Introduction

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Repolarization of cardiac ventricular myocytes is due mainly to outward potassium currents (Carmeliet, 1993). One of the most important currents is the delayed rectifier potassium current, I_K, which has both rapidly (I_Kr) and slowly activating components (Sanguinetti and Jurkiewicz, 1990). Activation of the rapid component of the delayed rectifier potassium current, I_Kr, initiates repolarization and terminates the plateau phase of the cardiac action potential. The human ether-a-go-go-related gene (HERG) (Curran et al., 1995; Sanguinetti et al., 1995; Trudeau et al., 1995) encodes the major protein underlying I_Kr, and mutations in the HERG gene account for chromosome 7-linked inherited long QT syndrome-2 (Keating, 1995; Sanguinetti et al., 1996; Viskin, 1999; Ficker et al., 2000). Patients diagnosed with long QT-2 syndrome present with prolonged QT intervals in the surface electrocardiogram and have a high risk for ventricular "torsade de pointes" arrhythmias, a cause of sudden cardiac death. Recently, a patient with homozygous premature truncation of HERG and therefore complete absence of I_Kr, has been reported by Hoorntje et al. (1999). This patient had severe cardiac arrhythmias, but there was no evidence of dysfunction in any other organ. Thus, the role of HERG in other functional systems is either limited or compensated during the development.

Class III antiarrhythmic drugs, such as dofetilide, amiodarone, or clofilium, have been shown to be potent inhibitors of HERG potassium channels (Kiehn et al., 1996, 1999b; Suessbrich et al., 1997). A block of I_Kr causes lengthening of the cardiac action potential, which produces a class III antiarrhythmic effect. Prolongation of cardiac refractoriness has been proposed as a mechanism to prevent atrial and ventricular arrhythmias resulting from re-entrant pathways (Singh and Nademanee, 1985). However, the therapeutical use of class III antiarrhythmic drugs is limited by their proarrhythmic potential: excessive prolongation of the cardiac action potential can lead to acquired long QT syndrome and life-threatening "torsade de pointes" arrhythmias (Napolitano et al., 1994; El-Sherif and Turitto, 1999).

In the search for novel antiarrhythmic drugs with reduced proarrhythmic risk, compounds with diverse electrophysiological effects have been tested. In particular, it has been suggested that combining potent inhibition of I_Kr and moderate calcium channel blockade might lead to a reduced risk of proarrhythmic side effects (Bril et al., 1996, 1998; Chouabe et al., 1998; Zhang et al., 1999; Noble and Colatsky, 2000). BRL-32872, a novel compound derived from the calcium and potassium channel antagonist verapamil (Zhang et al., 1999), was found to inhibit the delayed rectifier potassium current and the L-type calcium current in guinea pig ventricular cardiomyocytes (Bril et al., 1995). Furthermore, it has been demonstrated that BRL-32872 has a potent antiarrhythmic effect and induces fewer proarrhythmic events than the typical class III antiarrhythmic agent E-4031 in a dog model of programmed electrical stimulation-induced arrhythmias (Bril et al., 1996). In addition, Faivre et al. (1999) found that BRL-32872 does not cause early after depolarizations (EADs) in canine Purkinje fibers and suppresses EADs induced by clofilium, a selective inhibitor of the delayed rectifier potassium current (Suessbrich et al., 1997) in the same model.

The aim of the present study was to investigate the potential interaction of BRL-32872 with cloned HERG potassium channels heterologously expressed in Xenopus laevis oocytes and in the human cell line HEK 293. This approach revealed detailed insights into the biophysical mechanism of high-affinity HERG channel block by BRL-32872.

	Materials and Methods

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Molecular Biology. Procedures for in vitro transcription and oocyte injection have been published previously (Kiehn et al., 1999b). Briefly, HERG wild-type (Warmke and Ganetzky, 1994; GenBank accession no. hs04270) (a kind gift from M. T. Keating) and HERG S620T (Ficker et al., 1998) cRNAs were prepared with the mMESSAGE mMACHINE kit (Ambion, Austin, TX) using SP6 RNA polymerase after linearization with EcoRI (Roche Diagnostics, Mannheim, Germany). Stage V-VI defolliculated Xenopus oocytes were injected with 46 nl of cRNA per oocyte.

