AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 282: H1879-H1888, 2002; doi:10.1152/ajpheart.00952.2001
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Vol. 282, Issue 5, H1879-H1888, May 2002

Channels involved in transient currents unmasked by removal of extracellular calcium in cardiac cells

Regina Macianskiene1, Francesco Moccia1, Karin R. Sipido2, Willem Flameng1, and Kanigula Mubagwa1

1 Laboratory of Cardiac Cellular Research, Centre for Experimental Surgery and Anaesthesiology, and 2 Laboratory of Experimental Cardiology, University of Leuven, Leuven B-3000, Belgium


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In cardiac cells that lack macroscopic transient outward K+ currents (Ito), the removal of extracellular Ca2+ can unmask "Ito-like" currents. With the use of pig ventricular myocytes and the whole cell patch-clamp technique, we examined the possibility that cation efflux via L-type Ca2+ channels underlies these currents. Removal of extracellular Ca2+ and extracellular Mg2+ induced time-independent currents at all potentials and time-dependent currents at potentials greater than -50 mV. Either K+ or Cs+ could carry the time-dependent currents, with reversal potential of +8 mV with internal K+ and +34 mV with Cs+. Activation and inactivation were voltage dependent [Boltzmann distributions with potential of half-maximal value (V1/2) = -24 mV and slope = -9 mV for activation; V1/2 = -58 mV and slope = 13 mV for inactivation]. The time-dependent currents were resistant to 4-aminopyridine and to DIDS but blocked by nifedipine at high concentrations (IC50 = 2 µM) as well as by verapamil and diltiazem. They could be increased by BAY K-8644 or by isoproterenol. We conclude that the Ito-like currents are due to monovalent cation flow through L-type Ca2+ channels, which in pig myocytes show low sensitivity to nifedipine.

myocyte; channel; nonselective; pig


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IN CARDIAC CELLS, voltage-dependent A-type or transient outward K+ currents (Ito) are variably expressed in different species and in different regions of the myocardium. These currents are large in the ventricular cells of some species such as the mouse, rat, rabbit, or human (4), but are either absent or small in pig and guinea pig ventricular cells (15, 16, 23) or in cells from the septal subendocardial layers of the human hypertrophied left ventricle (3). In cells that lack macroscopic Ito, the removal of extracellular Ca2+ (Cao) has been reported to unmask a large "Ito-like" current carried by K+ (3, 16). However, the current is different from Ito because, besides its sensitivity to block by Cao and by other divalent cations such as extracellular Cd2+ and extracellular Co2+, it is insensitive to the standard Ito blockers 4-aminopyridine (4-AP) and to tetraethylammonium (TEA) (3, 16). Similarly, in cells where Ito is present, a decrease of Cao, in addition to modifying Ito, also induced an outward current component that was unaffected by 4-AP (6). This difference in pharmacological sensitivity and additional differences in the kinetics of inactivation and recovery from inactivation indicated that the current was due to channels distinct from those underlying normal Ito (3). However, the nature of the channels responsible for the 4-AP-insensitive current has not been clarified, with one study suggesting that they are due to L-type Ca2+ channels (30), whereas other studies not finding any evidence of this (3, 16).

Changes of Cao can affect channels in different ways. First, Cao changes cause a shift in the apparent voltage-dependent activation/inactivation of channels, an effect related to the screening of surface charges by divalent cations (14). Second, Cao removal can modify the ion selectivity of many channels, especially of Ca2+-permeable ones. For example, in cardiac and other cells, L-type and T-type Ca2+ channels become highly permeable to monovalent cations when Cao is decreased or removed (13, 19). Finally, Cao removal can also induce the opening of new channels, apparently not conductive in the presence of divalent cations (2, 17, 22, 28, 29, 31). Thus the time-dependent, Ito-like currents induced upon Cao removal could be due either to a modified behavior of known channels or to the unmasking of novel ones.

Because Cao removal can transform small Ca2+-permeable currents into large monovalent cation currents, it can unravel the presence of channels that conduct only mildly under normal conditions (21) but that could be important under pathological conditions. Thus it is important to characterize the conductance underlying Ito-like currents. Although Inoue et al. (16) observed that the Ito-like current induced in 0 Ca2+ could not be fully suppressed by 1 µM D600, the possibility that it is due to monovalent cation flow through L-type Ca2+ channels (30) cannot be completely excluded, because evidence exists for the presence, in some species, of Ca2+ channels with low sensitivity to Ca2+ antagonists (24). Therefore, in the present study, we examined the possibility that K+ outflux via L-type Ca2+ channels underlies the Ito-like currents observed upon removal of Cao.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Measurements were performed on pig isolated, single ventricular myocytes. The study was carried out in accordance with the Declaration of Helsinki and with institutional guidelines for the care and use of laboratory animals.

