INTRODUCTION

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THE ACTION POTENTIAL OF CARDIAC muscle is the result of complex interplay between ionic currents which would depolarize or repolarize the membrane. Rapidly activating and rapidly inactivating K⁺ currents as well as inwardly rectifying K⁺ currents play an important role in the repolarization of the action potential. In neuronal cells, these currents are known as A-type K⁺ channels. In cardiac tissue, they are known as transient outward K⁺ currents (I_to) because their original description did not make it clear whether they represented a conductance selective for K⁺ and/or an anionic channel. I_to1 is now acknowledged to be a K⁺-selective voltage current, whereas I_to2 is considered to reflect a Ca²⁺-activated Cl conductance (28). Membrane currents corresponding to I_to are widely distributed between species and regions of the heart (3). Their absence has been noted on a few occasions from the ventricle of the guinea pig (13, 14) and it is generally considered that the long plateau of the guinea pig ventricular action potential is the result. Notwithstanding this consensus, there have been two reports of "I_to-like" currents in isolated myocytes of the guinea pig ventricle (15, 20). An inwardly rectified I_to has been described by Li et al. (20), which may contribute to early repolarization but by virtue of its rectification and rapid inactivation permits the characteristic long plateau of the action potential. Inoue and Imanaga (15) described a different and to date a unique form of I_to. Two points underlie the unusual nature of this form of I_to. First, the current was insensitive to 4-aminopyridine (4-AP), which is generally considered to be a selective and characteristic blocker of I_to (16). Second, this current was inhibited by physiological concentrations of extracellular Ca²⁺, by 100 µM Cd²⁺, and by 100 µM of the organic Ca²⁺ channel antagonist D600. No physiological role could be given to this current, which is nevertheless included in reviews of the physiology of I_to in cardiac muscle (3, 27).

The L-type Ca²⁺ current (I_Ca,L) plays a predominant role in the translation of electrical into mechanical activity in cardiac muscle. This current shows inactivation that is the result of complex interactions between voltage- and Ca²⁺-dependent mechanisms (23). The most common method of separating these processes has been to employ Ba²⁺ as a charge-carrying cation through the Ca²⁺ channels because this would not activate the Ca²⁺-dependent inactivation mechanism (17). Recent studies (5, 6) have challenged this view. In 1987, Hadley and Hume (10) isolated the voltage-dependent mechanism of inactivation of I_Ca,L in isolated ventricular myocytes of the guinea pig by removing extracellular Ca²⁺ and recording the outward flux of intracellular K⁺ through L-type Ca²⁺ channels (29). This current was outward, and it was voltage gated, rapidly activating, and rapidly inactivating, as described by Inoue and Imanaga (15). The evidence that is presented here overwhelmingly supports the conclusion that the outward current revealed by the removal of extracellular Ca²⁺ represents the efflux of intracellular monovalent cations via L-type Ca²⁺ channels and not an A-type K⁺ current.

	METHODS

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Cell preparation. All animal experiments were conducted according to the ethical standards of the Ministère Français de l'Agriculture (license no. B37-261-4). Male guinea pigs (250-400 g) were killed by cervical dislocation and the hearts were removed. Single ventricular myocytes were isolated using collagenase and protease digestion as described elsewhere (19). Myocytes isolated from the left ventricle were aliquoted into 35-mm-diameter plastic petri dishes that served as experimental chambers. The storage solution consisted of the standard extracellular solution described below. Dishes that contained myocytes were kept on the laboratory bench and used within 6-8 h after isolation.

