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0022-3565/01/2982-598 –606$3.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics JPET 298:598–606, 2001 Vol. 298, No. 2 3646/918887 Printed in U.S.A. Suppression of Transient Outward Potassium Currents in Mouse Ventricular Myocytes by Imidazole Antimycotics and by Glybenclamide M. J. HERNANDEZ-BENITO,1 R. MACIANSKIENE,1 K. R. SIPIDO, W. FLAMENG, and K. MUBAGWA Laboratory of Cardiac Cellular Research, Centre for Experimental Surgery and Anaesthesiology (M.J.H.-B., R.M., W.F., K.M.), and Laboratory of Experimental Cardiology (K.R.S.), University of Leuven, Leuven, Belgium Received December 11, 2000; accepted April 26, 2001 This paper is available online at http://jpet.aspetjournals.org 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 This study was supported by Grant 0299.98 for FWO, the Flemish Foundation for Science. Published abstracts on parts of this work are as follows: Hernandez MJ, Sipido KR and Mubagwa K (1999) High sensitivity to 4-aminopyridine of the transient outward current in mouse ventricular myocytes. Biophys J 76:A88; Hernandez MJ, Sipido KR and Mubagwa K (1999) Outward currents in mouse cardiomyocytes. Pfluegers Arch 437:R9; Hernandez MJ, Sipido KR and Mubagwa K (1999) Identification of two transient outward current components in mouse cardiomyocytes by the imidazole antimycotics clotrimazole and miconazole. Pfluegers Arch 438:R31; and Macianskiene R, Moccia F, Sipido K and Mubagwa K (2001) Glybenclamide inhibits transient outward potassium currents in mouse ventricular myocytes. Pfluegers Arch, in press. 1 These authors contributed equally to this study. ABBREVIATIONS: 4-AP, 4-aminopyridine. 598 the noninactivating current. The effect did not reverse upon washout, was not induced by intracellular drug application, and occurred without a change of the steady-state inactivation. In the presence of glybenclamide Ito peak amplitude was reduced and its inactivation accelerated. In contrast to the antimycotics, glybenclamide suppressed both the fast and the slow components (IC50 of ⬇50 M), its effect was reversible, and was associated with a negative shift of the steady-state inactivation. These data demonstrate a pharmacological separation of Ito components using antimycotic drugs but not glybenclamide. (ⱕ50 M) 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-APsensitive 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., Downloaded from jpet.aspetjournals.org at ASPET Journals on May 10, 2017 ABSTRACT The whole-cell patch-clamp technique was used in adult mouse ventricular myocytes at 22°C to study the transient outward current (Ito) and its sensitivity to the antimycotics miconazole and clotrimazole, as well as to glybenclamide. Ito elicited by depolarizing steps from a holding potential of ⫺80 mV consisted of a fast inactivating component and a slowly inactivating component. In the presence of miconazole (IC50 of ⬇8 M) or clotrimazole, Ito peak amplitude was reduced and its inactivation accelerated, due to a selective suppression of the slow component, without an effect on the fast component or on Miconazole, Clotrimazole, and Glybenclamide Effects on Ito 1999), we wanted to further examine the effect of this drug and to compare them with those of the antifungal drugs. Materials and Methods Itotal ⫽ Ifast ⴱ exp共⫺t/fast兲 ⫹ Islow ⴱ exp(⫺t/slow) ⫹ I⬁ where Ifast and Islow are the amplitudes of the fast decaying and slowly decaying components, respectively, fast and slow their respective time constants, and I⬁ the magnitude of the time-independent (noninactivating) component. To measure steady-state inactivation, prepulses lasting 5 s were given from the holding level to various potentials (between ⫺120 and ⫹70 mV, in 10-mV steps) before depolarizing to a test potential of ⫹60 mV. Our preliminary experiments with prepulses lasting 0.4 to 2 s indicated that while such prepulses allowed steady-state inactivation of the fast component, longer prepulses were needed to allow steady-state inactivation of the