Electrophysiology and Statistics. Two-microelectrode voltage-clamp recordings from Xenopus laevis oocytes were carried out as published previously (Thomas et al., 1999). In brief, recordings were performed using a Warner OC-725A amplifier (Warner Instruments, Hamden, CT) and Pclamp software (Axon Instruments, Foster City, CA) for data acquisition and analysis. Microelectrodes had tip resistances ranging from 1 to 5 megaohms. The recording chamber was continually perfused.

Solutions and Chemicals. Voltage-clamp measurements of Xenopus oocytes were performed in a physiological potassium solution containing (in mM): 5 KCl, 100 NaCl, 1.5 CaCl₂, 2 MgCl₂, and 10 HEPES (pH 7.4 with NaOH). Current and voltage electrodes were filled with 3 M KCl solution. For whole-cell patch-clamp recordings from HEK 293 cells, electrodes were filled with the following solution (in mM): 100 K-aspartate, 20 KCl, 2.0 MgCl₂, 1.0 CaCl₂, 10 EGTA, 10 HEPES, and 40 glucose (pH 7.2 with KOH). The external solution for these experiments contained (in mM): 140 NaCl, 5.0 KCl, 1.0 MgCl₂, 1.8 CaCl₂, 10 HEPES, and 10 glucose (pH 7.4 with NaOH).

	Results

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BRL-32872 Blocks HERG Potassium Currents. Figure 1 shows the effects of BRL-32872 on HERG potassium channels expressed in Xenopus laevis oocytes. HERG currents were elicited by a 2-s depolarizing step to +20 mV, followed by a repolarizing step to 40 mV for 1.6 s to produce large, slowly decaying outward tail currents that are a characteristic of HERG potassium currents (Sanguinetti et al., 1995). The holding potential was 80 mV in all experiments performed in this study. This voltage protocol was repeated every 10 s during superfusion with the drug solution, and the amplitude of the current was monitored until no changes in current amplitude could be recorded for 3 min. After this monitoring period, test pulses were applied to determine the amount of block. HERG tail currents were blocked potently by BRL-32872 at nanomolar concentrations as shown in Fig. 1A. To study the concentration dependence of HERG current block by BRL-32872, inhibition of HERG peak tail currents was normalized to the respective control values and plotted as relative current amplitude in Fig. 1B (n = 5-6 oocytes at each concentration). The IC₅₀ for the block of tail currents was 241.4 nM.

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Inhibition of HERG channels expressed in Xenopus oocytes by BRL-32872. Representative current traces recorded from the same oocyte under control conditions and after perfusion with BRL-32872 (100 nM, 1 µM, and 100 µM) are displayed in panel A. Currents were elicited by a depolarizing pulse to +20 mV (2 s), and tail currents were recorded during a step to 40 mV (1.6 s). Current amplitudes were monitored during control and drug application periods with the same voltage protocol (0.1-Hz pulsing frequency) until steady-state conditions were reached. B, dose-response relationship for the effect of BRL-32872 on HERG peak tail currents. Error bars denote S.D. (n = 5-6 oocytes). The IC50 yielded 241.4 nM. C, time course of HERG tail current inhibition by 10 µM BRL-32872. Currents were measured as described above; but, for simplicity, not all current measurements are displayed. After a control period of 22 min, currents decreased rapidly upon perfusion with the drug solution, and steady-state block was reached after 2 min. Holding potential: 80 mV; bath: 5 mM K+.

