Introduction

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K⁺ currents that rapidly activate upon depolarization and then inactivate with time are a major component of the total repolarizing current in cardiac cells (Barry and Nerbonne, 1996). These currents constitute a potential target for the modulation of the cardiac electric activity by physiological or pathological conditions, and by pharmacological agents. In the mouse ventricular myocyte, the voltage-activated transient outward current is large (Benndorf et al., 1987; Wang and Duff, 1997; London et al., 1998a,b), making of this preparation a valuable cell model for studies on the underlying channels. Current evidence indicates that the transient outward K⁺ current is due to two or more distinct channels: 1) The time course of inactivation of the total outward current is complex and can be resolved into two or more exponentials; 2) While low 4-aminopyridine (4-AP) concentrations (50 µM) block a slowly inactivating component (Fiset et al., 1997; London et al., 1998a,b; Zhou et al., 1998; Xu et al., 1999b), higher concentrations are needed to inhibit the fast one; 3) Transgenic mice overexpressing Kv1.1N206Tag, a truncated potassium channel, show a significant reduction in the density of a rapidly activating, slowly inactivating, 4-AP-sensitive outward K⁺ current and a marked decrease in the level of Kv1.5 peptide (London et al., 1998a,b). Conversely, in Kv4.2W362F-expressing mice the fast inactivating component is lost, whereas the slowly inactivating component is maintained (Barry et al., 1998; Guo et al., 1999). Such observations indicate that the slow component is a Kv1.5 or a related channel. Based on the finding that clotrimazole suppresses voltage-dependent currents produced by a human cardiac Kv1.5 channel clone expressed in Xenopus laevis oocytes (Dumaine et al., 1998), as well as the voltage-dependent K⁺ current in pulmonary artery myocytes (Yuan et al., 1995), the maxi-K⁺ currents in ferret portal vein (Rittenhouse et al., 1997a), PC12 cells (Rittenhouse et al., 1997b), and carotid body cells (Hatton and Peers, 1996), one objective of the present study was to examine the effect of this antimycotic, and of its related congener miconazole on the transient outward currents in mouse cardiac myocytes, and to test whether they differentially block various current components. In addition, since a recent report shows that glybenclamide blocks transient outward currents (Schaffer et al., 1999), we wanted to further examine the effect of this drug and to compare them with those of the antifungal drugs.

	Materials and Methods

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Preparation of Mouse Ventricular Myocytes. The study has been carried out in accordance with the Declaration of Helsinki and with the institutional guides for the care and use of laboratory animals.

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Electrophysiological Recordings and Data Analysis. Membrane currents were measured as described before (Stengl et al., 1998) using the whole-cell patch-clamp technique (Hamill et al., 1981). Heat-polished borosilicate glass electrodes (horizontal puller; Zeitz Instrumente, Munich, Germany), with tip resistances of 0.5 to 1 M when filled with the internal solution, were used. The electrodes were connected to an Axopatch 200B amplifier (Axon Instruments, Foster City, CA), and a DigiData 1200 (Axon Instruments) interface controlled by the pClamp 5.5.1 software (Axon Instruments) was used to generate command pulses and acquire data. All experiments were carried out at room temperature (21-22°C). The holding potential was set at 80 mV. Under our experimental conditions (0.18 mM Ca²⁺ in the extracellular solution) a small inward current component that could be attributed to Ca²⁺ current, I_Ca, was detectable in a small percentage of cells but was negligibly small, especially at positive potentials (<100 pA). Because of the small magnitude of I_Ca and the fact that typical blockers of L-type Ca²⁺ channel also block I_to (Gotoh et al., 1991) we decided not to use any Ca²⁺ channel blocker.

Solutions and Drugs. The myocytes were superfused with a Tyrode's solution containing 135 mM NaCl, 5.4 mM KCl, 0.9 mM MgCl₂, 0.18 mM CaCl₂, 0.33 mM NaH₂PO₄, 10 mM HEPES, and 10 mM glucose; pH adjusted to 7.4 with NaOH. The internal solution contained 130 mM KCl or K-glutamate, 25 mM KCl, 1 mM MgCl₂, 5 mM Na₂ATP, 1 mM EGTA, 0.1 mM Na₂GTP, 5 mM HEPES; pH 7.25 (adjusted with KOH). 4-AP (Sigma) was made as a 1.5 M stock solution in distilled water (pH adjusted to 7.4 with HCl) and was added to the external solution to obtain the desired concentration. Solutions containing 4-AP were protected from light. Clotrimazole, miconazole, and glybenclamide were obtained from Sigma. Stock solutions were prepared by diluting an appropriate amount of compound in dimethyl sulfoxide. The dimethyl sulfoxide concentration in the perfusing solutions never exceeded 0.2% (v/v) and caused no effect of its own.