slower component. Assuming full inactivation after a 5-s prepulse to the positive potentials, the lowest current at ⫹60 mV following these prepulses was taken as baseline level. The total timedependent current was measured as difference between peak current following a given prepulse and this baseline level, and was normalized relative to the current following the most negative prepulse (⫺120 or ⫺100 mV). Similarly, the amplitudes of the fast (Ifast) and slow (Islow) components at ⫹60 mV following prepulses to various levels were obtained by exponential fitting and were normalized to the amplitude of the corresponding component following a prepulse to ⫺120 or ⫺100 mV. Normalized availability or inactivation curves were fitted using one single Boltzmann distribution function: availability ⫽ 兵1 ⫹ exp关共V ⫺ V1/ 2兲兴/k其 ⫺ 1 (or a sum of two such functions) where V1/2 stands for the potential of half-maximum inactivation, and k for the slope factor. Functions were fitted to data using Clampfit (Axon Instruments) or Origin (MicroCal, Northampton, MA). Average data are expressed as mean ⫾ S.E.M. Statistical comparison was made using a twotailed t test. 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 Different Kinetic Components of Ito. 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 M 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 M 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 (Ifast), of a slowly inactivating component (Islow), and a noninactivating component (I⬁). The whole time-dependent current (sum of Ifast and Islow) will be referred to as Ito. Ito magnitude decreased when prepulses were given before the depolarization to ⫹60 mV (Fig. 8A). With 5-s prepulses, the availability (or “inactivation”) curve could be fitted by a Downloaded from jpet.aspetjournals.org at ASPET Journals on May 10, 2017 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. Single ventricular myocytes were obtained from adult mice. The animals were heparinized (250 IU, given intraperitoneally) and anesthetized with sodium pentobarbitone (Nembutal; 150 –300 mg kg⫺1, given intraperitoneally). The excised heart was cannulated via its aorta, mounted on a Langendorff system and perfused at 37°C and at constant flow (2.5 ml/min) for 2 to 5 min with an oxygenated normal Tyrode’s solution. The heart was then perfused with Ca2⫹free Tyrode’s solution for 5 to 10 min, followed by a 15- to 20-min perfusion with a Ca2⫹-free Tyrode’s solution containing 0.14 mg ml⫺1 protease (type XIV; Sigma, St. Louis, MO) and 0.5 mg ml⫺1 collagenase (type A; Roche Molecular Biochemicals, Mannheim, Germany), and a 10-min washing perfusion with Tyrode’s solution in which the [Ca2⫹] was raised stepwise from 0.09 to 0.18 mM. The ventricle was cut into a few pieces in the 0.18 mM Ca2⫹ Tyrode’s solution and cells were dispersed by gentle mechanical agitation. The cells were stored in the same solution at room temperature (21–22°C). Ca2⫹-tolerant rod-shaped ventricular myocytes with clear striations were selected randomly for the electrophysiological studies. 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, ICa, was detectable in a small percentage of cells but was negligibly small, especially at positive potentials (⬍100 pA). Because of the small magnitude of ICa and the fact that typical blockers of L-type Ca2⫹ channel also block Ito (Gotoh et al., 1991) we decided not to use any Ca2⫹ channel blocker. The time-dependent decay of the outward currents was fitted by a sum of exponentials: 599 600 Hernandez-Benito et al. single Boltzmann distribution curve (see Figs. 5 and 8B). The potential for half-maximum inactivation (V1/2) was ⫺52 ⫾ 0.7 mV and the slope factor of the inactivation curve was 11.5 ⫾ 0.25 mV (n ⫽ 11). However, in experiments carried out at a preliminary stage of this study, in which 1 s or shorter prepulses were used, a sum of two Boltzmann equations (with V1/2 of ⫺53 and ⫺22 mV) was needed to fit the inactivation curve, presumably as a result of an incomplete inactivation of Islow. Under such