Introduction

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Vesnarinone is a phosphodiesterase (PDE) inhibitor and used for treatment of patients with congestive heart failure (Taira et al., 1984; Yamashita et al., 1984). Although most other PDE inhibitors improved the morbidity rate but not mortality rate of congestive heart failure patients, it was reported that vesnarinone improved both morbidity and mortality rates when orally administered at low doses (Feldman et al., 1993). In addition to PDE inhibition, vesnarinone prolongs the cardiac action potential duration by inhibiting the delayed outward K⁺ currents, which may be partly responsible for the positive inotropic and negative chronotropic effects of the drug (Taira et al., 1984; Iijima and Taira, 1987; Rapundalo et al., 1988; Asanoi et al., 1989). Therefore, this drug is thought to improve not only the contraction force but also the relaxation of the diseased heart. The delayed outward K⁺ current, I_K, initiates repolarization of the cardiac action potential and thus controls its duration. In most mammalian species, including guinea pig and humans, I_K is made up of two distinct components, the rapidly and slowly activating K⁺ currents, I_Kr and I_Ks, respectively (Sanguinetti and Jurkiewicz, 1990, 1991; Wang et al., 1994; Li et al., 1996). The ether-a-go-go related gene in humans (HERG) expresses a K⁺ channel current with biophysical characteristics similar to those of I_Kr (Sanguinetti et al., 1995; Trudeau et al., 1995). Although recently it was indicated that a minK-related peptide 1 (MiRP1) coassembles with HERG to reconstitute I_Kr, the kinetic properties of HERG current with and without the protein do not largely alter (Abbott et al., 1999). However, the coexpression of minK with KvLQT1 is mandatory to reconstitute I_Ks (Barhanin et al., 1996; Sanguinetti et al., 1996; Yang et al., 1997).

In this study, we examined the effects of vesnarinone on the currents flowing through HERG and KvLQT1/minK channels expressed in HEK293T cells. HERG current was inhibited by vesnarinone with an IC₅₀ of 1.1 µM, whereas KvLQT1/minK current was not significantly depressed by the drug even at 30 µM. These results suggest that vesnarinone would prolong the cardiac action potential by specifically inhibiting I_Kr. Detailed examination of the kinetics of the effect of vesnarinone has revealed that this compound inhibits HERG current in a voltage- and time-dependent manner. The recovery from inhibition at the resting level was relatively rapid, which caused the steady-state level of block to become more prominent at higher stimulation frequency. These properties could be explained exclusively by the voltage- and time-dependent kinetics of the drug-channel interaction.

	Materials and Methods

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Transfection and Cell Culture. HERG and human KvLQT1 cDNAs were kindly provided by Drs. M. T. Keating and M. C. Sanguinetti (University of Utah, Salt Lake City, UT). The human minK cDNA was obtained by polymerase chain reaction from genomic DNA of human whole blood (Murai et al., 1989). They were each subcloned into an expression vector pcDNA3 (Invitrogen, San Diego, CA). HEK293T cells were transfected with pcDNA3-HERG or cotransfected with pcDNA3-KvLQT1 and pcDNA3-minK by using LipofectAMINE (Life Technologies, Gaithersburg, MD) according to the manufacturer's instructions. Electrophysiological measurements were usually conducted 2 to 4 days after transfection.

Electrophysiological Recordings and Analysis. The channels expressed in HEK293T cells were recorded with the whole-cell variant of the patch-clamp method. Experiments were conducted at room temperature (22-25°C). A dish containing cells was placed on the stage of an inverted microscope and superfused continuously with the standard extracellular solution. The electrodes filled with the internal solution had a resistance of 2 to 5 M after coating with silicon and being fire polished. Currents were measured with a patch-clamp amplifier (Axopatch 200A; Axon Instruments, Inc., Foster City, CA) and monitored with an analog-storage oscilloscope (dual beam storage oscilloscope; Tektronix, Inc., Beaverton, OR). For subsequent analysis, currents were recorded on videocassette tapes by using a PCM converter (VR-10B; Instrutech Corp., Mineola, NY). For analysis, the data were reproduced, low-pass filtered at 1.0 kHz (3 decibels) with an 8-pole Bessel filter (Frequency Devices, Haverhill, MA), and digitized at 3 or 5 kHz with an analog-to-digital converter (ITC-16; Instrutech Corp.). These data were analyzed off-line on a computer (Macintosh Quadra 700; Apple Computer Inc., Cupertino, CA) with commercially available programs, i.e., Pulse Program (HEKA Electronik, Lambrecht, Germany) and Patch Analyst Pro (MT Corporation, Hyogo, Japan). A microperfusion system allowed local application and rapid change of the different experimental solutions. HEK293T cells per se expressed a voltage-dependent K⁺ current, which was insensitive to dofetilide. Because dofetilide (3 µM) completely suppressed HERG current (data not shown), we applied the drug to the cells at the end of each experiment. The HERG currents were defined as the dofetilide-sensitive components of the membrane currents in the HEK293T cells transfected with HERG cDNA. Results were expressed as mean ± S.E. Student's t test was used to compare differences between mean values, with a value of P < .05 being considered significant.