Preparation of pig ventricular myocytes. The methods used for the dissociation of pig cells have been described previously (26). After premedication with azaperone (4 mg/kg im) and atropine (0.35 mg/kg iv), the animals were anesthetized with pentobarbitone sodium (Nembutal; 5-15 mg/kg iv), intubated, and set on a respirator. They were heparinized (500 IU/kg iv) and killed with an overdose of Nembutal (100 mg/kg iv). After a thoracotomy, the heart was removed and placed into cold Tyrode solution. A piece of the left ventricular wall was excised with its supplying artery, cannulated, and perfused at 37°C and at constant pressure for 30 min with an oxygenated Ca2+-free Tyrode solution, followed by a 20- to 25-min perfusion with a Ca2+-free Tyrode solution containing 0.1 mg/ml protease (type XIV, Sigma) and 1.4 mg/ml collagenase (type A, Boehringer-Mannheim). After a 15-min washing perfusion with 0.18 mM Ca2+-Tyrode solution, the tissue was removed from the perfusion and cut into small pieces. Cells were dispersed by gentle mechanical agitation. Cao was raised stepwise to 0.5 and 1 mM, and cells were stored in the latter solution at room temperature (21-22°C). Ca2+-tolerant rod-shaped ventricular myocytes with clear striations were selected for the electrophysiological studies.

Electrophysiological recordings and data analysis. Membrane currents were measured as previously described (12, 27) using the whole cell patch-clamp technique (11). Heat-polished borosilicate glass electrodes (horizontal puller, Zeitz Instrumente; Munich, Germany) with tip resistances of 1-1.5 MOmega when filled with the internal solution were used. The electrodes were connected to an Axopatch 100A amplifier (Axon Instruments; Foster City, CA), and an analog-to-digital interface controlled by pCLAMP software (Axon Instruments) was used to generate command pulses and acquire data. All experiments were carried out at room temperature.

Currents were generated by 1-s step depolarizations given every 5 s from a holding potential of -80 mV. To determine the voltage-dependent activation of the time-dependent currents induced by Cao removal, we obtained the underlying conductance (G) at each potential by dividing the amplitude of these currents with the driving force as follows
G=(i<SUB>o</SUB><IT>−i<SUB>∞</SUB></IT>)<IT>/</IT>(<IT>V</IT><SUB>m</SUB><IT>−V</IT><SUB>rev</SUB>) (1)
where io and iinfinity are the currents at the beginning and at the end of the voltage step, respectively; Vm is the potential of the step; and Vrev is the reversal potential. Vrev was defined for each experiment as the potential where the time-dependent current changed polarity and was close to +8 mV when K+ was the major intracellular cation and +34 mV when Cs+ was the major cation (see RESULTS). The conductance at each potential was expressed relative to its maximum value at positive potentials. To measure steady-state inactivation, prepulses lasting 1 s were given to various levels (between -120 and +70 mV, in 10-mV steps) before depolarization to a test potential of +60 mV. Assuming full inactivation after a 1-s prepulse to the positive potentials, the lowest current at +60 mV after these prepulses was taken as the baseline level. Each time-dependent current obtained at +60 mV after a given prepulse was measured as the difference between peak current and this baseline level and was normalized relative to the maximum current (i.e., the current after a prepulse to -120 mV). Normalized activation or availability curves were fitted using one single Boltzmann distribution function as follows
activation or availability = {1 + exp[(<IT>V</IT>−V<SUB>1/2</SUB>)/<IT>k</IT>]}<SUP>−1</SUP> (2)
where V is the voltage, V1/2 is the potential of half-maximum activation or inactivation, and k is a factor describing the slope of the distribution curve.

In experiments carried out to study the effect of different concentrations of nifedipine, depolarizations to various levels to induce Ito-like current [in the absence of Cao and extracellular Mg2+ (Mgo)] or L-type Ca2+ current (ICa,L; in the presence of Cao and Mgo) were preceded by a 400-ms prepulse to -40 mV. In a few cases, the command voltage consisted of 4-s ramps, given every 10 s, from -120 to +80 mV and back to -120 mV. Currents were measured during the descending limb of the voltage ramp. The 2-s duration of the ascending limb allowed inactivation of the voltage-dependent Na+ current.

Functions were fitted to data using CLAMPfit (Axon Instruments) or Origin (Microcal; Northampton, MA). Averaged data are expressed as means ± SE. Statistical comparison was made using a two-tailed t-test.