BRL-32872 Blocks HERG Potassium Channels in the Open State and with Lower Affinity in the Inactivated State. To determine whether the channel is blocked in the closed or open and inactivated states, we activated currents by use of a protocol with a single depolarizing step to 0 mV for 30 s (Kiehn et al., 1999b). After having obtained the control measurement (Fig. 2A), we allowed 10 µM of the drug to wash in for 10 min while holding all channels in the closed state at 80 mV membrane potential. The first pulse after this equilibrium period showed no effect on the initial time course of current activation, but revealed a time-dependent block of HERG current that developed during the depolarizing step (Fig. 2A). Apparently, there is mainly a block of open or inactivated channels with no marked inhibition of closed channels. Subsequent depolarizing pulses at 10-s intervals showed no time-dependent component of block, thus indicating that BRL-32872 had not dissociated appreciably from the channel due to slow unbinding kinetics within this time frame.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   State-dependent block of open HERG channels by BRL-32872 in Xenopus oocytes. A, HERG currents were activated by a 30-s depolarizing voltage step to 0 mV from a holding potential of 80 mV. After having recorded the control measurement, the oocyte was held at 80 mV for 10 min during perfusion with the drug solution (10 µM BRL-32872). The control recording and the first 5 pulses (at 10-s intervals) measured immediately after the incubation period are displayed, illustrating that BRL-32872 blocks HERG channels mainly in the open state. Block of inactivated channels was assessed qualitatively by use of a modified voltage protocol. B, control measurement with an intervening step to 80 mV in the middle of the measurement and a short preceding equilibration pulse in comparison with a continuous 0 mV pulse. C, effect of BRL-32872 (10 µM) in the same cell. The currents at the 0 mV steps before and after the 80 mV intervening step were fitted exponentially, and fits were drawn into the graph. Note the gap (indicated by the arrow) between the fitted lines, which resulted from less block during the 80 mV step of the measurement. Test pulse with four steps in B and C: step 1, 80 mV (100 ms); step 2, 0 mV (1 s); step 3, 80 mV (1.5 s); and step 4, 0 mV (6 s). Bath: 5 mM K+.

The Biophysical Mechanism of HERG Channel Blockade by BRL-32872. We further elucidated the blockade of HERG channels during depolarization and questioned how HERG channels are blocked at the end of the cardiac action potential while recovering from inactivation. To answer this question, we applied a two-step protocol. During the first step from the holding potential of 80 mV to 80 mV (5 s) channels are mainly inactivated (Kiehn et al., 1999a). The fraction of open channels was increased during a second voltage step to 0 mV (5 s), before returning to 80 mV (Smith et al., 1996). The graphical overlay of a typical control measurement and the first pulse after incubation with 10 µM BRL-32872 for 10 min at 80 mV (without pulsing) is displayed in Fig. 3A. The inlet shows the beginning of the test pulse (Fig. 3A). After application of the drug, the current trace shows a time-dependent increase and reaches a peak amplitude larger than the control trace. Then, the current after drug application became smaller than the control current (n = 4).

View larger version (26K): [in this window] [in a new window]   Fig. 3.   The biophysical mechanism of HERG channel block by BRL-32872. Under control conditions, currents were mainly inactivated during 5-s pulses to 80 mV, followed by a step to 0 mV (5 s), where the fraction of open channels was increased. The control trace and the first recording after incubation with 10 µM BRL-32872 while holding the cell at 80 mV are superimposed in panel A. Note the larger initial peak current during the 80 mV step in the presence of the drug, compared with control conditions (arrow). A hypothetical model for the transition between closed (C1, Cn), open (O), and inactivated (I) states of the channel illustrates the changes in current course and peak amplitude induced by BRL-32872 (B, C). Currents are enhanced due to an increased transition rate from inactivated (I) to open (O) states (partly via C1), in combination with a decreased transition rate from open (O) to closed (C1) and inactivated (I) states (see text for details). Holding potential: 80 mV. Bath: 5 mM K+.

