Open Channel Block by KCB-328 [1-(2-Amino-4-methanesulfonamidophenoxy)-2-[N-(3,4-dimethoxyphenethyl)-N-methylamino]ethane Hydrochloride] of the Heterologously Expressed HumanEther-a-go-go-Related Gene K⁺ Channels

Materials and Methods

Expression of HERG and Current Recording in Xenopus Oocytes.

Stage V to VI oocytes were collected from the anesthetized femaleXenopus laevis (Carolina Biological Supply Co., Burlington, NC) through a small abdominal incision. The follicular membranes were then removed after collagenase treatment (type II; Sigma-Aldrich, St. Louis, MO). cRNA of HERG was synthesized from linearized cDNA using an in vitro transcription kit (Ambion, Austin, TX) and stored in 10 mM Tris-HCl, pH 7.4, at −80°C. Oocytes were then injected with 50 nl of cRNA (1 ng nl⁻¹). After injection, oocytes were cultured at 18°C in solution containing 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 5 mM HEPES, pH 7.55, supplemented with 5 mM pyruvate and 50 μg ml⁻¹ gentamicin sulfonate. Currents were recorded 2 to 4 days after injection. Water-injected oocytes showed insignificant amounts of currents from endogenous channels.

HERG currents were measured at room temperature (21–23°C) using a two-electrode voltage-clamp amplifier (OC-725B; Warner Instruments, Hamden, CT). Stimulation and data acquisition were controlled with Digidata 1200B and pClamp 6.0.3 (Axon Instruments, Union City, CA). Electrodes were filled with 3 M KCl and had a resistance of 1 to 2 MΩ for current-passing electrodes and a resistance of 4 to 6 MΩ for voltage-recording electrodes. Oocytes were then bathed in Ringer's solution containing 120 mM NaCl, 2.5 mM KCl, 1.1 mM CaCl₂, 1.0 mM EGTA, 1 mM MgCl₂, 10 mM HEPES, pH 7.2.

Pulse Protocols and Analysis.

The standard protocol to obtain current-voltage relationships and activation curves consisted of depolarizing steps in 10-mV increments from −70 to +40 mV for 4 s from a holding potential of −80 mV and repolarization to −40 mV for 6 s. Steps were repeated at 60-s intervals. The steady-state currents were obtained at the end of the 4-s depolarizations. Further details are given in the text and figure legends.

The voltage dependence of HERG current activation was determined for each oocyte by fitting peak values of tail current (I_tail) versus test potential (V_t) to the Boltzmann function:where I_tail^max is maximum tail current, V_1/2 is the voltage at which 50% of the channels are activated, and kis the slope factor. Concentration-response relationships for KCB-328-induced HERG block were fitted to a Hill equation.where I indicates current, [D] the drug concentration, n the Hill coefficient, and IC₅₀ the concentration necessary for 50% block. The data were expressed as mean ± S.E.M. All data were compared by Student's t test.

Drugs.

KCB-328 was kindly provided by C & C Research Laboratories (Kyunggi-do, Korea) and was dissolved in dimethyl sulfoxide.

Results

To examine whether KCB-328 blocks HERG, we studied the effect of KCB-328 on HERG current expressed in Xenopus oocytes. Figure2A shows a typical HERG current family elicited by depolarizing voltage steps ranging from −70 to +40 mV with 10-mV increments, with a return potential of −40 mV. InXenopus oocytes injected with the cRNA-encoding HERG, slowly rising currents were apparent on depolarizations. The relationship between the current at the end of the pulse and membrane potential showed the characteristic bell-shaped relationship of HERG, reflecting inward rectification at more positive potentials (Fig. 2B). Upon returning to repolarization at −40 mV, the large, slowly deactivating tail currents characteristic of HERG were observed. Tail current amplitude increased with depolarizing step from −40 to 0 mV and was then superimposed on further depolarizing steps to + 40 mV. KCB-328 (1 μM) inhibited both the outward and tail currents (Fig. 2A). The current-voltage relationship of the steady-state current at the end of the depolarizing step and peak tail current are depicted in Fig. 2, B and C, respectively, before and after exposure to 1 μM KCB-328 (n = 6). For control conditions, the threshold for activating HERG current was close to −40 mV, and full activation was obtained at a voltage near +10 mV. In the presence of 1 μM KCB-328, outward current at depolarizing step and tail current amplitude were reduced compared with the control. Steady-state current and peak tail current were recovered to more than 95% of the control level within 10 min after washout of the drug (Fig. 2, B and C).

