Introduction

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Vesnarinone inhibits phosphodiesterase (PDE) and has been used as a cardiotonic agent to treat chronic heart failure (Inoue et al., 1987; Asanoi et al., 1989; Feldman et al., 1993). Although PDE inhibitors greatly improve left ventricular function, most drugs with this activity have failed to decrease mortality in patients with heart failure. Vesnarinone is an exception and was reported to improve mortality rates when administered orally at a dose lower than that required to inhibit PDE or to have a positive inotropic action (Taira et al., 1984). Besides inhibiting PDE, vesnarinone inhibits potassium channels and prolongs the action potential duration (APD) of cardiomyocytes (Toyama et al., 1997; Yang et al., 1997). This additional action may contribute to the ability of vesnarinone to improve cardiac contractility.

We demonstrated previously that vesnarinone prolongs the APD of rabbit ventricular myocytes in a frequency-dependent manner (Toyama et al., 1997). This action was associated with frequency-dependent inhibition of the rapid delayed rectifier potassium channel, I_Kr. Recently, Katayama et al. (2000) reported that vesnarinone blocks HERG channels, but not the KvLQT1/minK current. These investigators found that the onset of HERG current inhibition by vesnarinone was faster and more prominent as the membrane potential became progressively depolarized. Channel block was modeled by a first-order reaction that was independent of channel gating (Katayama et al., 2000).

Channel inactivation or coexpression of HERG with MiRP1 subunits can modulate the potency of drug block of HERG. C-type inactivation of HERG channels can be removed through the simultaneous introduction of two point mutations (G628C, S631C) (Smith et al., 1996). Inactivation-removed HERG mutant channels are far less sensitive to block by most I_Kr blockers (Wang et al., 1996, 1997; Ficker et al., 1998; Lees-Miller et al., 2000). It was unlikely, given the study by Katayama et al. (2000), that vesnarinone block of HERG channels would be affected by the removal of inactivation, but this possibility has not been tested experimentally. The effect of MiRP1 on the block of HERG channels by vesnarinone has also not been investigated.

We recently used alanine-scanning mutagenesis of HERG to identify the specific binding site for the potent blocker MK-499 (Mitcheson et al., 2000a). Mutation to Ala of specific residues that line the inner cavity of the HERG channel reduced the potency of block and defined the high-affinity binding site for the drug. Recovery from the block of HERG channels by MK-499, as with many other drugs, is extremely slow. Vesnarinone is much less potent than MK-499, and recovery from channel block is much faster. Thus, we were interested to determine whether vesnarinone interacted with the same residues that line the inner cavity of HERG as we demonstrated previously for MK-499.

In this study, we use the oocyte expression system and a two-microelectrode voltage-clamp technique to investigate the voltage- and time-dependence of block, the role of channel inactivation, and the effect of MiRP1 coexpression on the potency of vesnarinone block of HERG channels. In addition, we used Ala-scanning mutagenesis to define the structural basis of HERG channel block by vesnarinone.

	Materials and Methods

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Site-Directed Mutagenesis. Mutations were introduced into the HERG K⁺ channel (Warmke and Ganetzky, 1994) with the use of site-directed mutagenesis as described previously (Sanguinetti and Xu, 1999). The cDNA expression construct in the pSP64 transcription vector (Promega, Madison, WI) and synthesis of cRNA were conducted as described previously (Sanguinetti and Xu, 1999). MiRP1 cDNA was kindly provided by S. A. N. Goldstein (Yale University, New Haven, CT).

cRNA Injection and Voltage Clamp of Oocytes. Isolation and maintenance of Xenopus laevis oocytes and injection with cRNA were performed as described previously (Stuhmer, 1992). X. laevis frogs were anesthetized by immersion in 0.2% tricane (Sigma, St. Louis, MO) for 15 to 30 min. Ovarian lobes were removed and digested with 2 mg/ml type IA collagenase (Sigma) in Ca²⁺-free ND96 solution for 1.5 h to remove follicle cells. Stage V and VI oocytes were injected with 10 ng of cRNA encoding wild-type (WT) HERG or G628C:S631C HERG. For HERG-MiRP1 coexpression studies, 2 ng of MiRP1 cRNA was coinjected with 10 ng of HERG cRNA. To study I_Ks, KVLQT1 (5 ng) and minK (1 ng) cRNA were coinjected into oocytes. Oocytes were cultured in Barth's solution supplemented with 50 µg/l gentamicin and 1 mM pyruvate at 18°C.