conditions, after application of 25 to 50 M 4-AP, one single Boltzmann distribution (V1/2 of ⫺52 mV) satisfactorily fitted the inactivation curve (data not shown), hence supporting the view that low 4-AP concentrations preferentially suppress a slow Ito component. Preferential Block of Islow by Miconazole and Clotrimazole. Figure 2, A and B, show the effect of miconazole (30 and 100 M) and clotrimazole (30 M), respectively, on Ito induced by steps to ⫹60 mV. The peak amplitude of Ito 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 M 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 endof-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) Downloaded from jpet.aspetjournals.org at ASPET Journals on May 10, 2017 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 M 4-AP are superimposed. B, difference between current traces in the absence and in the presence of 50 M 4-AP. Inset, semilogarithmic plot of the difference current. Notice one single (slow) component of inactivation. such as those of Fig. 2C are summarized in Fig. 2D, which confirm that 10 M miconazole decreased Ito (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 IK1 (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. The miconazole- or clotrimazole-sensitive currents decayed monoexponentially (insets of Fig. 2, A and B, respectively), and their time constants (865 and 1044 ms, respectively) were of the same order of magnitude compared with the time constants of the slow component of the control current (in the same cells: 1125 and 1361 ms, respectively) and with that of the 4-AP-sensitive current (see above; Fig. 1B). This suggests that antimycotics caused an inhibition of Islow and that the fast-decaying current remaining in the presence of the antimycotics is due to Ifast. The effect on channels underlying Islow could develop in the rested state, but similar results can also be obtained if, as a result of a channel block in the open or inactivated state (Carmeliet and Mubagwa, 1998), Islow is modified so as to decay with a time constant close to and indistinguishable from that of Ifast. We therefore tested whether the block of Islow is time-dependent during the voltage pulse, by isolating this component from the superimposed Ifast. Islow can be isolated by depolarizations to potentials greater than ⫺30 mV that are long enough to fully inactivate Ifast but short to only partly inactivate Islow. Figure 3A compares the effect of miconazole on the current at ⫹60 mV elicited after a prepulse to ⫺90 mV (Fig. 3Aa) or after a 1-s prepulse to ⫺30 mV (Fig. 3Ab). With the prepulse to ⫺30 mV, the fast component was fully inactivated and the transient component induced at ⫹60 mV was essentially due to Islow. Under these circumstances, the current at ⫹60 mV in the presence of miconazole did not show a fast decay (Fig. 3Ab; in contrast to the current generated following the prepulse to ⫺90 mV, Fig. 3Aa), suggesting the absence of a slowly developing open or inactivated channel block on this component. These data indicate that the antimycotic blocked Islow by interacting with the underlying channels in the rested state. To further examine the preferential effect on Islow, the effect of the antimycotics was tested in the presence of 50 M 4-AP to suppress this current. Figure 3B illustrates that when clotrimazole was applied on top of 50 M 4-AP, no effect was obtained on the fast-decaying current besides the further elimination of a small persisting slow component probably incompletely blocked by 4-AP. To quantitatively assess the extent of drug-induced change in the Ito components, the currents in the absence or in the presence of the antimycotics were fitted by a sum of two exponentials. The magnitude of each component under steady state (after 10 –20 min) in the presence of the drug was expressed relative to its magnitude in control conditions. The relative magnitude of the two Ito components in the presence of various miconazole concentrations is plotted in Fig. 4. This analysis indicated that the slow component was