Solutions and drugs. During measurements, the myocytes were superfused with a K+-free Tyrode solution containing (in mM) 135 NaCl, 5.4 CsCl, 0.9 MgCl2, 1.8 CaCl2, 0.33 NaH2PO4, 10 HEPES, and 10 glucose; pH was adjusted to 7.4 with NaOH. Nominally Ca2+-free or Mg2+-free solutions were made by simply omitting these ions from the standard solution. To further deplete Cao, 1 mM EGTA was added in nearly all experiments. The internal solution contained either K+ or Cs+ as the major cation. The K+-based internal solution contained (in mM) 130 potassium glutamate or KCl, 25 KCl, 1 MgCl2, 5 Na2ATP or MgATP, 1 EGTA, 0.1 Na2GTP, and 5 HEPES (pH 7.25, adjusted with KOH). In Cs+-based internal solution, potassium glutamate and KCl were replaced by cesium glutamate and CsCl, respectively (pH was adjusted with CsOH). When desired, 4-AP (3 mM), DIDS (200 µM), isoproterenol (Iso; 1 µM), propranolol (1 µM), nifedipine (1-100 µM), verapamil (50 µM), diltiazem (50 µM), BAY K-8644 (1 µM), or tetrodotoxin (TTX; 10 µM) were added to the external solution. Stock solutions of 4-AP (1.5 M, pH was adjusted to 7.4 with HCl) and diltiazem were prepared in distilled water, DIDS (100 mM) stock solutions were prepared in DMSO, and nifedipine (50 mM) or BAY K-8644 (0.5 mM) stock solutions were prepared in ethanol. Solutions containing 4-AP or nifedipine were protected from light. All drugs were from Sigma (Bornem, Belgium) except verapamil, which was from Knoll (Ludwigshafen, Germany).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cao removal induces time-independent and -dependent currents. Figure 1, A-C, shows currents recorded during 1-s depolarizations to potentials between -120 and +70 mV from the holding potential of -80 mV. The cell was internally dialyzed with a K+-containing solution but was superfused with a K+-free, Cs+-containing Tyrode solution to block inward K+ currents. TTX was added at 10 µM to block voltage-dependent Na+ channels. Under basal conditions (Fig. 1A), the inward currents induced at negative potentials were indeed very small. Outward currents at positive potentials were larger but practically time independent, i.e., there was no evidence of a large transient outward current. Upon removal of both Cao and Mgo (Fig. 1B), the magnitude of inward and outward currents was markedly increased. In addition, a time-dependent outward current developed at potentials greater than +20 mV. Further Cao chelation by 1 mM EGTA caused an additional increase of currents, especially of the time-dependent components (Fig. 1C). In the presence of EGTA, inward transients developed at membrane potentials between 0 and -40 mV (Fig. 1C, see also Figs. 3A, 6B, and 8B). Transients remained absent at potentials negative to -50 mV, suggesting the existence of a voltage threshold for the activation of time-dependent currents. All these changes were completely reversible and reproducible upon readmission of Cao and Mgo (data not illustrated, but see Fig. 7E) (22).


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  		Fig. 1.   Changes of membrane currents upon removal of extracellular Ca2+ (Cao) and extracellular Mg2+ (Mgo). A-C: current traces elicited by 1-s depolarizations to potentials between -120 and +70 mV (in 10-mV increments) from a holding potential (VH) of -80 mV. TTX (10 µM) was present. A, inset: voltage-pulse protocol. Horizontal dashed line, zero current level. A: basal conditions in the presence of 1.8 mM Cao and 0.9 Mgo; B: 0 Cao and 0 Mgo; C: 0 Cao and 0 Mgo with 1 mM EGTA. Notice the increase of currents and appearance of a time-dependent component upon removal of the divalent cations.

The changes in currents and the induction of time-dependent components are also apparent in the current-voltage relationships shown in Fig. 2. Currents measured at the beginning (peak; solid symbols) and at the end (steady state; open symbols) of the pulse under each condition are superimposed in Fig. 2, A-C. While in the presence of Cao and Mgo, the peak and steady-state currents were small and indistinguishable throughout the whole potential range (Fig. 2A); in the absence of Cao and Mgo (especially in the presence of EGTA), the currents were increased and clear differences appeared between the early and the late currents (Fig. 2, B and C). The Cao- and Mgo-sensitive currents were obtained by subtracting the total current in 1.8 Cao and 0.9 Mgo from those in 0 Cao and 0 Mgo (without or with EGTA) and are shown in Fig. 2D (for peak currents) and in Fig. 2E (for end of pulse or steady-state currents). The difference current obtained when using peak currents in the presence of EGTA (Fig. 2D, solid squares) showed a region of negative slope conductance between -50 and 0 mV, which was absent for the difference of end of pulse currents (Fig. 2E). All difference currents reversed at a potential close to 0 mV, indicating that the conductances induced in 0 Cao and 0 Mgo were permeable to a mixture of ions. Given that previous studies have shown that Cao and Mgo removal in cardiac cells induces 1) a permeability of L-type Ca2+ channels to monovalent cations (13, 19) and 2) a time-independent cation nonselective current (22, 30, 33), it is conceivable that the changes described above are due to a sum of these effects. We restricted further analysis to the time-dependent component. Figure 2F shows the difference between peak and end of pulse currents in the presence of EGTA. Except for the reversal potential and the potential of maximum current, the current-voltage relationship resembles that which is normally obtained for ICa,L. Results similar to those illustrated in Figs. 1 and 2 were obtained in a total of 17 cells dialyzed with KCl-based internal solution. In the following sections, we examine whether the Ito-like currents at very positive potentials can be entirely accounted for by a current flow through voltage-dependent L-type Ca2+ channels.