Experimental procedures. Plastic petri dishes that contained isolated myocytes were placed on the stage of an Olympus CK2 inverted microscope. Isolated myocytes were superfused with experimental solutions via a parallel pipes system lowered into the vicinity of the cells. Fluid flow was maintained by gravity from syringe barrel reservoirs and the exchange of solutions was achieved by manual displacement of the pipes. Solution exchange around the myocyte was estimated to be complete in 4-5 s. All experiments were conducted at room temperature (~23°C). Whole cell current- voltage (I-V)-clamp experiments were conducted with a patch-clamp amplifier (model 202A, Axon Instruments) in resistive feedback mode. Pipettes were fabricated from thin-walled borosilicate glass capillary tubes (Clark Electromedical Instruments; Pangbourne, UK) with a double-stage puller (model PB7, Narishige Instruments; Tokyo, Japan). Pipettes were coated with Sylgard (Dow Corning; Midland, MI) and then heat polished. The finished pipettes had a resistance of <2 M when filled with standard intracellular solution. Experimental voltage-clamp protocols and data acquisition were controlled with Acquis1 software (Dipsi Industrie; Chatillon, France) installed on a 386 personal computer. Data were filtered at either 1 or 2 kHz and acquired at 2 or 5 kHz, respectively. Cell capacitance and series resistance were compensated (~80%) with the Axon Instruments amplifier. Data analysis was performed with Acquis1 and Origin 4.1 (Microcal Software). Once the whole cell configuration of the patch-clamp cell current recording technique (11) had been achieved, the isolated myocytes were voltage clamped at 80 mV. Voltage-clamp protocols were delivered to the cells from this holding potential. Voltage-clamp protocols were preceded by voltage steps to either 40 or 50 mV to inactivate any residual Na⁺ current remaining after the application of 10 µM tetrodotoxin (TTX) and to inactivate any T-type Ca²⁺ current (2).

Experimental solutions. The standard extracellular solution used to fill the petri dishes and store myocytes before the experiments contained (in mM) 140 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, 10 glucose, and 10 HEPES, pH 7.4 with NaOH. Ten micromoles of TTX citrate salt (Alomone Labs; Jerusalem, Israel, or Latoxan; Valence, France) were added to this standard solution when it was used to superfuse cells during experiments. Calcium-free (0 calcium) extracellular solution contained 250 µM EGTA · NaOH, 3 mM MgCl₂, and no added Ca²⁺. Extracellular solution with 0.2 and 20 mM Ca²⁺ contained (in mM) 0.2 CaCl₂, 2.8 MgCl₂ or 20 CaCl₂, and 1 MgCl₂. The standard intracellular solution used to fill the patch pipettes contained (in mM) 140 KCl, 10 NaCl, 5 EGTA · KOH, 1.4 MgCl₂, 0.1 CaCl₂, 2 ATP-Mg²⁺, 10 glucose, and 10 HEPES, pH 7.3 with KOH. The estimated free concentrations of Mg²⁺ and Ca²⁺ in this solution were 1 mM and 1 nM, respectively. Cadmium, lanthanum, cobalt, and nickel were added to extracellular solutions as their chloride salts. Tetraethylammonium (TEA) chloride and 4-AP were added directly to extracellular solutions. Nifedipine was dissolved as a 10 mM stock solution in acetone and added to extracellular solutions to give a final concentration of 10 µM.

	RESULTS

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Figure 1 shows that the removal of extracellular Ca²⁺ unveiled a large time-dependent outward current. The major intracellular cation under these experimental conditions was K⁺ (see METHODS), and it seemed likely therefore that this outward current represented in very large part the efflux of K⁺ from the cell. This current was activated by voltage steps to +20 mV and more positive membrane potentials. This current closely resembled the extracellular Ca²⁺-inhibited A-type K⁺ current that had been described by Inoue and Imanaga (15) in the same preparation.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   A-type K+ current revealed by removal of extracellular Ca2+ in ventricular myocytes of the guinea pig. These raw cell current traces were recorded from one isolated ventricular myocyte, first in normal extracellular solution, which contained 2 mM Ca2+ (A), and subsequently in extracellular solution, which did not contain Ca2+ (B). Voltage steps were applied to the cell from a prior voltage of 40 mV to between +10 and +60 mV in 10-mV increments. Dotted traces show the 0-pA current level, and the time and current scales apply to both sets of traces.

The recordings of this transient outward current which are shown in Fig. 2, illustrate three points. First, this current was not blocked by the extracellular application of 4-AP (Fig. 2A). Second, this current was not blocked by the extracellular application of TEA (Fig. 2B). Third, this current showed voltage-dependent inactivation, and it was reduced by short prepulse voltage steps to between 60 and +20 mV (Fig. 2C).

View larger version (27K): [in this window] [in a new window]   Fig. 2.   K+ current blockers and the A-type K+ current. A and B: raw cell current traces were obtained from two isolated ventricular myocytes during voltage steps from a prior voltage of 40 mV to between 0 and +60 mV in 10-mV increments. Cell currents were each recorded in 0 calcium extracellular solution to reveal the A-type K+ current under control conditions (a) and after the addition of either 5 mM 4-AP (A,b) or 50 mM tetraethylammonium (TEA) (B,b). C: voltage-dependent inactivation of the A-type K+ current. These raw cell current traces were obtained from one myocyte in 0 calcium extracellular solution. Cell current records shown on the left were evoked by a voltage step to +60 mV after 50-ms prepulse voltage steps to between 60 and +20 mV in 10-mV increments. The graph to the right illustrates the peak () and steady-state () amplitudes of the test pulse currents. Dotted traces in cell current records indicate the 0-pA current level. The time and current scales apply to recordings obtained from each myocyte.