Data Analysis. Data are presented as mean ± S.E.M. unless otherwise specified. Statistical comparisons between the different experimental groups were obtained using analysis of variance. Differences were considered significant at p < 0.05. Concentration-response relationships were fit to the Hill equation to determine the drug concentration required for 50% inhibition (IC₅₀). A nonlinear least-squares curve-fitting program (Clampfit 6.0.4) was used to analyze the kinetics of current deactivation.

	Results

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Effects of Vesnarinone on Oocytes Expressing HERG or HERG + MiRP1 Subunits. The effects of vesnarinone on currents induced by the expression of HERG alone and with MiRP1 were determined. Currents were elicited during 2-s depolarizing pulses to potentials ranging from 80 to +50 mV from a holding potential of 90 mV. Tail currents were elicited by return of the membrane potential to 80 mV after each voltage step. Vesnarinone (10 µM) caused a similar decrease in the amplitude of outward currents during depolarization and tail current on repolarization in oocytes expressing HERG or HERG + MiRP1 subunits. The decreases in time-dependent current and tail current at 0 mV, respectively, were 35.0 ± 10.2% (n = 6) and 30.4 ± 12.5% for HERG alone and 37.7 ± 11.0% (n = 6) and 32.2 ± 12.5% for HERG with MiRP1. There were also nonsignificant differences in the time-dependent kinetics (Fig. 1A). The drug concentration required to block the current by 50% (IC₅₀) was determined from a plot of the peak time-dependent current recorded during a pulse to +0 mV versus drug concentration. The IC₅₀ value for HERG was 17.7 ± 2.5 µM (n = 6). The IC₅₀ value for HERG/MiRP1 was 15.0 ± 3.0 µM (n = 5), which was not significantly different (p > 0.5) from HERG alone (Fig. 1B).

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Effects of vesnarinone on HERG ± MiRP1 channel current and KvLQT1/minK channel current in X. laevis oocytes. A, representative HERG and HERG/MiRP1 channel currents recorded from oocytes before and after application of 10 µM vesnarinone. Voltage-clamp protocol is shown above current traces. B, concentration-response relationship for block of current recorded at the end of a 2-s depolarizing pulse to 0 mV for HERG () or HERG/MiRP1 (). IC50 values were 17.7 ± 2.5 µM (n = 6) for HERG and 15.0 ± 3.0 µM (n = 5) for HERG/MiRP1. C, representative current traces recorded from oocytes expressing KvLQT1 + minK subunits before and after application of 300 µM vesnarinone. D, I-V relationship for KvLQT1/minK currents measured at the end of a 7.5-s depolarization step (n = 5). Data were obtained before () and after () 300 µM vesnarinone.

KvLQT1/minK Currents. The effect of vesnarinone on the KvLQT1/minK current was examined. From a holding potential of 90 mV, 7.5-s pulses were applied to potentials ranging from 80 mV to +30 mV. Tail currents were elicited by return of the membrane potential to 80 mV. Vesnarinone at 300 µM affected neither the slowly activating outward current elicited during depolarization nor the tail currents at 80 mV (Fig. 1C). Similar results were obtained in five oocytes (Fig. 1D). Thus, vesnarinone blocks only one of the two major types of delayed rectifier K⁺ currents expressed in ventricular myocytes.

Voltage Dependence of Vesnarinone Block of HERG Channels. The effect of vesnarinone on currents recorded at voltages ranging from 70 to +50 mV are shown in Fig. 2. The I-V relationship peaked at 20 mV both before and after exposure of oocytes to 10, 30, and 100 µM vesnarinone (Fig. 2A). Drug-induced inhibition of the current during a 2-s pulse (Fig. 2A) or peak tail current (Fig. 2B) was concentration-dependent; however, there was no voltage-dependent inhibition of current when quantified as relative tail current amplitude (Fig. 2C). The relative tail current over the voltage range of 50 to +50 mV varied from 0.61 to 0.64 at 10 µM, 0.46 to 0.48 at 30 µM, and 0.22 to 0.25 at 100 µM.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Block of HERG channel current by vesnarinone. A, HERG channel I-V relationship before () and after application of 10 µM (), 30 µM (), and 100 µM () vesnarinone (n = 5). B, I-V relationship for peak tail currents. C, voltage-dependence for block of HERG tail current by vesnarinone. The ratio of tail currents in the presence and absence of vesnarinone was used as a measure of relative current.