	Results

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Different Kinetic Components of I_to. Figure 1A shows currents elicited by 5-s voltage steps from the holding potential (80 mV) to +60 mV, in the absence or in the presence of 50 µM 4-AP. The currents inactivated with time during the depolarizing step. Under control conditions, an initial rapid decrease was followed by a slow decay, and two exponentials were needed to satisfactorily fit the time course of the inactivation. At +60 mV _fast was 88.9 ± 6.80 ms and _slow was 1022 ± 45.27 ms (n = 47). These rates of decay did not change substantially with voltage >0 mV (data not shown). In the presence of 50 µM 4-AP the outward current was decreased in peak amplitude and decayed faster than in control, but the end-of-pulse current was practically unchanged. The 4-AP-sensitive current, i.e., the difference between the traces in control and in the presence of the drug, is presented in Fig. 1B, and the inset shows that its inactivation could be resolved by one exponential. Its amplitude (15.6 pA/pF) and time constant (716 ms) were of the same magnitude as those of the slow component of the total current (16.6 pA/pF, 931 ms), suggesting that there was a selective suppression of a slow component by 4-AP, with no or marginal effect on the fast component. These results are consistent with the findings of other studies (Fiset et al., 1997; London et al., 1998a,b; Zhou et al., 1998), which indicated a high sensitivity of the slow component of the transient current to 4-AP. For simplicity, we will assume below that the total outward current is made of a fast inactivating component (I_fast), of a slowly inactivating component (I_slow), and a noninactivating component (I). The whole time-dependent current (sum of I_fast and I_slow) will be referred to as I_to.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Separation of the two kinetic components by 4-AP. A, currents elicited at +60 mV from a holding potential (VH) of 80 mV in the absence and in the presence of 50 µM 4-AP are superimposed. B, difference between current traces in the absence and in the presence of 50 µM 4-AP. Inset, semilogarithmic plot of the difference current. Notice one single (slow) component of inactivation.

Preferential Block of I_slow by Miconazole and Clotrimazole. Figure 2, A and B, show the effect of miconazole (30 and 100 µM) and clotrimazole (30 µM), respectively, on I_to induced by steps to +60 mV. The peak amplitude of I_to was decreased and the current decayed faster in the presence of either drug. The time course of the drug effect is illustrated in Fig. 2C for a cell in which 10 µM miconazole was applied while giving voltage pulses consisting of a 1-s hyperpolarization to 120 mV followed by a 5-s depolarization to +60 mV. Peak current at +60 mV decreased progressively in the presence of miconazole, whereas there was no effect on the end-of-pulse current at +60 mV and only a marginal decrease at 120 mV (see Fig. 3Aa). On washout of miconazole the effect could not be reversed. Pooled data from experiments (n = 4) such as those of Fig. 2C are summarized in Fig. 2D, which confirm that 10 µM miconazole decreased I_to (peak current at +60 mV: 64 ± 4.4 pA/pF in control and 43 ± 6.9 pA/pF with drug; P < 0.05) while having no effect on the noninactivating current or on I_K1 (current at 120 mV: 23 ± 2.4 pA/pF in control, and 21 ± 3.5 pA/pF with drug; P > 0.05). However, with higher concentrations and prolonged treatments, decreases in these latter currents could be observed but were not further investigated in the present study.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Effect of miconazole and clotrimazole on outward currents. A and B, examples of traces obtained at +60 mV from a VH of 80 mV, in the absence or in the presence of miconazole (30-100 µM; A) or clotrimazole (30 µM; B). C, time evolution of currents recorded at 120 mV and at +60 mV (at peak and at the end of the pulse) in control conditions, during the application of 10 µM miconazole, and during washout of the drug. Different cells in A-C. D, mean and S.E.M. (n = 4) of data such as those in C, obtained at 120 mV and at +60 mV (at peak and at the end of a pulse) in control conditions and during the application of 10 µM miconazole. *P < 0.05 for miconazole versus control.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Effect of the antimycotics on isolated slow or fast components. A, effect of prepulses on the time dependence of the current in the presence of miconazole. Superimposed currents, obtained at +60 mV in control and in the presence of 30 µM miconazole, when the test pulse was preceded by a 1-s prepulse to either 90 mV (a) or 30 mV (b). Note absence of fast decaying component after the prepulse of 30 mV, both in the absence and presence miconazole. B, effect of clotrimazole on outward currents after application of 50 µM 4-AP to isolate Ifast. Current traces obtained at +60 mV in control (a), in the presence of 50 µM 4-AP (b), and in the presence of 30 µM clotrimazole added on top of 4-AP (c).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Concentration dependence of the effect of miconazole on the two kinetic components of Ito (Ifast and Islow), as resolved by exponential analysis. The magnitude of each component in the presence of a given drug concentration (Idrug) is expressed relative to its magnitude in the absence of the drug (Icontrol). Data were fitted with a Hill equation: Idrug/Icontrol=1  [1 + (IC50/D)n]1, where D is the drug concentration, IC50 is the half-maximum effective concentration, and n is the Hill coefficient.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Lack of effect of miconazole and clotrimazole on steady-state inactivation. A and B, availability curves in the absence () and in the presence () of 30 µM miconazole (A) or 30 µM clotrimazole (B). Data were fitted by Boltzmann distribution functions (under Materials and Methods).