The effect of extracellular Ca²⁺ on the transient outward current is shown in Fig. 3. In the absence of extracellular Ca²⁺ no inward current flow through Ca²⁺ channels was recorded and the transient outward current was visible (Fig. 3A). In the presence of 200 µM Ca²⁺, small inward currents through Ca²⁺ channels were visible and their amplitude increased when the extracellular Ca²⁺ concentration was increased to 2 mM (Fig. 3A). At the same time, the transient outward current was reduced in amplitude. Although the transient outward current evoked by a voltage step to +60 mV had been almost completely inhibited by 2 mM extracellular Ca²⁺ (Fig. 3A), it was found that this could be recovered by applying voltage steps to more positive voltages (Fig. 3B). Even in the presence of 20 mM Ca²⁺, a transient outward current could be evoked provided that sufficiently positive voltage steps were applied to the cells (Fig. 3B). The I-V relationships for the peak amplitude of the currents recorded in different concentrations of Ca²⁺ are shown in Fig. 3C. It is clear that inward currents and transient outward current existed, respectively, negative and positive to a voltage, which depended on the extracellular concentration of Ca²⁺.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   The effect of extracellular Ca2+ on the A-type K+ current and L-type Ca2+ current (ICa,L). Illustrated cell current records were obtained from two isolated ventricular myocytes (A and B). A: cell currents were evoked by voltage steps from 50 mV to between 0 and +60 mV in 10-mV increments. B: cell currents were evoked from 50 mV to between 0 mV and +80 or +100 mV in 10-mV increments. Cell currents were first recorded in extracellular solutions with variable amounts of Ca2+ and subsequently in the same extracellular solutions, which also contained 200 µM Cd2+. The traces represent Cd2+-difference currents obtained by digital subtraction of cell currents recorded in the presence of Cd2+ from those recorded in its absence. Dotted traces, 0-pA current level. The time and current scales apply to records obtained from each myocyte. A: extracellular solution contained either 0 (a), 200 µM (b), or 2 mM (c) Ca2+. B: extracellular solution contained either 2 mM (a) or 20 mM Ca2+ (b). C: peak current-voltage (I-V) relationships for Cd2+-difference currents. The symbols and bars represent the means ± SE values of data obtained from n different myocytes that had been exposed to 0 calcium (, n = 9), 200 µM Ca2+ (, n = 9), 2 mM Ca2+ (, n = 15), or 20 mM Ca2+ (, n = 6) in the extracellular solution.

Figure 4 illustrates the effects of a variety of L-type Ca²⁺ channel blockers on the transient outward current. Each inhibited the transient outward current whether these were inorganic cations such as La³⁺ (Fig. 4A), Co²⁺ (Fig. 4B), Ni²⁺ (Fig. 4C), and Cd²⁺ (Fig. 4D) or the organic Ca²⁺ channel blocking compound nifedipine (Fig. 4E). Each of these compounds also blocked I_Ca,L (Fig. 4F).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Effect of Ca2+ channel blockers on the A-type K+ current. Raw current traces were recorded from 5 different myocytes (A-E) with voltage steps from 40 mV to between 0 and +60 mV in 10-mV increments. The cells were bathed in 0 Ca2+ extracellular solution to reveal the A-type K+ current. In each cell, currents were first recorded under control conditions (a) and then after the addition of either 50 µM La3+ (A,b), 2 mM Co2+ (B,b), 2 mM Ni2+ (C,b), 200 µM Cd2+ (D,b), or 10 µM nifedipine (Nif; E,b) to the extracellular solution. Dotted traces, 0-pA current level, and the time and current scales apply to recordings obtained from each cell. F,a: inhibition of the A-type K+ current by Ca2+ channel blockers. The amplitude of the outward current evoked by a voltage step to +60 mV in 0 Ca2+ extracellular solution served as witness. F,b: inhibition of ICa,L by Ca2+ channel blockers. The amplitude of ICa,L evoked by a voltage step to 0 mV in normal extracellular solution, which contained 2 mM Ca2+, served as witness. The effects of the indicated compounds are expressed as relative to that caused by the application of 200 µM Cd2+ in the same cell. The columns and bars represent the mean ± SE values of data obtained from n different myocytes exposed to 50 µM La3+ (n = 5), 2 mM Co2+ (n = 8), 2 mM Ni2+ (n = 5), and 10 µM (NIF; n = 6).