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  		Fig. 2.   Effect of Cao and Mgo removal on current-voltage relations and determination of the divalent cation-sensitive currents. A-C: changes of membrane currents upon removal of Cao and Mgo. Currents were measured at the beginning (filled symbols) or at the end (open symbols) of the voltage pulse. A: basal conditions, in the presence of 1.8 mM Cao and 0.9 Mgo; B: 0 Cao and 0 Mgo; C: 0 Cao and 0 Mgo with 1 mM EGTA. D and E: difference currents obtained by subtracting currents under basal conditions (i.e., in the presence of 1.8 mM Cao and 0.9 Mgo) from those in the absence of Cao and Mgo. D: Peak currents obtained by measuring at the beginning of the voltage pulse; E: end-of-pulse currents; F: difference currents obtained by subtracting end-of-pulse currents from peak currents in 0 Cao and 0 Mgo with 1 mM EGTA.

Voltage-dependent activation and inactivation. If both outward and inward time-dependent currents are due to the same channels, then one single activation curve should be obtained from conductances calculated from outward and inward current transients. We therefore determined the voltage dependence of activation by dividing the amplitude of the time-dependent component by the driving force, i.e., by the difference between the step voltage and the observed reversal potential (Vm - Vrev). Figure 3A shows typical traces used to determine activation, and Fig. 3C shows the average activation curve (n = 10). In each individual experiment, the activation could be described by one single Boltzmann distribution curve. V1/2 was -23.9 ± 3.7 mV, and the slope factor was -9.2 ± 1.2 mV. We also determined the voltage-dependent inactivation by depolarizing to a constant voltage (+60 mV) after 1-s prepulses to various levels between -120 and +70 mV. The magnitude of the transient outward current depended on the prepulse level (Fig. 3B), and the inactivation in each experiment could also be described by a single Boltzmann distribution curve, with a V1/2 of -57.8 ± 1.7 mV and a slope of 13.0 ± 1.1 mV (n = 17). The average inactivation curve is shown in Fig. 3D (see also Fig. 5B).


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  		Fig. 3.   Voltage-dependent activation and inactivation of the time-dependent currents. A: time-dependent components of the currents elicited by 1-s depolarizations to potentials between -120 and +70 mV (in 10-mV increments) from a VH of -80 mV. Notice the reversal of the current transients at potentials below +10 mV. Horizontal dashed line, zero current level. B: current traces obtained at +60 mV after 1-s prepulses to various levels in 0 Cao and 0 Mgo with 1 mM EGTA. C: voltage-dependent activation obtained (see METHODS) from data such as those displayed in A (n = 10). D: availability (or inactivation) curve obtained by normalizing data such as those in B (n = 17). The fitting lines in C and D are Boltzmann distribution curves drawn using the mean of parameters obtained in each experiment.

After currents reached their peak level, the currents induced by depolarization decayed to a steady state. Two exponentials were needed to fit the current decay. At +70 mV, the time constants of decay (tau ) were as follows: tau fast = 29.5 ± 2.2 ms and tau slow = 205.9 ± 25.7 ms (n = 17). The time constants did not vary substantially at potentials between +20 and +70 mV (at +20 mV: tau fast = 29.6 ± 3.2 ms and tau slow = 303.2 ± 66.1 ms, n = 17, P > 0.05 vs. data at +70 mV). Recovery from inactivation was investigated using a two-pulse protocol in which the interval between the two pulses was varied between 100 and 3,000 ms (Fig. 4A). Recovery followed a biexponential time course, with time constants of 54 ms (65% of total amplitude) and 672 ms (35% of total) (Fig. 4B). Similar results were obtained in five cells.


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  		Fig. 4.   Time course of the recovery of outward currents from inactivation. A: superimposed currents obtained by a double-pulse protocol with a varying interval between the two pulses. VH was -80 mV; test potential was +60 mV. Pulses were given every 5 s. Horizontal dashed line, zero current level. B: amplitude of the transient current during the second pulse expressed relative to the current during the first pulse plotted as a function of the interval between the pulses. Data were calculated from the tracings in A. Insets: voltage-pulse protocol (left) and semilogarithmic plot of 100 minus %recovery (right).

Insensitivity to DIDS and to 4-AP. Because Cl- channels can constitute an important component of Ito, we tested the possibility that the transient outward currents are due to ion flow via these channels. Figure 5A shows that the time-dependent currents induced in 0 Cao and 0 Mgo were resistant to 200 µM DIDS. In addition, in 11 cells, decreasing internal Cl- from 157 to 27 mM (Cl- replaced by glutamate) did not cause a negative shift of Vrev, as would be expected if the currents were carried by Cl-. These results are in agreement with previous data (30) and suggest that the Ito-like currents were not due to Cl- channels and that Cl- movement did not contribute to the currents. The addition of 3 mM 4-AP alone or on top of DIDS (Fig. 5A, right) was also without effect (n = 2). The voltage-dependent inactivation (Fig. 5B) or activation was also not affected by the drugs. Together, these results exclude the possibility that Ito-like currents are simply due to an altered selectivity of classic K+- or Cl--conducting Ito channels.


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  		Fig. 5.   Lack of effect of DIDS or 4-aminopyridine (4-AP) on transient currents. A: current traces elicited by 1-s depolarizations to potentials between -40 and +70 mV (in 10-mV increments) from a VH of -80 mV during superfusion with 0 Cao and 0 Mgo with 1 mM EGTA. Left: absence of any other drug; middle, presence of 200 µM DIDS; right, presence of 3 mM 4-AP added on top of DIDS. Horizontal dashed line, zero current level. B: inactivation curves in the presence and in absence of the drugs, same cell as in A.