selectively decreased by miconazole. The half-maximum inhibitory concentration (IC50) for the slow component was 7.0 M (nHill ⫽ 0.95). Time constants and amplitudes of the fast and slow components, as well as of the offset (noninactivating Miconazole, Clotrimazole, and Glybenclamide Effects on Ito 601 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 M 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 M 4-AP to isolate Ifast. Current traces obtained at ⫹60 mV in control (a), in the presence of 50 M 4-AP (b), and in the presence of 30 M clotrimazole added on top of 4-AP (c). component), under control conditions and in the presence of 30 M miconazole or 30 M clotrimazole are presented in 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. Table 1. Changes in the magnitude of the fast component were absent or relatively less marked compared with the effect on the slow component. Although a complete concen- Downloaded from jpet.aspetjournals.org at ASPET Journals on May 10, 2017 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 M; A) or clotrimazole (30 M; 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 M 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 M miconazole. *P ⬍ 0.05 for miconazole versus control. 602 Hernandez-Benito et al. TABLE 1 Effect of miconazole, clotrimazole, and glybenclamide on fast and slow current components Components of Ito were resolved by fitting the current decay with a sum of two exponentials (Materials and Methods). Data are given as mean ⫾ standard error of the mean. Amplitude Time Constants n Ito component control drug control pA/pF drug ms Miconazole (30 M) fast slow offset 30.8 ⫾ 5.00 31.0 ⫾ 4.00 15.4 ⫾ 2.63 28.4 ⫾ 3.36 7.3 ⫾ 1.64* 17.4 ⫾ 6.92 103 ⫾ 20.3 1145 ⫾ 100.2 32 ⫾ 5.2* 988 ⫾ 190.7 6 Clotrimazole (30 M) fast slow offset 15.3 ⫾ 3.89 15.4 ⫾ 1.12 11.6 ⫾ 2.75 15.8 ⫾ 3.78 10.4 ⫾ 2.50* 11.1 ⫾ 3.09 104 ⫾ 18.7 1291 ⫾ 39.7 58 ⫾ 12.1* 1771 ⫾ 256.4 4 Glybenclamide (50 M) fast slow offset 25.5 ⫾ 3.40 24.2 ⫾ 4.22 15.7 ⫾ 1.95 11.0 ⫾ 1.29* 10.6 ⫾ 1.98* 12.9 ⫾ 1.84 102 ⫾ 14.7 1167 ⫾ 82.2 61 ⫾ 7.0* 1405 ⫾ 187.1 9 * P ⬍ 0.05 versus control using two-tailed paired t test. Fig. 5. Lack of effect of miconazole and clotrimazole on steady-state inactivation. A and B, availability curves in the absence (E) and in the presence (F) of 30 M miconazole (A) or 30 M clotrimazole (B). Data were fitted by Boltzmann distribution functions (under Materials and Methods). tion potential due to the different intracellular solution composition: KCl for miconazole experiments and K-glutamate for clotrimazole). Due to the lack of reversibility of the miconazole effect (Fig. 2C), we tested whether the drug was acting on an internal site by adding 30 M miconazole to the patch pipette solution. The slow component of Ito was not suppressed in three cells dialyzed with the drug in this way, and the cells still responded to extracellularly applied miconazole (data not shown). This result excludes the possibility that the miconazole effect was due to an interaction with some intracellular site from which it was difficult to remove the drug by extracellular washout. Block of Both Ifast and Islow by Glybenclamide. Given the reported acceleration of Ito inactivation by glybenclamide (Schaffer et al., 1999), we examined whether the drug acts in the same way as the antimycotics by preferentially suppressing Islow. Figure 6A illustrates the effect of glybenclamide (100 M) 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 IK1 (Fig. 6, C and D). In contrast, the drug caused a decrease of the peak amplitude of Ito 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 M 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 Ito peak occurred in the absence of significant effect on the current (largely IK1) at negative potentials or on the noninactivating current at depolarized levels (current at ⫺120 mV: ⫺30 ⫾ 3.0 pA/pF in control, Downloaded from jpet.aspetjournals.org at ASPET Journals on May 10, 2017 tration-effect curve was not established for clotrimazole, it decreased the slow