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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.,
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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
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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.
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)
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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-
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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,
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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
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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.
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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
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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.
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