accumulation processes of the SR, rather than by Ca2+ circulation between anatomically distinct loading and release compartments. The fast initial component of transsarcolemmal Ca2+ current would trigger Ca2+ release, whereas the slow component would load the SR with an amount of Ca2+ available for release during the subsequent tension transients.     

Fluctuations in membrane current driven by intracellular calcium in cardiac Purkinje fibers

Spontaneous oscillatory fluctuations in membrane potential are often observed in heart cells, but their basis remains controversial. Such activity is enhanced in cardiac Purkinje fibers by exposure to digitalis or K-free solutions. Under these conditions, we find that voltage noise is generated by current fluctuations that persist when membrane potential is voltage clamped. Power spectra of current signals are not made up of single time-constant components, as expected from gating of independent channels, but are dominated by resonant characteristics between 0.5 and 2 HZ. Our evidence suggests that the periodicity arises from oscillatory variations in intracellular free Ca that control ion movements across the surface membrane. The current fluctuations are strongly cross-correlated with oscillatory fluctuations in contractile force, and are inhibited by removing extracellular Ca or exposure to D600. Chelating intracellular Ca with injected EGTA also abolishes the current fluctuations. The oscillatory mechanism may involve cycles of Ca (or Sr) movement between sarcoplasmic reticulum and myoplasm, as previously suggested for skinned cardiac preparations. Our experiments in intact cells indicate that changes in surface membrane potential can modulate cytoplasmic Ca oscillations in frequency and perhaps amplitude as well. A two-way interaction between surface membrane potential and intracellular Ca stores may be a common feature of heart, neuron, and other cell types.     

Kinetic analysis of single sodium channels from canine cardiac Purkinje cells

Single sodium channel events were recorded from cell-attached patches on single canine cardiac Purkinje cells at 10-13 degrees C. Data from four patches containing two to four channels and one patch with one channel were selected for quantitative analysis. The channels showed prominent reopening behavior at voltages near threshold, and the number of reopenings declined steeply with depolarization. Mean channel open time was a biphasic function of voltage with the maximum value (1-1.5 ms) occurring between -50 and -40 mV and lower values at more and at less hyperpolarized levels. Inactivation without opening was also prominent near threshold, and this occurrence also declined with depolarization. The waiting time distributions and the probability of being open showed voltage and time dependence as expected from whole-cell current studies. The results were analyzed in terms of a five-state Markovian kinetic model using both histogram analysis and a maximum likelihood method to estimate kinetic parameters. The kinetic parameters of the model fits were similar to those of GH3 pituitary cells (Horn, R., and C. A. Vandenberg. 1984. Journal of General Physiology. 84:505-534) and N1E115 neuroblastoma cells (Aldrich, R. W., and C. F. Stevens. Journal of Neuroscience. 7:418-431). Both histogram and maximum likelihood analysis implied that much of the voltage dependence of cardiac Na current is in its activation behavior, with inactivation showing modest voltage dependence.     

Membrane currents following activity in canine cardiac Purkinje fibers.

Recent experiments in canine Pukinje fibers (Gadsby and Cranefield, 1979) have shown that following a period of sodium loading in K+-free solution a slowly decaying outward current is observed. This current has been attributed to the activity of the electrogenic Na+-K+ exchange pump. In the present paper we show that similar slowly decaying outward currents are observed following prolonged periods of overdrive with action potentials or with brief depolarizing voltage clamp pulses. The dependent of the prolonged outward current on the duration and frequency of the preceding period of overdrive and on the potential following overdrive is reported. We also present results which indicate that a large portion of this current can be induced by phasic Na+ loading through the fast-inward channel.     

Rapid ionic modifications during the aequorin-detected calcium transient in a skinned canine cardiac Purkinje cell

A microprocessor-controlled system of microinjections and microaspirations has been developed to change, within approximately 1 ms, the [free Ca2+] at the outer surface of the sarcoplasmic reticulum (SR) wrapped around individual myofibrils (0.3-0.4 micron radius) of a skinned canine cardiac Purkinje cell (2.5-4.5 micron overall radius) at different phases of a Ca2+ transient. Simultaneously monitoring tension and aequorin bioluminescence provided two methods for estimating the peak myoplasmic [free Ca2+] reached during the spontaneous cyclic Ca2+ release from the SR obtained in the continuous presence of a bulk solution [free Ca2+] sufficiently high to overload the SR. These methods gave results in excellent agreement for the spontaneous Ca2+ release under a variety of conditions of pH and [free Mg2+], and of enhancement of Ca2+ release by calmodulin. Disagreement was observed, however, when the Ca2+ transient was modified during its ascending phase. The experiments also permitted quantification of the aequorin binding within the myofibrils and determination of its operational apparent affinity constant for Ca2+ at various [free Mg2+] levels. An increase of [free Ca2+] at the outer surface of the SR during the ascending phase of the Ca2+ transient induced further release of Ca2+. In contrast, an increase of [free Ca2+] during the descending phase of the Ca2+ transient did not cause further Ca2+ release. Varying [free H+], [free Mg2+], or the [Na+]/[K+] ratio had no significant effect on the Ca2+ transient during which the modification was applied, but it altered the subsequent Ca2+ transient. Therefore, Ca2+ appears to be the major, if not the only, ion controlling Ca2+ release from the SR rapidly enough to alter a Ca2+ transient during its course.     

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).     