Block of Both I_fast and I_slow by Glybenclamide. Given the reported acceleration of I_to inactivation by glybenclamide (Schaffer et al., 1999), we examined whether the drug acts in the same way as the antimycotics by preferentially suppressing I_slow. Figure 6A illustrates the effect of glybenclamide (100 µM) on currents induced by a 1-s step to 120 mV followed by a 5-s depolarization to +60 mV. Glybenclamide caused no or marginal changes of the current during the hyperpolarizing pulse, suggesting that it had no or little effect on I_K1 (Fig. 6, C and D). In contrast, the drug caused a decrease of the peak amplitude of I_to induced at +60 mV and accelerated its inactivation, but had no effect on the noninactivating current at the end of the pulse. Figure 6B shows the glybenclamide-sensitive current, i.e., the difference current obtained by subtracting the trace in the presence of glybenclamide from the control trace. The inset illustrates that the difference current could not be resolved by one single exponential (_fast = 120 ms, _slow = 965 ms; compared with _fast = 116, _slow = 1047, for the control tracing), hence indicating that the glybenclamide effect could not be readily attributed to the selective suppression of a current component that decays monoexponentially. The time course of the glybenclamide effect is illustrated in Fig. 6C. The effect developed rapidly, reached a steady-state within 2 to 4 min with 50 µM glybenclamide, and could be readily reversed on drug washout from the extracellular solution. Figure 6C and pooled data from nine experiments in Fig. 6D, further illustrate that the effect on I_to peak occurred in the absence of significant effect on the current (largely I_K1) at negative potentials or on the noninactivating current at depolarized levels (current at 120 mV: 30 ± 3.0 pA/pF in control, 28 ± 3.1 pA/pF during exposure to the drug; P > 0.05, n = 9).

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Effect of glybenclamide on outward currents. A, superimposed currents, obtained in control and in the presence of 100 µM glybenclamide, during a test pulse to +60 mV preceded by a 1-s prepulse to 120 mV. VH = 80 mV. B, glybenclamide-sensitive current, measured as difference between current traces in the absence and in the presence of 100 µM glybenclamide. Inset, semilogarithmic plot of the difference current. Notice more than one component of inactivation. C, time evolution of currents recorded at 120 mV and at +60 mV (at peak and at the end of the pulse) in control conditions, during the application of 50 µM glybenclamide, and during washout of the drug. D, mean and S.E.M. (n = 9) of data such as those in C, obtained at 120 mV and at +60 mV (at peak and at the end of a pulse) in control conditions and during the application of 50 µM glybenclamide. *P < 0.05 for miconazole versus control.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Concentration dependence of the effect of glybenclamide on the two kinetic components of Ito, as resolved by exponential analysis. The magnitude of each component in the presence of a given drug concentration is expressed relative to the magnitude in the absence of the drug. Data were fitted with a Hill equation (see legend to Fig. 4).

View larger version (21K): [in this window] [in a new window]   Fig. 8.   Negative shift of the Ito steady-state inactivation by glybenclamide. A, current traces obtained at +60 mV following 5-s prepulses to various levels in control conditions (left), in the presence of 50 µM glybenclamide (middle), and after washout of the drug (right). B, availability curves in the absence () and in the presence () of 50 µM glybenclamide. Voltage clamp protocol as in A. Data were fitted by Boltzmann functions (under Materials and Methods).