component by 32% at 30 M (n ⫽ 4). To further examine the possibility of a block in the inactivated state, we analyzed the effect of the antimycotics on the steady-state inactivation of Ito. Figure 5 shows that the inactivation curve was unchanged in the presence of either 30 M miconazole (Fig. 5A; n ⫽ 4) or 30 M clotrimazole (Fig. 5B; n ⫽ 4). For the data with miconazole, the potential of half-maximum inactivation (V1/2) was ⫺51.9 ⫾ 1.3 and ⫺53.7 ⫾ 1.3 mV (P ⬎ 0.05) before and during application of the drug. Similarly, there was no significant shift in V1/2 in the presence of clotrimazole. (The differences in V1/2 between miconazole and clotrimazole data are explained by the junc- Miconazole, Clotrimazole, and Glybenclamide Effects on Ito 603 ⫺28 ⫾ 3.1 pA/pF during exposure to the drug; P ⬎ 0.05, n ⫽ 9). To assess the extent of drug-induced change in the Ito components, the currents at ⫹60 mV before drug application or in the presence of glybenclamide were fitted by a sum of two exponentials and one constant. The magnitude of the two Ito components in the presence of various glybenclamide concentrations is plotted in Fig. 7. The data indicate that glybenclamide concentration dependently decreased both the fast and the slow components of Ito, and that the IC50 values for the effect on both components were of the same magnitude (38 – 49 M glybenclamide). Time constants and amplitudes of the fast and slow components, as well as of the offset (noninactivating component), under control conditions and in the presence of 50 M glybenclamide are also included in Table 1. We also examined whether glybenclamide interacts with the inactivated channel. Figure 8A shows current traces recorded at ⫹60 mV following 5-s prepulses to various levels, in control conditions, in the presence of 50 M glybenclamide, and after washout of the drug. In the presence of glyben- clamide prepusles to ⫺60 mV and to ⫺50 mV caused a more marked depression of Ito, and this effect was reversible on drug washout. Figure 8B shows that the inactivation curve was shifted in the negative direction in the presence of glybenclamide: V1/2 of inactivation was ⫺52 ⫾ 1.3 mV in baseline conditions and was shifted to ⫺67 ⫾ 5.1 mV during application of glybenclamide (n ⫽ 6). These data indicate that the effect of glybenclamide were due, at least in part, to an enhancement of Ito inactivation. Effects on Action Potential. Given the prominent effects of either the antimycotics or glybenclamide on Ito, 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 M) without change in the resting potential (⫺81.4 ⫾ 1.60 mV). This effect was completely reversed with 5 to 10 min of drug Downloaded from jpet.aspetjournals.org at ASPET Journals on May 10, 2017 Fig. 6. Effect of glybenclamide on outward currents. A, superimposed currents, obtained in control and in the presence of 100 M 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 M 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 M 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 M glybenclamide. *P ⬍ 0.05 for miconazole versus control. 604 Hernandez-Benito et al. washout. Miconazole (n ⫽ 5) or clotrimazole (n ⫽ 1), each at 10 M, 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 The presence of distinct components of the inactivating outward current (here simply called Ito) 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 Ito was absent (London et al., 1998a,b). This prompted the authors to suggest that the slow component (which they called Islow) is encoded by Kv1.5. Barry et al. (1998) found that in cardiomyocytes from Kv4.2W362F mice the fast inactivating Ito (Ifast) 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, Islow was absent, hence demonstrating that Kv1.4 or a related protein is the molecular correlate of Islow. In the present study we have corroborated the presence of two Ito components. The decay of Ito 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 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 M glybenclamide (middle), and after washout of the drug (right). B, availability curves in the absence (E) and in the presence (F) of 50 M glybenclamide. Voltage clamp protocol as in A. Data were fitted by Boltzmann functions (under Materials and Methods). 