Time-Dependent Inhibition by Vesnarinone. Time-dependent changes of HERG block by vesnarinone were evaluated by two protocols. First, an envelope of tail currents was used as a measure of onset of drug block. HERG tail currents were generated by applying depolarizing pulses from 40 to +40 mV in 20-mV steps of variable duration (50-1000 ms) from a holding potential of 90 mV. Tail currents elicited by repolarization to 80 mV were measured before and after application of vesnarinone at 30 and 100 µM. In the absence of drug, tail currents increased as the pulse duration was prolonged and reached a plateau level (Fig. 3A, left). In the presence of 100 µM vesnarinone, tail currents decreased as the pulse was lengthened (Fig. 3A, right). The ratio of tail current amplitudes measured in the presence and absence of drug was plotted as a function of pulse duration (Fig. 3A, bottom). The time course of decay of relative tail current was well fitted with a single exponential function. The onset of HERG block by 30 µM vesnarinone developed with a time constant () of 372 ± 76 ms at +40 mV (n = 4). The time constants for onset of block (_onset) were faster at 100 µM (98 ± 33 ms at +40 mV, n = 4) than at 30 µM, but were voltage-independent (Fig. 3B), as were the steady-state values for block (Fig. 3C).

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Onset of HERG channel current block by vesnarinone using an envelope of tails protocol. A, voltage-clamp pulse protocol and representative recordings of HERG current before and after exposure of an oocyte to 100 µM vesnarinone (top). Bottom, a plot of the onset of current block expressed as relative tail current. The time-dependent decay in relative tail current was fitted to a single exponential function with a time constant of 126 ms. The relative steady state value of current was 0.21. B, onset for decay of relative tail current plotted as a function of voltage after application of 30 µM () and 100 µM () vesnarinone (n = 4). C, steady-state value of relative tail current (n = 4).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Onset of HERG channel current block by vesnarinone assessed during depolarizing voltage steps. A, representative recordings of HERG currents elicited by 2-s pulses to 20 mV before and after 30 and100 µM vesnarinone. E-4031 at 20 µM was applied at the end of the experiment to completely block HERG current. B, onset of current block by 100 µM vesnarinone during depolarizing voltage steps to the indicated voltage. Residual current remaining after complete block of HERG with E-4031 was subtracted from currents before and after vesnarinone at 100 µM. Then, relative tail currents were estimated by the ratio of these currents. The time constants for development of block of HERG current was 345 ms at +20 mV, 376 ms at 0 mV, 438 ms at 20 mV, and 456 ms at 40 mV. C, time constants for the onset of HERG current block at different voltages for 30 µM () and 100 µM () vesnarinone (n = 5). D, steady-state value of the relative tail current determined from single exponential fit of relationships shown in B (n = 5).

Recovery from Block. Recovery from HERG channel block by vesnarinone was evaluated at different membrane potentials using a paired-pulse protocol. A conditioning pulse to +50 mV was applied for 2 s to induce steady-state block. The potential was then clamped to 60, 90, or 120 mV for a variable time (1-29 s, in 2-s increments), followed by a 50-ms test pulse to +50 mV, then returned to 70 mV to assess recovery from block (Fig. 5A). Before drug, the amplitude of the tail current was relatively unchanged as the duration of the conditioning pulse was prolonged (Fig. 5A, top). However, in the presence of 100 µM vesnarinone, the tail current amplitude was progressively increased with longer conditioning pulses and reached a steady state within 13 to 15 s (Fig. 5A, bottom). The recovery of the tail current was fitted by a single exponential function. The time constants (_recov) varied little at the three potentials studied, from 9.1 ± 2.3 s at 60 mV (n = 4) to 8.6 ± 1.6 s at 90 mV (n = 4) to 9.7 ± 2.0 s at 120 mV (n = 4) (Fig. 5B). Thus, the recovery of HERG current from block by vesnarinone was not significantly affected by membrane voltage.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Recovery from block of HERG channel current by vesnarinone at hyperpolarized holding potentials. A, voltage-clamp pulse protocol and representative recordings for an oocyte in which the recovery was assessed at 90 mV. B, time constants for recovery from block of HERG current (n = 4).

Block of Inactivation-Removed Mutant HERG Channels. The potency for many blockers is usually greater for WT HERG than inactivation-removed HERG mutant channels. Therefore, we determined the concentration-dependence for block of G628C/S631C HERG channel current by vesnarinone.