HERG S620T Mutant Channels Show Reduced Sensitivity to BRL-32872 Blockade. Previous studies have shown that the S620T mutation in HERG almost abolishes C-type inactivation and modifies block of HERG channels by antiarrhythmic drugs (Suessbrich et al., 1997; Ficker et al., 1998). Because we observed that BRL-32872 block is reduced by HERG channel inactivation, drug effects were tested on the HERG S620T channel under the same conditions as previously described for the HERG wild-type (WT) channel (see Fig. 1A). Figure 4A displays typical current traces recorded from HERG S620T channels during the test pulse at +20 mV (compare with Fig. 1A). HERG S620T peak tail currents were only blocked by 50.2 ± 9.5% at a BRL-32872 concentration that caused an almost complete block of HERG WT channels (10 µM), and the concentration dependence analysis resulted in an IC₅₀ value of 12.4 µM (Fig. 4B; n = 6 cells studied at each concentration), which is 51 times higher compared with the IC₅₀ obtained from HERG WT channels in Xenopus oocytes (241.4 nM).

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Inactivation-deficient HERG S620T mutant channels are less sensitive to BRL-32872 block. Representative current traces recorded from one oocyte expressing HERG S620T channels under control conditions and after application of 10 µM, 100 µM, and 300 µM BRL-32872 are displayed in panel A. Note the loss of current inactivation during the test pulse at +20 mV (compare with Fig. 1A). B, concentration dependence of BRL-32872 inhibition of HERG S620T channels. Percent peak tail current block values were fit with a Hill equation and gave an IC50 value of 12.4 µM. Error bars denote S.D., and six cells were studied at each concentration. Protocol, plot, and experimental conditions are the same as in Fig. 1.

BRL-32872 Does Not Markedly Affect Inactivation of HERG Channels. We investigated the effects of BRL-32872 on HERG current inactivation by testing whether the rate of inactivation was affected by the drug. Pulses were applied to 40 mV for 900 ms where channels are partially open but mostly inactivated. A brief repolarization to 100 mV for 16 ms caused rapid recovery from inactivation without marked deactivation. During a second depolarizing pulse (150 ms) to different voltages ranging from 60 mV to 40 mV (increment 20 mV), large, rapidly inactivating currents were produced. The holding potential was 80 mV. Inactivating currents were recorded before (Fig. 5A) and after equilibration of the block with 300 nM BRL-32872 (Fig. 5B) by current monitoring. Single exponential fits to the large inactivating currents yielded the time constants of inactivation at different voltages. In a set of four cells, no significant changes in the time constant for HERG channel inactivation were observed (Fig. 5C).

View larger version (25K): [in this window] [in a new window]   Fig. 5.   BRL-32872 does not markedly affect HERG current inactivation. The time constant of channel inactivation was assessed by 900-ms pulses to 40 mV, followed by a brief depolarization at 100 mV (16 ms). Variable voltage steps ranging from 60 mV to 40 mV (150 ms; increment 20 mV) were consecutively applied to evoke inactivating currents (A). The holding potential was 80 mV. Typical current measurements recorded before (A) and after incubation with 300 nM BRL-32872 for 10 min (B) are displayed. Panel C reflects the corresponding inactivation time constants obtained from single-exponential fits to the large inactivating current traces (n = 4). Panels D and E show measurements of the steady-state inactivation at constant 20 mV after various potentials from 120 to 30 mV (increment 10 mV). The normalized current amplitude at the beginning of the inactivating current at 20 mV is displayed in panel F, giving the steady-state inactivation curve. There was only a small shift of 3.9 mV from 74.2 mV to 78.1 mV in this representative experiment. Error bars denote S.D.; bath: 5 mM K+.