10% of myocytes randomly dispersed from left and right ventricles. In our study, V1/2 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 V1/2 values obtained by different groups difficult. In the study of Zhou et al. (1998) V1/2 for the slow component was ⫺35 mV but they included 1 mM Ca2⫹, 1 mM Mg2⫹, and 2 mM Co2⫹ in the extracellular solution. V1/2 for a fast component was ⫺24 mV in the studies of Xu et al. (1999a,b), while for the slow component (IKslow) they found a biphasic inactivation curve with V1/2 values of ⫺73 and ⫺19 mV in the presence of 1 mM Ca2⫹, 2 mM Mg2⫹, and 5 mM Co2⫹. In contrast, a V1/2 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 Downloaded from jpet.aspetjournals.org at ASPET Journals on May 10, 2017 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). Miconazole, Clotrimazole, and Glybenclamide Effects on Ito 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 Islow 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 Ito 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 Ito,1 and IKur (Schaffer et al., 1999). In contrast to the decrease of the end-of-pulse current (interpreted as an effect on Iss) 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 Islow or IKur. The noted acceleration of Ito 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 Ito 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 IK-ATP channels is only obtained at submicromolar concentrations (Schaffer et al., 1999). Since the IC50 for the effect on Ito is about 50 M, plasma concentrations usually reached under well controlled therapy are unlikely to cause cardiac complications due to an effect on Ito. Many ion channels (swelling-activated, cAMPactivated, 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, Ito 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 IK-TP is desired. Acknowledgments We thank Patricia Holemans and Dr. F Moccia for assistance in the study. References Barry DM and Nerbonne JM (1996) Myocardial potassium channels: electrophysiological and molecular diversity. Annu Rev Physiol 58:363–394. Barry DM, Xu H, Schuessler RB and Nerbonne JM (1998) Functional knockout of the transient outward current, long-QT syndrome, and cardiac remodeling in mice expressing a dominant-negative Kv4 alpha subunit. Circ Res 83:560 –567. Benndorf K, Markwardt F and Nilius B (1987) Two types of transient outward currents in cardiac ventricular cells of mice. Pfluegers Arch 409:641– 643. Benndorf K and Nilius B (1988) Properties of an early outward current in single cells of the mouse ventricle. Gen Physiol Biophys 7:449 – 466. Downloaded from jpet.aspetjournals.org at ASPET Journals on May 10, 2017 slow component (IC50 of ⬇ 3 M) 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 Ito 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 Ito of mouse myocardial cells. The magnitude of Ito 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 Ito (Islow) as is seen in the presence of micromolar 4-AP concentrations. The IC50 (7 M) for Islow inhibition by miconazole is nearly one order of magnitude lower than for the ketoconazole inhibition of Kv1.5 expressed in oocytes (105 M). 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 Islow 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 Ito (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 Ifast. 