Experimental evidence that might distinguish between a K⁺-selective A-type current and I_Ca,L was sought. It was decided to test whether the A-type K⁺ current revealed by the removal of extracellular Ca²⁺ might be permeant to Cs⁺. Figure 5 illustrates transient outward current carried by K⁺ and Cs⁺, which were revealed by the removal of extracellular Ca²⁺. In both cases, transient outward currents were clearly visible notwithstanding that in these experiments the intracellular solution contained 40 mM TEA (see METHODS). The current carried by Cs⁺ (Fig. 5B) was much smaller than that carried by K⁺ (Fig. 5A). The current carried by Cs⁺ was activated by voltage steps more positive than +30 mV, whereas the current carried by K⁺ was activated by voltage steps more positive than +10 mV (Fig. 5C). The voltage dependence of inactivation of the currents carried by K⁺ and Cs⁺ were compared (Fig. 5D). In these experiments, the test pulse of the double-pulse voltage-clamp protocol was to +80 mV to enhance the reliability of the measurement of the current carried by Cs⁺. There was no difference in the availability-voltage (A-V) relationships for transient outward currents carried by K⁺ and Cs⁺ (Fig. 5D). This strongly suggested that they represented current flow through the same channels.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   K+ and Cs+ permeation of the A-type K+ current. Cells were bathed in 0 calcium extracellular solution to reveal the A-type K+ current, and cell currents were evoked by voltage steps applied from 50 mV to between +10 and +80 mV in 10-mV increments in two different myocytes with either K+-based solutions (A) or Cs+-based solutions (B). Illustrated cell records represent Cd2+-difference currents obtained as described in Fig. 3. I-V (C) and availability-voltage (A-V) (D) relationships were obtained with a double-pulse voltage- clamp protocol, which consisted of 1,000-ms prepulse voltage steps to between 50 and +80 mV in 10 mV-increments, followed by a test pulse voltage step to +80 mV. The symbols and bars represent the means ± SE values of data obtained from n different myocytes bathed in either K+-based solutions (, n = 6) or Cs+-based solutions (, n = 7). The data in D were normalized to the test pulse current amplitude recorded after a prepulse voltage step to 50 mV, and the traces represent fits of the Boltzman equation to the data with half-inactivation voltage (V0.5) values of 29 and 29 mV and slopes of 4.5 and 4.4 mV for K+- and Cs+-based solutions, respectively.

Further evidence for the association between the transient outward current and I_Ca,L was looked for. Both transient outward current (Figs. 2C and 5D) and I_Ca,L (23) show voltage-dependent inactivation. Figure 6 compares the A-V relationships of transient outward current recorded at +80 mV and I_Ca,L recorded at +10 mV. Both A-V relationships were U shaped with identical voltage dependence. It is considered to be extremely unlikely that the Ca²⁺ current and a different and I_to would show identical voltage dependence of inactivation.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   A-V relationships of ICa,L and A-type K+ currents. A-V relationships were obtained with the double-pulse voltage-clamp protocol described in Fig. 5 and a similar analysis was applied. Data were normalized to those obtained after a prepulse to 60 mV. Experiments were conducted in normal extracellular solution, which contained 2 mM Ca2+. The A-V relationships of ICa,L recorded with a test pulse voltage step to +10 mV (, n = 8) were compared with those of the A-type K+ current recorded with a test pulse voltage step to +80 mV (, n = 6). Symbols and bars represent means ± SE values of data obtained from n different myocytes. The traces represent fits of the Boltzman equation to between the maximum and minimum values of the data with the following characteristics: test pulse +10 mV, V0.5 22 mV, and; slope 5.0 mV, and test pulse +80 mV, V0.5 22 mV, and slope 5.8 mV.