BRL-32872 Has No Effect on HERG Channel Activation. The effect of BRL-32872 on HERG current-voltage (I-V) relationship was investigated under isochronal recording conditions. Oocytes were clamped at a holding potential of 80 mV. Depolarizing pulses were applied for 2 s to voltages between 80 and +80 mV in 10 mV increments, and tail currents were recorded during a constant repolarizing step to 60 mV for 1.6 s. Families of current traces from one cell are shown for control conditions and after exposure to 300 nM BRL-32872 in Fig. 6. The currents activated at potentials greater than 50 mV, reached a peak at 10 mV, and then decreased at more positive potentials due to inactivation (Sanguinetti et al., 1995; Smith et al., 1996; Spector et al., 1996), giving the I-V relationship its typical bell-shaped appearance (Fig. 6C). The peak tail current, measured during the repolarizing second step of the voltage protocol, increased with voltage steps from 40 mV to +20 mV and then plateaued for test pulse potentials positive to +20 mV (Fig. 6D). HERG currents at the end of the test pulse to 0 mV were reduced by 45.2 ± 17.8% (n = 4), and peak tail currents were blocked by 45.1 ± 7.7% (n = 4). Figure 6D displays peak tail currents normalized to their respective peak values as a function of the test pulse potential, resulting in isochronal activation curves. BRL-32872 caused no significant change in the half-maximal activation voltage V_1/2 of 2.3 ± 2.6 mV from 21.2 ± 2.9 mV to 23.5 ± 3.4 mV (n = 4).

View larger version (36K): [in this window] [in a new window]   Fig. 6.   Application of BRL-32872 has no effect on HERG activation kinetics. Control measurement (A) and the inhibitory effects of 300 nM BRL-32872 (B) in one representative oocyte. C, the resulting current amplitude at the end of the test pulse as a function of the preceding test pulse potential under control conditions and after incubation with 300 nM BRL-32872. The maximum current amplitude at 0 mV is reduced in the measurement with BRL-32872 by 53.5%. D, activation curves, i.e., the normalized inverted peak tail current amplitudes as a function of the test pulse potential during the first step of the voltage protocol, recorded under isochronal conditions. No significant changes in the half-maximal activation potential V1/2 were apparent (V1/2 = 2.5 mV). Voltage protocol in panels A and B: holding potential 80 mV, test pulse 80 to 80 mV (2 s) in 10 mV increments, return pulse constant 60 mV (1.6 s). Bath: 5 mM K+.

Block of HERG Channels by BRL-32872 Is Not Voltage-Dependent. To address the question whether HERG current block by BRL-32872 varies with voltage, we applied the following methodical approach. Since mainly open channels were blocked and because unblocking was extremely slow, only one experiment at each potential could be carried out with one individual oocyte. Activating currents were elicited by 35-s depolarizing pulses ranging from 50 mV to +80 mV from a holding potential of 80 mV, and peak inward tail currents were recorded during a second step to 120 mV (400 ms). First, control currents were recorded. Then, oocytes were superfused with the drug solution (1 µM BRL-32872) while holding the cell at constant 80 mV for 10 min, where HERG channels are in the closed state. After this, the measurement at the test pulse potential was obtained. Typical recordings for 40 mV and 80 mV are shown in Fig. 7, A and B. Percent inhibition of the peak tail currents was plotted as a function of the preceding test pulse potential (Fig. 7C; n = 6-7 cells studied at each potential). BRL-32872 application reduced the currents throughout all voltages, but the degree of blockade was not significantly different at the potentials tested, indicating that block by the drug was not voltage-dependent.

View larger version (11K): [in this window] [in a new window]   Fig. 7.   BRL-32872 block of HERG currents is not voltage-dependent. Comparison of typical current traces recorded from a cell after a long depolarizing pulse to 40 mV (A) and +80 mV (B), before and after exposure to 1 µM BRL-32872, illustrates that no significant changes of HERG channel block could be observed at different potentials. C, fraction of blocked control peak tail currents as a function of various test pulse potentials. Data are expressed as the mean ± S.D., and n = 6-7 cells were studied at each potential. Voltage protocol: peak tail currents were measured during a repolarizing step to 120 mV (400 ms) following a test pulse to potentials ranging from 50 to +80 mV (35 s). Holding potential: 80 mV; bath: 5 mM K+.