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 605 606 Hernandez-Benito et al. of maxi-K currents in ferret portal vein smooth muscle cells by the antifungal clotrimazole. Am J Physiol 273:C45–C56. Rittenhouse AR, Vandorpe DH, Brugnara C and Alper SL (1997b) The antifungal imidazole clotrimazole and its major in vivo metabolite are potent blockers of the calcium-activated potassium channel in murine erythroleukemia cells. J Membr Biol 157:177–191. Schaffer P, Pelzmann B, Bernhart E, Lang P, Machler H, Rigler B and Koidl B (1999) The sulphonylurea glibenclamide inhibits voltage dependent potassium currents in human atrial and ventricular myocytes. Br J Pharmacol 128:1175–1180. Stengl M, Carmeliet E, Mubagwa K and Flameng W (1998) Modulation of transient outward current by extracellular protons and Cd2⫹ in rat and human ventricular myocytes. J Physiol (Lond) 511:827– 836. Thomas GP, Karmazyn M, Zygmunt AC, Antzelevitch C and Narayanan N (1999) The antifungal antibiotic clotrimazole potently inhibits L-type calcium current in guinea-pig ventricular myocytes. Br J Pharmacol 126:1531–1533. Wang L and Duff HJ (1997) Developmental changes in transient outward current in mouse ventricle. Circ Res 81:120 –127. Xu H, Barry DM, Li H, Brunet S, Guo W and Nerbonne JM (1999a) Attenuation of the slow component of delayed rectification, action potential prolongation, and triggered activity in mice expressing a dominant-negative Kv2 alpha subunit. Circ Res 85:623– 633. Xu H, Li H and Nerbonne JM (1999b) Elimination of the transient outward current and action potential prolongation in mouse atrial myocytes expressing a dominant negative Kv4 alpha subunit. J Physiol (Lond) 519:11–21. Yuan XJ, Tod ML, Rubin LJ and Blaustein MP (1995) Inhibition of cytochrome P-450 reduces voltage-gated K⫹ currents in pulmonary arterial myocytes. Am J Physiol 268:C259 –C270. Zhou J, Jeron A, London B, Han X and Koren G (1998) Characterization of a slowly inactivating outward current in adult mouse ventricular myocytes. Circ Res 83: 806 – 814. Address correspondence to: K. Mubagwa, Center for Experimental Surgery and Anaesthesiology, University of Leuven, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. E-mail: [email protected] Downloaded from jpet.aspetjournals.org at ASPET Journals on May 10, 2017 Carmeliet E and Mubagwa K (1998) Antiarrhythmic drugs and cardiac ion channels: mechanisms of action. Prog Biophys Mol Biol 70:1–72. Chen Y and Woosley RL (1993) Ketoconazole blocks potassium currents in feline heart (Abstract). Circulation 88:I-38. Coley KC and Crain JL (1997) Miconazole-induced fatal dysrhythmia. Pharmacotherapy 17:379 –382. Dumaine R, Roy ML and Brown AM (1998) Blockade of HERG and Kv1.5 by ketoconazole. J Pharmacol Exp Ther 286:727–735. Fainstein V and Bodey GP (1980) Cardiorespiratory toxicity due to miconazole. Ann Intern Med 93:432– 433. Fiset C, Clark RB, Shimoni Y and Giles WR (1997) Shal-type channels contribute to the Ca2⫹-independent transient outward K⫹ current in rat ventricle. J Physiol (Lond) 500:51– 64. Gotoh Y, Imaizumi Y, Watanabe M, Shibata EF, Clark RB and Giles WR (1991) Inhibition of transient outward K⫹ current by DHP Ca2⫹ antagonists and agonists in rabbit cardiac myocytes. Am J Physiol 260:H1737–H1742. Guo W, Xu H, London B and Nerbonne JM (1999) Molecular basis of transient outward K⫹ current diversity in mouse ventricular myocytes. J Physiol (Lond) 521:587–599. Hamill OP, Marty A, Neher E, Sakmann B and Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cellfree membrane patches. Pfluegers Arch 391:85–100. Hatton CJ and Peers C (1996) Effects of cytochrome P-450 inhibitors on ionic currents in isolated rat type I carotid body cells. Am J Physiol 271:C85–C92. Huijgens PC, Boeijinga JK and van der Meer J (1975) Possibly cardiotoxic action of miconazol injection fluid. Ned Tijdschr Geneeskd 119:1549 –1551. London B, Jeron A, Zhou J, Buckett P, Han X, Mitchell GF and Koren G (1998a) Long QT and ventricular arrhythmias in transgenic mice expressing the N terminus and first transmembrane segment of a voltage-gated potassium channel. Proc Natl Acad Sci USA 95:2926 –2931. London B, Wang DW, Hill JA and Bennett PB (1998b) The transient outward current in mice lacking the potassium channel gene Kv1.4. J Physiol (Lond) 509:171–182. Monahan BP, Ferguson CL, Killeavy ES, Lloyd BK, Troy J and Cantilena LR Jr (1990) Torsades de pointes occurring in association with terfenadine use. J Am Med Assoc 264:2788 –2790. Rittenhouse AR, Parker C, Brugnara C, Morgan KG and Alper SL (1997a) Inhibition