Solutions and Chemicals. For whole-cell recording, the pipettes were filled with "internal" solution containing 140 mM KCl, 2 mM MgCl₂, 5 mM K₂ATP, 5 mM EGTA-KOH, and 5 mM HEPES-KOH (pH 7.3 with KOH). The bath was perfused with a control bath solution containing 136.5 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.53 mM MgCl₂, 5.5 mM glucose, and 5.5. mM HEPES-NaOH (pH 7.4 with NaOH). Vesnarinone was a gift from Otsuka Pharmaceutical Co. (Tokyo, Japan) and dissolved at 30 mM in dimethyl sulfoxide. Dofetilide was a gift from Pfizer Pharmaceutical Inc. (New York, NY) and dissolved at 10 mM in dimethyl sulfoxide. Other chemicals and materials were obtained from commercial sources. Dimethyl sulfoxide, at the concentrations used herein and which never exceeded 1:10,000, had no significant effect on any of the parameters measured in these studies (n = 3).

	Results

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Effects of Vesnarinone on KvLQT1/minK and HERG Channel Currents. We first compared the effects of vesnarinone on the K⁺ currents in HEK293T cells expressing KvLQT1/minK channels (Fig. 1A) and HERG channels (Fig. 1B). The cells expressing KvLQT1/minK channels were voltage clamped at 80 mV and voltage steps (7.5-s duration) were applied to potentials between 80 and +40 mV in 20-mV increments. The tail currents were recorded on repolarization to 50 mV. Under control conditions, voltage steps evoked a slowly activating outward K⁺ current (Fig. 1Aa). The threshold potential was ~20 mV, and the amplitude of the currents increased linearly as the membrane was depolarized. The amplitude of the tail current increased with depolarization and reached a maximum value after a voltage step to +40 mV. These properties are the same as those previously reported for KvLQT1/minK channels (Barhanin et al., 1996; Sanguinetti et al., 1996). The KvLQT1/minK channel currents were unaffected by the application of vesnarinone (1 ~10 µM) to the bath. Even in the presence of 30 µM vesnarinone, the K⁺ current was only slightly depressed without significant alteration of the activation time course (Fig. 1Ab). The amplitudes of peak tail current recorded after voltage steps to +40 mV in the control condition and in the presence of 30 µM vesnarinone were 280.6 ± 34.7 and 251.7 ± 29.0 pA, respectively (n = 4).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Effects of vesnarinone on KvLQT1/minK and HERG channel currents. A, representative traces of KvLQT1/minK currents in control conditions (a) and in the presence of 30 µM vesnarinone (b). KvLQT1/minK current was only slightly reduced by 30 µM vesnarinone. B, representative traces of HERG current in control conditions (a) and in the presence of 1 µM vesnarinone (b). HERG current was inhibited by ~50% by 1 µM vesnarinone. C, vesnarinone inhibited HERG tail current in a concentration-dependent manner. Tail current was elicited following the 4-s depolarizing pulse to +20 mV. The line represents fitting of the data with a Hill equation with an IC50 of 1.06 µM and Hill coefficient of 1.22.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   HERG I-V relationships in the absence () and presence of 1, 3, and 10 µM vesnarinone (, , and , respectively). A, HERG current amplitude at the end of the voltage step was plotted against membrane potential. B, I-V relationships of the peak tail current amplitude were fit with the Boltzmann function (lines). C, voltage-dependent block of HERG current by vesnarinone. The tail current amplitude in the presence of vesnarinone is shown relative to that under control conditions.

Voltage Dependence of Vesnarinone Block of HERG Channel Currents. The current-voltage relationship at the end of the 4-s depolarizing voltage steps was plotted in the absence and presence of 1, 3, or 10 µM vesnarinone (Fig. 2A). In the control, the HERG current increased with depolarization from ~50 mV and reached a peak at 0 mV. Due to fast C-type inactivation, it decreased with further depolarization. In the presence of vesnarinone, the current level was suppressed at potentials more positive than 40 mV in a concentration-dependent fashion. The peak of the current-voltage (I-V) curve also slightly shifted to the left, suggesting that the effect of vesnarinone may be more potent at more depolarized potentials.