Effects on Action Potential. Given the prominent effects of either the antimycotics or glybenclamide on I_to, one expects a slowing of the ventricular repolarization in the presence of these drugs. Action potentials were recorded in nine cells with the patch electrode under the same experimental conditions used for voltage clamp, except that the external solution contained 1.8 mM Ca²⁺. In three cells, the action potential duration at 50% repolarization (15.2 ± 3.71 ms under control conditions at 1-Hz pacing) was prolonged by 55 ± 12.0% in the presence of glybenclamide (100 µM) without change in the resting potential (81.4 ± 1.60 mV). This effect was completely reversed with 5 to 10 min of drug washout. Miconazole (n = 5) or clotrimazole (n = 1), each at 10 µM, caused a progressive increase in the delay between stimulus and action potential upstroke, a decrease of action potential amplitude (hence precluding a quantitative assessment of its effect on action potential duration), and induced a complete loss of excitability despite maintained resting potential.

	Discussion

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The presence of distinct components of the inactivating outward current (here simply called I_to) in mouse ventricular myocytes was first reported by Benndorf et al. (1987) and Benndorf and Nilius (1988), who observed two to three channel populations with different conductances and kinetics in cell-attached patches. Recently, more evidence has been presented for the existence of distinct channels at the molecular level. In myocytes of transgenic mice overexpressing a truncated Kv1.1 channel, the expression of Kv1.5 was markedly depressed and the slowly inactivating component of I_to was absent (London et al., 1998a,b). This prompted the authors to suggest that the slow component (which they called I_slow) is encoded by Kv1.5. Barry et al. (1998) found that in cardiomyocytes from Kv4.2W362F mice the fast inactivating I_to (I_fast) was eliminated, suggesting that members from the Kv4 family underlie the fast component. More recently, Xu et al. (1999a,b) reported four different components, and Guo et al. (1999) found that in ventricular myocytes from mice with a targeted deletion of the Kv1.4 gene, I_slow was absent, hence demonstrating that Kv1.4 or a related protein is the molecular correlate of I_slow.

In the present study we have corroborated the presence of two I_to components. The decay of I_to could be resolved into two exponentials with time constants differing by one order of magnitude as also found by previous studies (Benndorf et al., 1987; Wang and Duff, 1997; London et al., 1998a,b; Zhou et al., 1998). The two components were present in all our cells, although Xu et al. (1999a,b) found no fast component in 10% of myocytes randomly dispersed from left and right ventricles.

In our study, V_1/2 of the inactivation curve was about 50 mV. Differences in experimental conditions (concentration of divalent cations, presence of junction potential) among the various studies make a comparison of V_1/2 values obtained by different groups difficult. In the study of Zhou et al. (1998) V_1/2 for the slow component was 35 mV but they included 1 mM Ca²⁺, 1 mM Mg²⁺, and 2 mM Co²⁺ in the extracellular solution. V_1/2 for a fast component was 24 mV in the studies of Xu et al. (1999a,b), while for the slow component (I_Kslow) they found a biphasic inactivation curve with V_1/2 values of 73 and 19 mV in the presence of 1 mM Ca²⁺, 2 mM Mg²⁺, and 5 mM Co²⁺. In contrast, a V_1/2 of 66 mV has been reported in day 1 neonatal mouse, in which only the fast component is present (Wang and Duff, 1997). Thus, even after correction for divalent cation effects (Stengl et al., 1998) and for junction potentials major differences remain between different studies.

Channel-selective drugs are a useful tool for separating various channel populations. As also found by others (Fiset et al., 1997; London et al., 1998a,b; Zhou et al., 1998; Xu et al., 1999b), low 4-AP concentrations in our study blocked the slow component (IC₅₀ of  3 µM) while leaving intact a fast component. Millimolar concentrations were needed to block this fast component (data not shown). This differential sensitivity to 4-AP between fast and slowly inactivating components is an additional argument to support the view that I_to is composed of at least two distinct channels.

Based on the finding that ketoconazole blocks Kv1.5 expressed in X. laevis oocytes (Dumaine et al., 1998), we expected an effect of two ketoconazole-related fungicides, miconazole or clotrimazole, on native I_to of mouse myocardial cells. The magnitude of I_to in the presence of either drug was decreased and the inactivation was apparently accelerated. It is shown that the effect of the antimycotics can be attributed to a selective suppression of a slow component of I_to (I_slow) as is seen in the presence of micromolar 4-AP concentrations. The IC₅₀ (7 µM) for I_slow inhibition by miconazole is nearly one order of magnitude lower than for the ketoconazole inhibition of Kv1.5 expressed in oocytes (105 µM). In this study we did not use ketoconazole. If similar potencies are assumed for the antimycotics, the observed disparity in IC₅₀ values may be accounted for by a genuine difference between native I_slow and Kv1.5, or by an influence on drug sensitivity of the expression system (oocyte for Kv1.5) used. This effect of the antimycotics is important not only from a theoretical point of view but also from a clinical standpoint.