Cs+ can substitute for K+ in carrying outward currents. We examined the Cs+ permeability of the conductance underlying the Ito-like currents by substituting internal K+ with Cs+. Figure 6 shows that Ito-like currents similar to those observed with internal K+ (Fig. 6, A and B) were also present with internal Cs+ (Fig. 6, D and E). The currents carried by Cs+ also decayed biexponentially toward a steady state. The time constants (at +70 mV: tau fast = 28.1 ± 1.6 ms and tau slow = 291.0 ± 29.4 ms, n = 14) were similar to those obtained with internal K+, suggesting that they reflected an intrinsic property of the channel. This result indicates that either K+ or Cs+ was able to serve as a charge carrier for the outward Ito-like current. However, with internal Cs+, the current transients reversed at a more positive potential. Current-voltage relationships are plotted in Fig. 6, C and F. Vrev was shifted from +7.5 ± 1.0 mV in 15 cells dialyzed with K+ to +34.1 ± 2.1 mV in 14 cells dialyzed with Cs+. Similar results were obtained when Na+ was completely omitted from the internal solution (Na2ATP replaced by MgATP), i.e., in conditions where either K+ (Vrev = +16.8 ± 8.3 mV, n = 2) or Cs+ (Vrev = +40.7 ± 7.8 mV, n = 3) was the only permeant intracellular cation.


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  		Fig. 6.   Cs+ as a charge carrier of "transient outward K+ channel current (Ito)-like" currents. A, B, D, and E: current traces elicited by 1-s depolarizations to potentials between -120 and +70 mV (in 10-mV increments) from a VH of -80 mV. A and D: basal conditions; B and E: 0 Cao and 0 Mgo with 1 mM EGTA; A and B: K+ as the major internal cation; D and E: Cs+ as the major internal cation. Horizontal dashed line, zero current level. C and F: current-voltage relationships with K+ (C) or Cs+ (F) as the major intracellular cation. Notice the reversal potential (Vrev) = +8 mV with intracellular K+ and +34 mV with intracellular Cs+.

beta -Adrenergic stimulation of the outward currents. Figure 7 illustrates the effect of applying 1 µM Iso on the transient currents induced by the removal of extracellular divalent cations. With either K+ (data not illustrated) or Cs+ (Fig. 7, A-C) as the charge carrier, the amplitude of the peak currents was increased in the presence of Iso at potentials between -40 and +70 mV (at -10 mV: increase from -4.0 ± 0.6 pA/pF in the presence of EGTA alone to -5.7 ± 2.1 pA/pF after the addition 1 µM Iso, n = 7). The time course of the current decay during depolarizations was slowed in the presence of Iso (see Fig. 7E, inset). The Iso effect could be reversed by adding propranolol (1 µM) on top of the agonist (Fig. 7D, see also Fig. 7E). In contrast, there was little or no effect on the currents at potentials negative to -40 mV, i.e., Iso changed the time-dependent currents while not having an effect on the time-independent currents induced by Cao and Mgo removal. Figure 7E shows the time evolution of currents measured at +80 and -80 mV using voltage ramps (see METHODS). Whereas Iso largely increased the current at +80 mV, it had practically no effect on the current at -80 mV. Thus Iso exerted an effect only at potentials at which the transients could contribute to the currents generated by the ramp.


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  		Fig. 7.   Increase of the "Ito-like" currents by beta -adrenergic receptor stimulation. A-D: current traces elicited by 1-s depolarizations to potentials between -120 and +70 mV (in 10-mV increments) from a VH of -80 mV. A: basal conditions; B: 0 Cao and 0 Mgo with 1 mM EGTA; C: 0 Cao and 0 Mgo with 1 mM EGTA + 1 µM isoproterenol (Iso); D: 0 Cao and 0 Mgo with 1 mM EGTA + 1 µM Iso + 1 µM propranolol (Propr). Cs+ was the major internal cation. E: effect of Iso (1 µM) and propranolol (1 µM) on membrane currents at +80 mV and at -80 mV. K+ was the major internal cation. Currents were measured during the descending limb of 4-s ramps from -120 to +80 mV and back to -120 mV (see METHODS). Nif, nifedipine (100 µM); Ver, verapamil (50 µM). Inset: time-dependent components of the currents recorded during step depolarizations in the experiment illustrated in E. Horizontal dashed line, zero current level.

Inhibition by Ca2+ antagonists and stimulation by Ca2+ agonists. The voltage dependence and kinetics of activation and inactivation and the response to beta -adrenergic stimulation are strikingly similar to those of ICa,L. Therefore, we hypothesized that the transient currents were due to a flow of monovalent cations in these channels. However, in apparent contradiction with this hypothesis, the Ito-like currents could still be observed in experiments where 10 µM nifedipine was used in an attempt to block L-type Ca2+ channels (see Fig. 6, A and B). One explanation could be that the Ca2+ channels were incompletely blocked. We therefore tested the effect of increasing the drug concentration. Figure 8 shows that increasing the nifedipine concentration to 100 µM largely but often incompletely suppressed the time-dependent currents. In additional experiments, we used a prepulse to -40 mV before depolarization to various levels in an attempt to enhance the nifedipine effect. In Fig. 8E (open symbols), the current transients recorded using this protocol in the presence of increasing nifedipine concentrations (1-100 µM) are plotted as a function of the membrane potential (n = 6, Cs+ in internal solution). The data suggest that the potency of nifedipine to suppress the currents was low, the IC50 being 2 µM for currents measured at -10 mV (Fig. 8F, solid symbols).