Frequency Dependence of Block. HERG block by BRL-32872 was frequency-dependent, as shown in Fig. 8. BRL-32872 (1 µM) was washed into the bath, and HERG channels were rapidly activated by a depolarizing step to 20 mV for 300 ms followed by a repolarizing step to 40 mV (300 ms) to elicit outward tail currents, before returning to the holding potential of 80 mV. Pulses were applied at intervals of 1, 2, 4, or 10 s to n = 5 to 6 oocytes, with each cell studied only at one frequency. The development of current reduction was plotted versus time (Fig. 8), and the resulting level of steady-state block is a measure for the frequency dependence of block. Block was use-dependent with a stronger steady-state level of block at higher frequencies. The time course of the development of block did also depend on the frequency of channel activation (Fig. 8).

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Frequency dependence of BRL-32872 block in Xenopus oocytes. HERG channels were activated with a test pulse to 20 mV (300 ms) from a holding potential of 80 mV, and outward tail currents were recorded during a repolarizing step to 40 mV (300 ms) before returning to the holding potential. Trains of pulses were applied at intervals of 1, 2, 4, and 10 s under control conditions and in the presence of 1 µM BRL-32872 until steady-state block was achieved. The resulting mean relative tail current amplitudes obtained from n = 5 to 6 oocytes are plotted versus time. The amount of steady-state block was lower when pulses were applied at lower stimulation frequencies compared with higher frequencies. Therefore, block was frequency-dependent. Bath: 5 mM K+.

BRL-32872 Blocks HERG Channels in a Human Cell Line. To demonstrate BRL-32872 block of HERG in human cells, we expressed HERG potassium channels heterologously in HEK 293 cells. Channels were activated by a 2-s depolarization to +20 mV, and outward tail currents were recorded during a step back to 40 mV for 1.6 s (Fig. 9). During the wash-in of the drug, we applied the protocol as described (frequency 0.1 Hz) until the block reached a maximum. The tail currents were blocked by BRL-32872 in a concentration-dependent manner. The IC₅₀ value for the BRL-32872 block of peak HERG tail currents under these conditions was 19.8 nM (n = 3-6 cells). In six control experiments, a period of 20 min had no significant effect on the HERG current amplitude (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 9.   BRL-32872 blockade of HERG channels expressed in human HEK 293 cells. A, typical whole-cell patch-clamp recordings from one HEK cell under control conditions and after application of 10 nM and 100 µM BRL-32872, where currents were blocked by 43.0% (10 nM) and 97.7% (100 µM), respectively. B, dose-response curve for inhibition of HERG peak tail currents in HEK 293 cells, yielding an IC50 value of 19.8 nM (n = 3-6 cells; error bars denote S.D.). Protocols and conditions are the same as in Fig. 1. Bath: 5 mM K+.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The results of our study demonstrate that BRL-32872 is a potent inhibitor of HERG potassium channels heterologously expressed in Xenopus laevis oocytes and HEK 293 cells. Blockade of HERG expressed in HEK cells by BRL-32872 displayed an IC₅₀ value of 19.8 nM, which is almost identical to the IC₅₀ for I_Kr block in guinea pig cardiomyocytes (28 nM) reported by Bril et al. (1995). In contrast, our experiments with HERG channels expressed in Xenopus oocytes revealed an IC₅₀ of 241.4 nM for the BRL-32872 block. This discrepancy is due to specific properties of the different expression systems. In particular, higher concentrations of drugs are necessary in Xenopus oocyte experiments when applied to the extracellular surface of whole oocytes. For example, the block of HERG by dofetilide gave an IC₅₀ that was 20-fold higher when the drug was applied to the bath, compared with the application of the drug to the internal surface of the membrane in inside-out membrane patches (Kiehn et al., 1996). One explanation for this observation is that the viteline membrane and the yolk reduce the actual concentration of drugs at the cell membrane. Nevertheless, the Xenopus oocyte expression system is a suitable preparation for investigations of the mechanism of block.