Time Dependence of Effects of Vesnarinone on HERG Currents. The activation level of the HERG current was assessed from the amplitude of the tail currents at 60 mV after voltage steps to +20 mV with various durations (0.08-5.12 s). In the control, the tail current amplitude increased with prolonging the duration of the voltage step. Its time course was in parallel with the increase of the HERG current during the voltage step (Fig. 3Aa). In the presence of 1 µM vesnarinone, the activation of HERG, which was assessed by the tail current amplitude, increased in the beginning but decreased gradually as the duration of the voltage step was prolonged (Fig. 3Ab). Therefore, the decrease of the HERG current during the voltage pulse to +20 mV was due to channel inhibition by the drug.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Time-dependent block of HERG current by vesnarinone. A, voltage-clamp protocol is shown schematically at top. Families of traces were obtained in control conditions (a) and in the presence of 1 µM vesnarinone (b). B, relative tail current amplitude in the presence of vesnarinone with reference to the control was plotted against test pulse duration and fitted by a single exponential curve (line) with a time constant of 0.89 s.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Voltage dependence of the time courses of vesnarinone-induced inhibition. A, superimposed traces recorded during voltage steps to 20 (top) and +20 mV (bottom) in control conditions and the presence of 1 µM vesnarinone. The development of block was increased as the pulse potential was depolarized. B, ratios of currents recorded in the presence and the absence of vesnarinone could be fitted by single exponential curves (solid lines). The decay time constant () and the steady-state ratio at the end of the depolarizing step (Po) decreased with depolarization until they saturated at 0 to +20 mV.

View larger version (33K): [in this window] [in a new window]   Fig. 5.   Analysis of vesnarinone block of HERG channel currents. A and B, steady-state ratios (Po) and the decay time constants () were estimated from the fitted curves in the presence of 1, 3, and 10 µM vesnarinone (, , and , respectively) as shown in Fig. 4. C and D, unbinding rate constants () and the apparent binding rate constants (*) were calculated from Po and : Po = /( + *),  = 1/( + *). The magnitude of  was almost constant with a mean value of 0.49 ± 0.02 (C, solid line). When we assumed * as [D], the magnitude of  could be fitted by a Boltzmann equation with V1/2 of 16.5 mV and k of 12.6: * =  × [D],  = 4.1 × 105/{1 + exp [(16.5  V)/12.6]}. The limits of S.E.M. did not exceed 50% and is not depicted for the sake of clarity.

1

Recovery from Block and Use Dependence of Vesnarinone. We examined recovery from vesnarinone-induced inhibition with a double-pulse voltage-clamp protocol (Fig. 6A). The amplitude of the tail current was examined on repolarization to 80 mV after the second voltage step. When the interval was 0 s, the tail current was suppressed by vesnarinone (1 µM) by 45.4 ± 1.4% (n = 4). When the interval was 5.12 s, it was suppressed by 16.9 ± 4.8% (n = 4). As shown in Fig. 6B, the amplitude of the tail current in the presence of 1 µM vesnarinone increased with a time constant of 1.75 s. This is similar to the value of 1/( + *) at 80 mV of 1.91 s, which was calculated from the voltage-dependent kinetics of vesnarinone block obtained in Fig. 5. This suggests that the fast recovery from vesnarinone block at 80 mV is derived from the same process as the inhibition of HERG current at depolarized potentials.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Recovery of HERG channels from vesnarinone block at 80 mV. A, double-pulse voltage clamp is illustrated schematically. Below are superimposed traces of tail currents recorded in control conditions () and in the presence of 1 µM vesnarinone () with interpulse intervals of 0 or 2.56 s. B, ratios of tail current in the presence of the drug to the control conditions were plotted against the interpulse intervals. Recovery from block was observed in the presence of vesnarinone (). The solid line was obtained by fitting a single exponential function with a time constant of 1.75 s, which was very similar to that of the time constant calculated from the kinetic analysis (1.91 s).

View larger version (27K): [in this window] [in a new window]   Fig. 7.   Use-dependent block of HERG current was tested at intervals of 1, 3, and 12 s (, , and , respectively). The voltage-clamp protocol is illustrated schematically. Ratios of peak tail current in the presence of 1 µM vesnarinone compared with those of the control were plotted against time after the first pulse. The final amount of steady-state block was lower at lower frequencies than at higher frequency. The limits of S.E.M. did not exceed 5% and are not depicted for the sake of clarity.

	Discussion

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In this study, the effects of vesnarinone on HERG and KvLQT1/minK channel currents were examined in a mammalian cell line. We found that the drug specifically inhibited the HERG current in a concentration-dependent manner. This is consistent with the reports in native cardiac myocytes that vesnarinone inhibits I_K (Iijima and Taira, 1987; Toyama et al., 1997). In rabbit cardiac myocytes, Toyama et al. (1997) showed that vesnarinone exhibited time-dependent block of I_K at the depolarized potential of +10 mV ( = 0.36 s), rapid recovery from block at a holding potential of 75 mV ( = 1.87 s) and an increase of the block with increasing frequency of stimulation (0.2-2 Hz). All of these properties were reproduced on the HERG current in this study.