Drugs that block ion channels often alter the voltage dependence of channel kinetics. Therefore, we analyzed the voltage dependence of activation of the peak amplitude of the decaying tail currents in the absence or presence of KCB-328 (1 μM) (Fig. 2C). The voltage dependence of HERG activation was determined using 4-s test pulses to potentials ranging from −70 to +40 mV. Peak amplitudes of tail currents were measured at −40 mV, plotted as a function of test potential, and fitted with a Boltzmann function. In this case, we used the data at the partial range (from −70 to +10 mV) because the relative current decreased at more positive potential. In the control experiment, the activation curve had a midpoint of −20.5 ± 1.1 mV and a slope factor of 6.6 ± 0.4 mV (n = 6), which is similar to values reported by other investigators (Wang et al., 1997). Addition of KCB-328 (1 μM) resulted in a decrease of the tail current amplitudes. However, there was no significant change in voltage dependence of the activation curve (n = 6, data not shown). When the concentration of KCB-328 was increased, the amplitude of the peak tail current was decreased, indicating that the maximum conductance of HERG channels is decreased by KCB-328. It was also noticed that in the presence of KCB-328, tail current did not reach the steady-state level but declined at more positive potentials, indicating that the blocking effect is more pronounced at positive potential.

This result may suggest that the effect of KCB-328 is voltage-dependent, and we tested this possibility in Fig.3. We compared the decrease of tail current by KCB-328 at different potentials and found a higher degree of block at more positive voltages (Fig. 3, A and B). At −30 mV, KCB-328 (3 μM) reduced peak tail current amplitude by 41 ± 5% (n = 6, P < 0.01), whereas at +30 mV, it reduced peak tail current amplitude by 74 ± 3% (n = 6, P < 0.01). Dose-response relationships obtained at −30, 0, and +30 mV are demonstrated in Fig.3C. Data were fitted with a Hill equation, and IC₅₀ values for KCB-328 block of HERG current were obtained at different potentials. IC₅₀values at −30, −20, −10, 0, +10, +20, +30, and +40 mV were 7.6 ± 0.5, 4.8 ± 0.4, 3.2 ± 0.3, 2.1 ± 0.3, 1.7 ± 0.2, 1.4 ± 0.2, 1.3 ± 0.1, and 1.2 ± 0.1 μM, respectively (n = 6). These data indicate that KCB-328 block of HERG current is voltage-dependent.

To determine possible mechanisms for KCB-328 block of HERG current, the extent of KCB-328 block was measured on repetitive depolarizations (Fig. 4). After the control currents were measured, the membrane potential was held at −80 mV to keep the channel in the closed conformation during wash-in of 3 μM KCB-328. The first depolarization to 0 mV after 10 min at −80 mV yielded little reduction in current (by 17.0 ± 4.6% of the amplitude of peak tail currents before KCB-328, n = 6). Subsequently, block developed on a pulse-by-pulse basis (30-s interval), requiring an additional 20 pulses for 10 min to achieve steady-state block. These results indicate that HERG block of KCB-328 requires the channel activation.