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  		Fig. 8.   "Ito-like" currents can be suppressed by high concentrations of nifedipine. A-D: current traces elicited by 1-s depolarizations to potentials between -120 and +70 mV (in 10-mV increments) from a VH of -80 mV. A: basal conditions in the presence of 1.8 mM Cao and 0.9 Mgo; B: 0 Cao and 0 Mgo with 1 mM EGTA; C: 0 Cao and 0 Mgo with 1 mM EGTA + nifedipine (100 µM); D: 0 Cao and 0 Mgo with 1 mM EGTA + nifedipine (100 µM) + verapamil (50 µM). K+ was the charge carrier. Horizontal dashed line, zero current level. E: current-voltage relationships at different nifedipine concentrations (n = 6). Cs+ was the charge carrier. The voltage protocol consisted of a prepulse to -40 mV before depolarizations to various levels. F: concentration-effect curves at -10 mV. Filled symbols, monovalent cation current; IC50 was 2 µM. Open symbols, L-type Ca2+ current.

We also tested the effect of other Ca2+ channel modulators. Figure 9, A-C, shows the effect by adding verapamil (50 µM), diltiazem (50 µM), or BAY K-8644 (1 µM) on the time-dependent current induced by Cao and Mgo removal at various potentials. Examples of original traces at +60 mV are also included as insets. Verapamil (Fig. 9A, see also Fig. 8, D and E, solid symbols, for verapamil added on top of 100 µM nifedipine) and diltiazem (Fig. 9B) suppressed, whereas BAY K-8644 (Fig. 9C) increased, both the inward and the outward time-dependent currents. Similar results obtained in different cells are summarized in Fig. 9D.


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  		Fig. 9.   Effect of other Ca2+ channel modulators on "Ito-like" currents. A-C: "Ito-like" current-voltage relationships in control conditions (open circle ), during removal of divalent cations (0 Cao and 0 Mgo with 1 mM EGTA; ), and after addition of drugs in the continued absence of divalent cations (). Insets: current traces elicited by 1-s depolarizations to +60 mV from a VH of -80 mV. A: verapamil (50 µM); B: diltiazem (DTZ; 50 µM); C: BAY K-8644 (1 µM). Cs+ was the charge carrier. D: means and SE of densities of the time-dependent currents (measured at +60 mV) in control conditions and during the removal of divalent cations in the absence or presence of the various drugs.

The above data suggest that L-type Ca2+ channels in pig myocytes show low sensitivity to nifedipine. Because all measurements were made on currents carried by monovalent cations, we also examined whether the same low sensitivity was present when Ca2+ is the charge carrier. Figure 8F (open symbols) shows the effect of various nifedipine concentrations on ICa,L, also measured at -10 mV using the above voltage protocol (holding potential = -80 mV, prepulse to -40 mV before depolarizations, n = 2-7). ICa,L was also blocked by nifedipine only at high concentrations, and its sensitivity to the drug was similar to that obtained with monovalent charge carriers.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the present report, we show that time-independent and -dependent currents are induced when perfusing cardiac cells with divalent cation-free solutions. The time-independent currents have been previously well characterized as due to a monovalent cation-permeable channel, specifically induced in the absence of extracellular divalent cations, and not involving Ca2+ channels (22, 29-31, 33). Because the nature of the channels underlying the time-dependent currents has remained unclear (3, 16, 30), we examined whether these currents can be accounted for by a flow of monovalent cation through L-type Ca2+ channels.

Activation and inactivation. As discussed in the introduction, the removal of extracellular divalent cations modifies the activation and inactivation of voltage-dependent channels and changes the selectivity of Ca2+ channels. In addition, extracellular divalent cations also appear to directly gate a number of channels. Thus Cao-inhibitable, cation-permeable nonselective pathways have been found in various tissue types and species, although their role under physiological or pathophysiological conditions remains unknown. The underlying channels include connexin hemichannels, transient receptor protein channels, polycystin channels, NSC1, etc. (18, 22). Currents mediated by most of these proteins are time independent and are present at all potentials. Only hemichannels mediate voltage- and time-dependent currents, but their gating kinetics (time-dependent increase) are different from those of the transient currents obtained in the present study (8).