We analyzed the details of the kinetic properties of vesnarinone-induced inhibition of HERG current, which could be described as a first order reaction between the drug-free and drug-bound channels. The reaction would occur independently of the gating of the HERG channel because the time course of the effect of vesnarinone during voltage steps could be fitted by single exponential curves. The binding rate constant of the drug to the channel increased as the membrane was depolarized, whereas the unbinding rate constant remained the same irrespective of the membrane potential. The recovery from block- and frequency dependence of the inhibition could be approximated from the voltage- and concentration dependence of the rate constants. This may indicate that this drug can access and leave the channel irrespective of the state of channel gating, suggesting that this drug uses the hydrophobic pathway to access the channel. This is in contrast to the properties of classical I_Kr blockers, such as dofetilide and E-4031 (Carmeliet, 1992; Snyders and Chaudhary, 1996; Spector et al., 1996). These drugs are so-called open channel blockers. They mainly use the hydrophilic pathway and thus enter and leave the HERG channel pore only when the channel is open. Because of this property, the recovery from drug-induced channel inhibition is very slow and the steady-state inhibition is the same irrespective of the stimulation frequency. This is thought to be one of the reasons why the classical I_Kr blockers show reverse frequency dependence in evoking prolongation of the cardiac action potential. Because vesnarinone inhibited the HERG current more prominently as the stimulation frequency was increased, this drug may not exhibit reverse frequency dependence. In addition, the kinetics of the drug effects may indicate a 1:1 interaction between the drug and the site, which was consistent with a Hill coefficient (~1) in the concentration-dependent curve. Because it is difficult to reach a definitive conclusion merely from the electrophysiological data, further studies examining the binding of the drug to the channel are needed.

Vesnarinone inhibited the HERG current more effectively as the membrane was depolarized. Because vesnarinone exists mostly in a neutral form at physiological pH (Shimizu et al., 1984), this voltage dependence cannot be explained by an interaction between a charged blocker and a binding site in the channel pore and other possibilities should be considered. Because the voltage dependence of the binding rate constant, V_1/2 of 16.5 mV and k of 12.6, and that of HERG activation, V_1/2 of 22.9 mV and k of 8.94, were similar, there may be some correlation between the two. However, because the drug-induced inhibition of the current could be described by a first order reaction that was independent of gating, the activation gating of the HERG channel itself seems not to be involved. Furthermore, it is likely that the drug exclusively uses the hydrophobic pathway to access to the channel. To explain these properties of vesnarinone-inhibition, we propose that it is possible that a HERG channel whose activation gate is open has a conformation that allows the drug to bind to the channel. This hypothesis would explain why the voltage dependence of the binding rate constant of the drug to the channel coincides with that of activation gating of the HERG channel, without direct interaction of the drug with the channel gate. Further studies are needed to examine the validity of this hypothesis.

Recently, it was shown that E-4031 inhibits the channels formed by coassembly of MiRP1 with HERG with a biphasic time course similar to that in native cardiac I_Kr channels, whereas this drug inhibits the channels formed by HERG alone in a monophasic time course (Abbott et al., 1999). However, the kinetics of vesnarinone inhibition of the homomeric HERG channel current obtained in this study was very similar to that reported in I_Kr in native cardiac myocytes (Toyama et al., 1997). Therefore, it is expected that the properties of vesnarinone inhibition of HERG current would not be significantly altered by coexpression of MiRP1.

Concentrations of vesnarinone in clinical use range from 4.9 to 10.1 mg/l, i.e., from 12.4 to 25.5 µM (Feldman et al., 1988), and 80% of vesnarinone are bound by plasma proteins (Miyamoto and Sasabe, 1984). The IC₅₀ value for vesnarinone inhibition of HERG channels is ~1 µM. The IC₅₀ for vesnarinone inhibition of PDE is reported to be ~300 µM (Taira et al., 1984). Therefore, the plasma concentration of the drug in clinical use is not high enough to cause PDE inhibition but sufficient to inhibit I_Kr and the expressed HERG current. This suggests that in the clinical use the inhibition of I_K, but not an increase of the inward Ca²⁺ current, may be mainly responsible for the inotropic action of this drug (Iijima and Taira, 1987).

In summary, this study showed that the prolongation of the cardiac action potential induced by vesnarinone can be attributed to its block of HERG channels, but not KvLQT1/minK channels. The characteristics of this compound are not associated with reverse use dependence, which might be beneficial to the antiarrhythmic effect. In addition, kinetic analysis indicates that vesnarinone may access the HERG channel via the hydrophobic pathway and recognize the open-state conformation. This might be the reason why vesnarinone shows a voltage dependence of its effect in spite of its uncharged form.