The slowly inactivating current accounts for about 35 to 40% of the peak total current, and for more than 50% of I_to (Table 1). Its kinetic properties and high sensitivity to 4-AP, miconazole, and clotrimazole support recent views that this current is due to Kv1.5 or a related channel. Since intracellularly applied miconazole was without effect, the drug does not act via an intracellular mechanism (e.g., an inhibition of cytochrome P450-dependent processes) but probably interacts directly with the channel at a site accessible from the extracellular side of the membrane. The mechanism of block was not clarified in the present study. However, it is unlikely that the drugs cause a fast open/inactivated channel block (Carmeliet and Mubagwa, 1998) since no time-dependent change was obtained when the slow current component was isolated after full inactivation of the fast component. Similarly, the absence of an effect on steady-state inactivation suggests that there is no preferential action on the inactivated channels. Thus, our data suggest an effect on the rested state. The noted decrease of the fast time constant in the presence of the drugs (Table 1) could indicate some effect of the antimycotics on I_fast. Such an effect cannot be completely excluded, especially with prolonged drug application and at high drug concentrations. However, a substantial overestimation of _fast can be caused by limitations in accurately determining this parameter in the presence of a substantial slow component by the least-square algorithm used.

Imidazole antimycotics such as ketoconazole, clotrimazole, and miconazole have been associated with QT interval prolongation and torsades de pointes (Monahan et al., 1990). A few reports have attributed miconazole-induced cardiac arrhythmias to a rapid intravenous administration with insufficient dilution (Huijgens et al., 1975; Fainstein and Bodey, 1980; Coley and Crain, 1997). The mechanism underlying the antimycotic-induced rhythm changes is unknown. Clotrimazole has been shown to inhibit L-type calcium current in guinea pig ventricular myocytes (Thomas et al., 1999), but this effect is unable to account for the QT prolongation. A short report suggested that ketoconazole also has a direct blocking effect on the delayed rectifier and transient outward currents in feline ventricular myocytes (Chen and Woosley, 1993), but these effects on K⁺ currents have not been examined in detail. Our study indicates that I_slow is a potential target to explain the toxic effects of the drugs. Although under our experimental conditions the antimycotics readily suppressed action potentials, indicating that the effects on other (Na⁺, Ca²⁺) channels may play a more critical role in the arrhythmogenesis, in conditions where excitability is not fully suppressed, the I_to suppression by antimycotics is likely to contribute to the arrhythmogenesis by causing action potential prolongation and increasing action potential dispersion in the ventricle. An effect on human ether-a-go-go-related gene-related native currents (Dumaine et al., 1998) could constitute an additional factor, but it was not examined in the present study.

As for glybenclamide, its effects on outward currents have been reported in human cardiomyocytes (Schaffer et al., 1999). In atrial cells, peak amplitude was decreased and inactivation was accelerated, as also found in our study on mouse ventricular cells. These results were interpreted as caused by a suppression of I_to,1 and I_Kur (Schaffer et al., 1999). In contrast to the decrease of the end-of-pulse current (interpreted as an effect on I_ss) in atrial cells, we did not find such an effect in mouse ventricular cells. However, since short (0.3-s) pulses were used in the atrial study, it is possible that the end-of-pulse current contained noninactivated I_slow or I_Kur. The noted acceleration of I_to inactivation could result from a selective suppression of a slow component with no effect on the fast component. This seems not to be the case for glybenclamide, for which the exponential analysis indicates an effect on both slow and fast components. In the present study, we show that glybenclamide shifts the inactivation curve of I_to in the negative direction, hence suggesting that it interacts with channels in the inactivated state.

It is now established that glybenclamide acts on many channels, and that its selectivity for I_K-ATP channels is only obtained at submicromolar concentrations (Schaffer et al., 1999). Since the IC₅₀ for the effect on I_to is about 50 µM, plasma concentrations usually reached under well controlled therapy are unlikely to cause cardiac complications due to an effect on I_to. Many ion channels (swelling-activated, cAMP-activated, Ca²⁺-activated, etc.; Schaffer et al., 1999) on which glybenclamide acts at high concentrations do not play a role in the basal cardiac electrical activity. In contrast, I_to participates to the normal repolarization of the cardiac action potential, hence the potential effect of glybenclamide on this current requires that higher concentrations be avoided both under conditions where a selective effect on I_K-TP is desired.