Time-dependent outward but not time-dependent inward currents were induced upon nominal removal of Cao and Mgo. Because our nominally divalent cation-free solutions contain free Ca2+ concentrations of 3-10 µM, as measured using a Ca2+-sensitive electrode, the absence of time-dependent inward currents indicates that the contaminating Cao was sufficient to block any inward monovalent cation movement through the channels. Similar results were obtained in rat atrial myocytes by Youm et al. (30), who kept Mgo at 0.5 mM while removing Cao. Additional divalent cation chelation by EGTA was necessary to induce time-dependent inward currents. In the presence of EGTA, time-dependent currents were only present at potentials positive to -50 mV, suggesting voltage-dependent activation. In addition, there was an apparent reversal potential because inward transients generated at potentials near the threshold level were replaced by large outward transients at more positive potentials. We tested whether both inward and outward transients were generated by the same channels by determining the voltage-dependent activation. For this purpose, we used the current transient instead of the total current to calculate the voltage-activated conductance at each potential. The use of the total current was inappropriate because it contains the voltage- and time-independent component also induced under conditions of 0 Cao and 0 Mgo (22, 29-31, 33). However, our method is strictly valid only if there is no persisting current due to the conductance responsible for the time-dependent currents. Experiments in which high nifedipine concentrations or verapamil were used to suppress the time-dependent current (see below and Fig. 8) indicate that when this component was completely eliminated, there was little or no effect on the steady-state current. This is consistent with the steady-state current being essentially made of a voltage-independent, nifedipine-insensitive, nonselective component carried by different channels (22, 29-31, 33). The voltage-dependent activation could be described by a single Boltzmann distribution curve, as expected if a single population of channels is responsible for all transients. In this study, we also determined the voltage-dependent steady-state inactivation and obtained a half-maximal availability at -58 mV. Bailly et al. (3) also noted a threshold (-20 mV) for activation of the Ito-like current in human left ventricular cells but did not determine the voltage dependence of activation. In addition, they noted that the inactivation followed a biexponential time course and was half-maximal at -30 mV. Differences in divalent cation composition of the solutions used may partly account for the different V1/2 values in their and our studies. V1/2 values for activation or inactivation as well as the potential (-20 mV) of maximum current are more negative for the Ito-like current than for ICa,L. However, this finding cannot be used as evidence for a difference in the channels underlying both currents because the values for the Ito-like current might result from a shift caused by the removal of the surface charge screening effect of Cao. In the present study as well as in previous studies, recovery from inactivation was biphasic, with time constants similar to those measured for the repriming of ICa,L.

Monovalent cation currents through L-type Ca2+ channels usually display little or slow inactivation, because there is no contribution of intracellular Ca2+ to inactivation (1, 9). The remaining voltage-dependent mechanism of inactivation is supposed to play a major role only at very positive potentials. In the present study, even inward currents inactivated relatively fast, suggesting that either the voltage-dependent mechanism is particularly marked in pig cells or that the marked negative shift of voltage-dependent inactivation in the absence of all divalent cations also affected its kinetics.

Selectivity to monovalent cations and block by divalent cations. The transient outward currents were supported by either internal K+ or internal Cs+. With internal K+ and external Cs+, Vrev of the current was nearly +8 mV, also indicating that the current was due to a flow of different cations. The large outward time-dependent currents were likely due to an efflux of intracellular K+ or Cs+, whereas the inward transients were probably due to external Na+ and Cs+. Either Na+ or Cs+ was also shown to support Ito-like currents attributed to ion flow via L-type Ca2+ channels in rat atrial cells (30). Readmission of Ca2+ and Mg2+ always suppressed the transient currents, suggesting that divalent cations were unable to cross the channels. Bailly et al. (3) noted that the currents were also suppressed by external Co2+ and suggested that other divalent cations could have a similar blocking effect. Because with either K+ or Cs+ as the major intracellular cation Vrev was closer to the equilibrium potential (E) of Na+ (ENa = 66 mV) than to EK or ECs, the data indicate that the permeability (P) of Na+ (PNa) was larger than either PK or PCs. In the present study, Vrev was changed from +8 to +34 mV when replacing internal K+ by Cs+ while keeping the same external solution. This shift indicates that PK was higher than PCs, as also suggested by the finding by others that currents carried by Cs+ were of one-third the amplitude of those carried by K+ (3). We used the Goldman-Hodgkin-Katz equation (14) and the magnitude of change in Vrev upon substituting K+ by Cs+ as the internal cation (with Na+-free internal solution) to determine the permeability ratio for Cs+ vs. K+ (PCs/PK). Similarly, PNa/PCs was obtained from the Vrev of experiments where Cs+ was the only intracellular monovalent cation. These calculations are justified because Cl- or other anions do not permeate the channel, as indicated by the lack of an effect of changing the Cl- chemical gradient on the Ito-like currents (3). Values of 0.3 for PCs/PK and 5.4 for PNa/PCs were obtained. Because of limitations in the diffusion of the pipette cations into the cell, these calculated values may not be quantitatively accurate, but they allow us to make a qualitative comparison. Given that Inoue and Imanaga (16) observed that internal TEA did not support the current, a permeability sequence of PNa > PK > PCs PTEA can be established. The sequence follows the Eisenmann series XI of atomic radii, as also found by others for the flow of monovalent cations through L-type Ca2+ channels (13).