View larger version (33K): [in this window] [in a new window]   Fig. 6.   Block of inactivation-removed (G628C/S631C) HERG channel current by vesnarinone. A, voltage-clamp pulse protocol and representative currents recorded from oocytes before and after application of 10 µM vesnarinone. B, concentration-response relationship for block of current measured at the end of a 2-s depolarization to 0 mV. IC50 value was 10.7 ± 3.7 µM (n = 5). C, I-V relationship for currents measured at the end of a 2-s pulse to the indicated voltage before () and after 3 µM (), 10 µM (), 30 µM (), and 100 µM () vesnarinone (n = 5). D, block of step current by vesnarinone is not voltage-dependent. The ratio of step currents in the presence of vesnarinone relative to control currents in C was calculated and plotted again.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Onset and recovery of G628C/S631C HERG channel current block by vesnarinone assessed during and after depolarizing voltage steps. A, representative recordings of G628C/S631C HERG currents elicited by 2-s pulses to +50 mV before and after 100 µM vesnarinone. E-4031 at 20 µM did not delete the time-dependent outward current. Therefore, relative step currents were estimated by the ratio of G628C/S631C HERG currents before and after application of vesnarinone. B, time constants for the onset of G628C/S631C HERG current block at different voltages for 30 µM () and 100 µM () vesnarinone (n = 5). *P < 0.05 versus values of 100 µM vesnarinone at +50 mV. C, steady-state value of the relative tail current determined from single exponential fit of relationships shown in B (n = 5). D, recovery from block of G628C/S631C HERG channel current by vesnarinone at hyperpolarized holding potentials. Voltage-clamp pulse protocol and representative recordings were shown for an oocyte where the recovery was assessed at three different voltages. E, time constants for recovery from block of G628C/S631C HERG current by 100 µM vesnarinone (n = 4).

Alanine-Scanning Mutagenesis to Define Binding Site. Alanine-scanning mutagenesis was used to determine residues important for block of the HERG channel by vesnarinone as reported previously (Mitcheson et al., 2000a). We mutated to alanine individual residues of S6 (L646-Y667) and the few residues of the pore helix (L622-V625) predicted to line the channel cavity and inner pore regions (Fig. 8A) based on homology with the solved crystal structure of the KcsA channel (Doyle et al., 1998). The sensitivity to block by a single concentration of vesnarinone (180 µM, 10 times IC₅₀) was determined for each mutant channel. Currents were measured during repetitive 5-s depolarizing steps to 0 mV, applied at a frequency of 0.166 Hz. Vesnarinone at 180 µM caused approximately 85% inhibition of WT HERG current and was used to assess each mutant channel. Examples of WT and three mutant HERG channel currents recorded before and after block by vesnarinone had reached a steady-state level are shown in Fig. 8B. Note that V659A HERG channel currents differ from WT because they deactivate very slowly, and a proportion of the channels are open at the 90-mV holding potential (Mitcheson et al., 200a). Consequently, an instantaneous component of current is observed upon depolarization. As before, T623A and G648A HERG channels are more inactivated than other channels; therefore, as described previously, the currents conducted by these channels were measured using a 96 mM KMES extracellular solution to elicit larger currents (Mitcheson et al., 200a). Vesnarinone reduced the current magnitude of most mutant channels to an extent similar to that measured for WT HERG (Fig. 8C). However, three channels with Ala missense mutations located in the S6 domain (G648A, F656A, and V659A) and three channels with a mutation located in the base of the pore helix (T623A, S624A, and V625A) were less sensitive to block by 180 µM vesnarinone. However, in contrast to all other drugs that have been investigated, there was little effect of mutating Y652.

View larger version (27K): [in this window] [in a new window]   Fig. 8.   Alanine-scanning mutagenesis of HERG to define binding sites for vesnarinone. A, sequence of the pore helix and inner helix for the KcsA channel (Doyle et al., 1998) and the equivalent residues in the pore helix and S6 transmembrane domain of the HERG K+ channel. Shaded residues of KcsA are predicted to face the inside of the inner channel pore. The region of HERG analyzed by Ala-scanning mutagenesis is dot-underlined. B, block of WT and mutant HERG channel current in oocytes by vesnarinone. HERG channel currents recorded before (Con) and after (Drug) achieving steady-state block of current with 180 µM vesnarinone. From a holding potential of 90 mV, currents were elicited during 5-s pulses to 0 mV applied repetitively at 0.166 Hz. C, normalized current (Ivesnarinone/Icontrol) measured after steady-state block by 180 µM vesnarinone (n = 4  5; error bars, +S.E.M.). N.T., residues that were not tested; N.E., mutant channels that lacked functional expression.