The present study was designed to analyze the biophysical mechanism of HERG channel block by BRL-32872 to understand better the class III antiarrhythmic properties of this novel compound. One important finding of this study was that BRL-32872 blocks HERG channels with high affinity in the open state, although weaker blockade of inactivated channels cannot be excluded. A pronounced shift in the half-maximal inactivation voltage, as reported for other HERG current inhibitors (Wang et al., 1999; Tie et al., 2000) could not be observed. The half-maximal inactivation voltage was only slightly shifted by 3.9 mV. Unblocking upon repolarization, which allows HERG channels to become available for opening, occurred very slowly. As a consequence, the block was use-dependent, with the block being stronger at higher stimulation frequencies.

Our results revealed an interesting phenomenon that illustrates the strong state dependence of the HERG channel block by BRL-32872. At very positive inactivating potentials (80 mV; Fig. 3C) in the presence of the drug, channels appear to have to open before the drug binds to the channel. Block occurs mainly to the small fraction of open HERG channels, forcing more channels to change their conformation from inactivated to open. This results in an additional outward current due to a markedly increased amount of open channels upon strong depolarization at 80 mV in the presence of BRL-32872 (see Fig. 3C). In addition to this, the drug molecule inside the channel pore on its way to the putative binding site might prevent the open channel from closing and from inactivating, which could result in an increased fraction of open channels, consecutively causing a larger initial current amplitude compared with control conditions.

The slow rate of unblocking may be due to a trapping mechanism of the drug at its binding site (Mitcheson et al., 2000). The finding of reduced block in the inactivation-deficient HERG S620T mutant demonstrates that channel inactivation has an enhancing effect on the open channel block by BRL-32872 (Numaguchi et al., 2000). Inactivation might be required for trapping of the drug molecule and for high-affinity drug binding, as reported previously for other class III antiarrhythmic drugs (Suessbrich et al., 1997; Ficker et al., 1998).

As a result of the outcome of the survival with oral d-sotalol SWORD trial (Waldo et al., 1996), where treatment with the pure class III antiarrhythmic drug d-sotalol caused an increase in mortality, recent research has focused on drugs controlling re-entrant ventricular tachyarrhythmias by having ancillary properties. BRL-32872 has been shown to produce pronounced antiarrhythmic efficacy in several arrhythmia models with fewer adverse effects than pure class III antiarrhythmic substances (Bril et al., 1995, 1998; Gout et al., 1995; Faivre et al., 1999). This is probably due to its electrophysiological profile with a combination of potent block of potassium channels (HERG) and moderate inhibition of L-type calcium channels (Bril et al., 1995; Noble and Colatsky, 2000). In guinea pig cardiomyocytes, the L-type calcium channel was 100-fold less sensitive to BRL-32872 than I_Kr (Bril et al., 1995). This is consistent with the observation that the increase in action potential duration in papillary muscle preparations due to high-affinity BRL-32872 block of I_Kr has been limited with increasing concentrations of the drug, when calcium current inhibition counterbalances the action potential prolongation. This dual activity produces a bell-shaped dose-response relationship (Bril et al., 1995). Similar mechanisms could also account for the relatively low incidence of "torsade de pointes" arrhythmias in the clinical use of the antiarrhythmic drug amiodarone (Hohnloser et al., 1994), since amiodarone is known to exhibit not only HERG potassium current blocking properties (Kiehn et al., 1999b), but also class I (Mason et al., 1984), class II (Polster and Broeckhuysen, 1976), and class IV antiarrhythmic action (Yabek et al., 1986).

In conclusion, the present results show that BRL-32872 is a high-affinity antagonist of cloned HERG potassium channels. The fact that the drug has the ability to block both potassium and calcium currents renders it a potentially useful action potential modulator. The pharmacological action with moderate calcium channel block counterbalancing excessive QT prolongation by strong potassium channel block possibly prevents the occurrence of "torsade de pointes" arrhythmias. Thus, BRL-32872 could serve as a promising starting point for more effective modern antiarrhythmic therapy with low proarrhythmic potential.