Modulation by pharmacological agents. In contrast to the standard Ito, which is sensitive to block by 4-AP, the Ito-like current induced in 0 Cao and 0 Mgo was insensitive to this blocker. In the present study, we show that all classes of L-type Ca2+ channel antagonists (represented by nifedipine, verapamil, and diltiazem) were able to suppress, whereas the agonist BAY K-8644 increased, the current. We obtained an IC50 of 2 µM for nifedipine, a value many orders of magnitude larger than the reported nanomolar binding affinity for dihydropyridine receptors. This apparent low affinity was obtained despite precautions to prepare nifedipine freshly and to carefully protect the nifedipine-containing solutions from light. One explanation for the low sensitivity to nifedipine is to assume that the measured currents are not via L-type Ca2+ channels but via other voltage-dependent channels. The relatively slow time course of inactivation, the voltage range of activation, and the insensitivity to 10 µM TTX make it unlikely that the currents are due to a flow via Na+ channels. Although 10 µM TTX might not have been sufficient to completely eliminate voltage-dependent Na+ channels in the presence of Cao and Mgo (see persistence of small fast inward currents in Fig. 1A), any possible contribution of these channels was further decreased by the expected shift of the inactivation curve to more negative potentials in the absence of Cao and Mgo (see disappearance of fast inward currents in Fig. 1, B and C). The "high" threshold for activation of the transient currents and their sensitivity to Iso also make it unlikely that they were due to monovalent cation flow through T-type Ca2+ channels. Finally, although nifedipine has been shown to block a variety of native or expressed K+ channels with low affinity (10, 32), extracellular divalent cations are not known to block Ito [except from a shift of the voltage-dependent activation and inactivation (12, 27)], and Ito is not increased by beta -adrenergic receptor stimulation. We are therefore left with the hypothesis that they are due to L-type Ca2+ channels.

The low affinity to block by nifedipine could be due to various factors. First, the effect of dihydropyridines is to some extent state dependent and is enhanced by inactivation; hence, the affinity increases when the holding potential is less negative (see Ref. 7). In our experiments, cells were held at -80 mV, a factor that decreases the probability of an interaction with inactivated channels. However, to obtain the dose response to nifedipine, we used a protocol consisting of a depolarization to -40 mV before induction of the Ito-like current or ICa,L. Second, because of the interaction with specific channel states induced by depolarization, the block by nifedipine is also to some extent use dependent. Given that in our experimental protocol pulses were repeated every 5 s, this could allow any block developing during the depolarization to fully recover during the interval between pulses, hence avoiding any accumulation of block. Our data therefore could indicate that in pig ventricular myocytes nifedipine interacts relatively poorly with the rested state. A similar but less marked decreased affinity has been found for ventricular myocytes of other species, where nifedipine or nitrendipine block ICa,L with an IC50 of 0.3-0.7 µM in steady-state conditions (5, 20, 25). Shen et al. (25) observed in guinea pig ventricular myocytes that 10-30 µM nifedipine failed to fully suppress ICa,L when given up to 30 s before pulse but that the block could be enhanced by prolonged drug exposure and by depolarization. The low sensitivity in our study is not due to the loss of an allosteric action caused by Ca2+ binding when permeating the channel because ICa,L was similarly blocked with low potency. The relative resistance to block by nifedipine could imply that there exist interspecies differences at the level of the L-type Ca2+ channels, affecting the a1C or the associated subunits.

This low sensitivity to nifedipine may be of practical importance. The drug is used mainly for its vascular effect in the treatment of hypertension and angina. The higher affinity of this drug for vascular smooth cells is usually related to their depolarized state, which favors binding to inactivated channels. This study shows that a state of low affinity to nifedipine may be inherent to some cardiac L-type Ca2+ channels and may protect the myocardium from unwanted negative inotropic action at therapeutic concentrations. In experimental settings, either high concentrations of nifedipine or other antagonists are needed to completely block L-type Ca2+ channels before excluding their contribution to the process under study.

In conclusion, based on 1) the occurrence of activation and inactivation of the Ito-like current in a voltage range close to that of activation and inactivation of L-type Ca2+ channels, 2) the pharmacological sensitivity to nifedipine, verapamil, diltiazem, and BAY K-8644 as well as to beta -adrenergic receptor stimulation, and 3) a similar ion selectivity sequence for monovalent cations, we conclude that the Ito-like current unmasked by the removal of extracellular divalent cations is due to monovalent current flow via L-type Ca2+ channels. Although some properties of the current (low sensitivity to nifedipine and fast inactivation of monovalent currents) argue against typical L-type Ca2+ channels and might be invoked to propose a different channel, the putative new channel should have a pharmacological and biophysical profile largely similar to that of L-type Ca2+ channels.


    ACKNOWLEDGEMENTS

We thank Virginie Bito and Dr. F. Heinzel for supplying the isolated myocytes.


    FOOTNOTES

This study was supported by grants from the Belgian Flemish Foundation for Scientific Research.

Address for reprint requests and other correspondence: K. Mubagwa, Centre for Experimental Surgery and Anaesthesiology, Katholieke Universiteit Leuven, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium (E-mail: kanigula.mubagwa{at}med.kuleuven.ac.be).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpheart.00952.2001

Received 2 November 2001; accepted in final form 28 December 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 282(5):H1879-H1888
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