	Discussion

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State-Dependent Block of HERG Channels by Vesnarinone. Our findings suggest that vesnarinone preferentially blocks HERG channels in the open state. Recovery from block of the HERG current was readily achieved when the membrane was held for a sufficient time at a negative potential. There was no significant decrease in current magnitude if a single, short, depolarizing pulse was subsequently applied, indicating very low affinity of the drug for HERG channels in the rested state. However, a decrease of HERG channel current developed gradually during a maintained depolarization, indicating that for drug-induced block to occur, the channels must first open. Moreover, we found that the drug was equally effective in blocking WT and inactivation-removed (G628C/S631C) HERG channels. Most drugs that block HERG channels have been shown to be far less potent in blocking inactivation-removed channels compared with WT channels (Wang et al., 1996, 1997; Ficker et al., 1998; Lees-Miller et al., 2000).

MiRP1 Does Not Affect Block of HERG Channels by Vesnarinone. Coexpression of MiRP1 with HERG subunits was reported to increase the rate of channel deactivation and increase the potency of channel block by E-4031, an experimental class III antiarrhythmic agent (Abbott et al., 1999). Unlike HERG alone, HERG/MiRP1 channels exhibited tonic block by E-4031. In addition to its affect on HERG, MiRP1 has also been shown to alter the gating of KvLQT1 channels (Tinel et al., 2000). Therefore, we determined whether MiRP1 might affect the block of HERG by vesnarinone. Under the conditions of our study, we found that MiRP1 did not alter the kinetics of HERG current, nor did it affect the potency of block by vesnarinone. Coexpression of MiRP1 with HERG was also recently reported not to affect channel block by dofetilide (Weerapura et al., 2000).

Binding Site for Vesnarinone Is within the Inner Cavity of the HERG Channel. Alanine-scanning mutagenesis of the HERG subunit revealed that the binding site for vesnarinone shares important similarities with that previously defined for MK-499. Channels containing a single Ala mutation at any one of the three amino acids located at the base of the pore helix (T623, S624, and V625), or of three amino acids located in the S6 domain (G648, F656, and V659), were significantly less sensitive to block by vesnarinone than WT HERG channels. Given the sequence alignment with the KcsA channel, it is clear that all of these residues face the inner cavity of the HERG channel. Mutation of these same residues was also found to decrease the potency of MK-499, although the magnitude of effects was different. For example, mutation of T623 and S624 to Ala had less effect on block by MK-499 than it did for vesnarinone, and the V659A mutation reduced block by vesnarinone more than by MK-499. A striking difference between the two drugs was the finding that Y652 was an important binding site for MK-499 but not for vesnarinone. Y652 is one of two aromatic residues unique to the eag channel family that is important for high-affinity drug binding (Mitcheson et al., 2000a). The lack of interaction of vesnarinone with Y652 might be responsible, in part, for the reduced potency of this drug. Another reason for the decreased potency of vesnarinone might relate to the inability of the drug to be trapped by closure of the activation gate, which can occur with charged drugs such as MK-499 (Mitcheson et al., 2000b). Our previous study also indicated that unlike block by MK-499, block of HERG channels by terfenadine and cisapride were not much affected by mutation of either V625 or G648 to Ala. The finding that vesnarinone also seems to interact with G648 and with residues at the base of the pore helix indicates that such interaction is not unique to methanesulfonanilides, as speculated previously (Mitcheson et al., 2000a).

Clinical Implications. Most potent blockers of I_Kr (e.g., dofetilide, E4031) prolong cardiac APD in a reverse-frequency-dependent manner: the faster the heart rate, the less the prolongation of APD (Carmeliet, 1992; Sedgwick et al., 1992; Jurkiewicz and Sanguinetti, 1993). The mechanism of this frequency profile is uncertain, but is not caused by frequency-dependent block of I_Kr (Jurkiewicz and Sanguinetti, 1993). Whatever the mechanism, it is commonly believed that reverse-frequency-dependent changes in APD may be proarrhythmic because of excessive prolongation of repolarization with bradycardia. A frequency-dependent lengthening of APD such as that caused by vesnarinone would theoretically provide protection against tachyarrhythmias and reduce the risk of proarrhythmia associated with bradycardia (Hondeghem and Snyders, 1990).