A comparison of transient outward currents in canine cardiac Purkinje cells and ventricular myocytes

Although abnormalities in Purkinje cell (PC) repolarization are important causes of cardiac arrhythmias, the detailed properties of repolarizing currents in PCs are incompletely understood. We compared transient outward K(+) current (I(to)) in single PCs from canine false tendons with midmyocardial ventricular myocytes (VMs). I(to) reactivation was biexponential, with a similar rapid-phase time constant (30 +/- 5 and 35 +/- 4 ms for VM and PC, respectively) but a large, slow component in PCs with a much greater time constant than VM (1,427 +/- 70 vs. 181 +/- 24 ms, P < 0.001). Tetraethylammonium had no effect on VM I(to) but reversibly inhibited PC I(to) (IC(50) = 2.4 +/- 0.4 mM). PC I(to) was also more sensitive to 4-aminopyridine (IC(50) = 50 +/- 7 vs. 526 +/- 49 microM in VM, P < 0.0001). H(2)O(2) slowed I(to) inactivation in PCs but did not affect VM I(to). We conclude that PC I(to) shows significant differences from VM I(to), with some features, such as tetraethylammonium sensitivity, that have been reported in neither cardiac I(to) of atrial or ventricular myocytes nor cloned K(+) channel subunits (Kv1.4, Kv4.2, or Kv4.3) known to participate in cardiac I(to).     

Photoalteration of calcium channel blockade in the cardiac Purkinje fiber.

Organic compounds that block calcium channel current (calcium antagonists) are important tools for the characterization of this channel. However, the practically irreversible nature of this block restricts the usefulness of this group of drugs. In this paper, we investigate the influence of light on calcium channel blockade by several organic compounds. Our results show that inhibition of calcium channel current by two dihydropyridine derivatives that contain an o-nitro moiety (nisoldipine and nifedipine) can be rapidly reversed by illumination. The energy range important to this reaction is for light wavelengths between 320 and 450 nm. Calcium channel inhibition by two other dihydropyridine derivatives (nicardipine and nitrendipine) as well as by D600, is not modulated by illumination. These results indicate that the photosensitivity of certain dihydropyridine calcium channel blockers make these compounds useful as reversible blockers of this channel.     

Extracellular [K+] fluctuations in voltage-clamped canine cardiac Purkinje fibers.

Membrane currents and extracellular [K+] were measured in canine Purkinje strands during voltage-clamp steps to plateau or diastolic potentials. Extracellular [K+] increased during step depolarizations and decreased during step hyperpolarizations. On hyperpolarization, the largest fraction of the K+ depletion occurred during the initial 500 ms of the voltage-clamp step and was correlated with a potassium depletion current, the id. A slower component of the depletion also occurred on hyperpolarization and had a time constant consistent with cylindrical diffusion of potassium within the Purkinje strands. On depolarization, there is an accumulation of K+ that is correlated with the plateau current ix. On termination of depolarizing test pulses, the K+ accumulation decays with a time course similar to the ix tail current. Surprisingly, no accumulation of K+ occurred during the arrhythmogenic transient inward current, TI, suggesting that the selectivity of this current should be reevaluated.     

The angiotensin AT2 receptor modulates T-type calcium current in non-differentiated NG108-15 cells.

We report here that angiotensin II (AII) and the AT2 receptor-selective ligand, CGP 42112, modulate the T-type calcium current in non-differentiated NG108-15 cells, which express only AT2 receptors. Both peptides decrease the T-type calcium current at membrane potentials above -40 mV and shift the current-voltage curve at lower potentials with maximal effect between 5 and 10 min after application. These data describe a new cellular response to AII and suggest that the AT2 receptor mediates certain neurophysiological actions of this hormone.     

The relationship among intracellular sodium activity, calcium, and strophanthidin inotropy in canine cardiac Purkinje fibers

The role of sodium and calcium ions in strophanthidin inotropy was studied by measuring simultaneously the electrical, mechanical, and intracellular sodium ion activities in electrically driven cardiac Purkinje fibers under conditions that change the intracellular sodium or calcium level (tetrodotoxin, strophanthidin, high calcium, and norepinephrine). Tetrodotoxin (TTX; 1-5 X 10(-6)M) shifted the action potential plateau to more negative values, shortened the action potential duration, and decreased the contractile tension and the intracellular sodium ion activity (aiNa). The changes in tension and in aiNa caused by TTX appear to be related since they had similar time courses. Strophanthidin (2-5 X 10(-7)M) increased tension and aiNa less in the presence of TTX, and, for any given value of aiNa, tension was less than in the absence of TTX. Increasing extracellular calcium (from 1.8 to 3.3-3.6 mM) or adding norepinephrine (0.5-1 X 10(-6)M) increased tension and decreased aiNa less in the presence than in the absence of TTX. When two of the above procedures were combined, the results were different. Thus, during the increase in aiNa and tension caused by strophanthidin in the presence of TTX, increasing calcium or adding norepinephrine increased tension markedly but did not increase aiNa further. In a TTX-high calcium or TTX-norepinephrine solution, adding strophanthidin increased both tension and aiNa, and the increase in tension was far greater than in the presence of TTX alone. The results indicate that: (a) the contractile force in Purkinje fibers is affected by a change in aiNa; (b) a decrease in aiNa by TTX markedly reduces the inotropic effect of strophanthidin, possibly as a consequence of depletion of intracellular calcium; (c) increasing calcium influx with norepinephrine or high calcium in the TTX-strophanthidin solution produces a potentiation of tension development, even if aiNa does not increase further; and (d) when the calcium influx is already increased by high calcium or norepinephrine, strophanthidin has its usual inotropic effect even in the presence of TTX. In conclusion, the positive inotropic effect of strophanthidin requires that an increase in aiNa be associated with suitable calcium availability.