	DISCUSSION

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The evidence presented here clearly indicates that an A-type K⁺ current does not exist in isolated ventricular myocytes of the guinea pig. This study therefore comforts the numerous observations that conventional I_to is absent from the guinea pig ventricle (3, 27). The removal of extracellular Ca²⁺ causes a negative shift of the reversal potential between the influx of Ca²⁺ and the efflux of monovalent cations through L-type Ca²⁺ channels (29). If Mg²⁺ and Ca²⁺ are removed from the extracellular medium, the L-type Ca²⁺ channels permit the passage of monovalent cations both inward as well as outward through the membrane (29). When Mg²⁺ is retained in the extracellular medium, it blocks the influx of extracellular monovalent cations but, being impermeant, it does not affect the apparent reversal potential of the channel currents (8). The blockage of monovalent cation efflux by extracellular Mg²⁺ is relieved by the driving force of cation efflux through the Ca²⁺ channels (12). In these circumstances, an outwardly rectified and rapidly inactivating current carried by K⁺ may then be recorded. This current flows through Ca²⁺ channels.

Inoue and Imanaga (15) described block of their A-type K⁺ current with extracellular 4-AP and intracellular TEA. In this study, 5 mM 4-AP, which is known to block I_to in other cardiac myocytes (16), had no effect (Fig. 2A). Inoue and Imanaga (15) only succeeded in blocking the current when 4-AP replaced all extracellular cations. It is possible that under these conditions the effect might be nonspecific. In a similar manner, intracellular TEA blocked the current only when it replaced all intracellular cations (15). TEA is not permeant through L-type Ca²⁺ channels (22, 29) and therefore the loss of the transient outward current under these conditions can be explained by the absence of a permeant cation for I_Ca,L rather than the block of a K⁺ current. A comparison of the I-V curves in Figs. 3C and 5C show that 40 mM intracellular TEA was associated with a smaller transient outward current. This reduction in the amplitude of the current also coincided with the reduction of the concentration of intracellular K⁺ from 140 (Fig. 3C) to 100 mM (Fig. 5C). The transient outward current carried by Cs⁺ was smaller than that carried by K⁺ (Fig. 5) and this resulted from the lesser permeation of I_Ca,L by Cs⁺ compared with that of K⁺ (22, 29).

Transient outward K⁺-selective currents exist in cardiac myocytes of a variety of species, including humans (3, 27). These currents serve to accelerate repolarization of the cardiac action potential and may play an important role in the adaptation of the action potential to alteration of cardiac rhythm. Extracellular divalent cations have been shown to influence I_to in cardiac myocytes (1, 4). These effects have been largely confined to alterations of the voltage dependence of the current, probably by influencing the membrane surface charge. These cations did not block I_to. Extracellular cations, such as La³⁺, Cd²⁺, Co²⁺, and Ni²⁺, blocked the apparent K⁺ current in this study with an equal efficacy as their block of I_Ca,L. Organic Ca²⁺ channel blockers have been reported to influence I_to (9, 18). They did not block it with an efficacy equivalent to their effect on I_Ca,L, which was shown here with nifedipine.

The range of voltages for the activation of I_Ca,L and I_to overlap. To study one, the other must be blocked. It is now customary to record I_Ca,L in isolated myocytes infused with Cs⁺-rich solutions from the recording electrode (24). Studies of I_to usually block I_Ca,L, often with Cd²⁺ and/or a dihydropyridine Ca²⁺ channel antagonist. The inwardly rectified I_to described by Li et al. (20) was recorded in this manner; therefore, this was clearly separated from the transient outward flux of cations through L-type Ca²⁺ channels, which was described here. A simple alternative might have been thought to be to exclude Ca²⁺ from the extracellular medium. The results of this study suggest that this might not be appropriate. Nakayama and Fozzard (26) studied I_to in isolated Purkinje fiber myocytes. Those authors occluded contamination of their recordings of outward K⁺ currents by excluding Ca²⁺ from the extracellular medium. They reported significant effects of -adrenergic stimulation on these K⁺ currents. The currents were increased and their decay was reduced by the suppression of a rapid phase of inactivation and the enhancement of a slow phase of inactivation. These results are unique for a I_to in cardiac myocytes, although similar results have been described concerning the effects of -adrenergic stimulation on the I_Ca,L (7, 25).

A recent study of transient outward current superimposed on a nonselective background current evoked by the chelation of Ca²⁺ and the reduction of extracellular Mg²⁺ in isolated ventricular myocytes of the pig (21) reached very similar conclusions to those formed here. These outward currents represent the efflux of intracellular monovalent cations via L-type Ca²⁺ channels. This phenomenon is therefore not confined to the ventricle of the guinea pig.