To directly test whether channel activation is required for the slowly developing block by KCB-328, the onset of open-channel block by a high concentration of KCB-328 (100 μM) was assessed using a single 20-s pulse to 0 mV (Fig. 5). The oocyte was held at −80 mV for 10 min during exposure to 100 μM KCB-328. During the first depolarizing step in the presence of KCB-328, the current was initially activated as in the control but subsequently displayed a time-dependent decline with a time constant of 1.6 s. With this paradigm, the time constant for onset of block averaged 1.7 ± 0.3 s (n = 6) for 100 μM KCB-328. The complete suppression of the subsequent tail current confirmed that all HERG current had been blocked during this single depolarizing step (Fig.5A). To evaluate whether stronger depolarizations would enhance block, we clamped the membrane potential at +80 mV (Fig. 5B). Interestingly, unlike the time-dependent decline of current at 0 mV in the presence of 100 μM KCB-328, the current at + 80 mV declined initially and then plateaued. It resumed its exponential time course when the potential was restored to 0 mV. This apparent delay of block was quantified by the horizontal shift of the exponential fits (Fig. 5B). The average delay was 1.8 ± 0.6 s (n = 6) with the 2-s intervening depolarization. These results indicate that the strong inactivation of the channel prevented HERG block of KCB-328.

Figure 6 illustrates the frequency-independent block of HERG currents by KCB-328. Twenty repetitive 100-ms depolarizing pulses of +10 mV from a holding potential of −80 mV were applied at three different frequencies of 1, 3, and 10 Hz. In the presence of KCB-328 (10 μM), the peak amplitudes of tail currents measured at −40 mV decreased by 56 ± 5% (n = 3), 57 ± 3%, and 59 ± 6% (n = 3) at 1, 3, and 10 Hz, respectively, in a frequency-independent manner.

Discussion

We have shown that KCB-328 blocks HERG current expressed inXenopus oocytes. KCB-328 has been demonstrated to be highly effective against a variety of arrhythmias in various animals (Xue et al., 1998a,b). It also prolonged APD and inhibited I_K (Lee et al., 1998). It is well known that heterologously expressed HERG currents share pharmacological and biophysical properties with I_Kr (Kiehn et al., 1995; Sanguinetti et al., 1995; Spector et al., 1996; Wang et al., 1997). The characteristics of the current recorded in the present study correspond to HERG current: slow current activation at negative potentials, large, long-lasting tail currents on repolarization, strong inward rectification and sensitivity to class III methanesulfonanilides such as MK-499 and dofetilide. Considering all of these, KCB-328 may induce APD prolongation through block of HERG as do class III methanesulfonanilides such as dofetilide and MK-499 do (Carmeliet, 1992; Snyders and Chaudhary, 1996; Spector et al., 1996). The present study is the first to characterize the interaction between KCB-328 and the HERG channels. The major finding of the present study is that KCB-328 blocks primarily the activated (open) HERG channels in a time-, voltage-, and concentration-dependent manner. This finding provides an ionic mechanism for the antiarrhythmic effect of KCB-328. In other words, all of these indicate that HERG is one of targets of KCB-328.

To clarify biophysical mechanism of HERG block by KCB-328, we analyzed the effect of KCB-328 on HERG current using various pulse protocols. The results suggest that KCB-328 block preferentially the activated HERG channels. HERG was not significantly blocked before activation because the first depolarizing pulse to 0 mV after holding at −80 mV for 10 min in the presence of KCB-328 (3 μM) reached a peak amplitude of ∼75% as shown in Fig. 4. Furthermore, KCB-328-induced HERG block was prevented when the channel was rectified or inactivated at +80 mV (Fig. 5B). At low concentrations, block subsequently developed in a pulse-dependent manner but time-dependent block was not apparent (Fig.4). To directly demonstrate a time-dependent blockade during a single depolarization, high concentrations (> 30 μM) of KCB-328 were needed as shown in Fig. 5. These results provide direct evidence for time-dependent open channel block of KCB-328, although it remains to be determined whether KCB-328 is a pure open-channel blocker of HERG. Additionally, KCB-328-induced block was not frequency-dependent (Fig.6), which may be due to a partly irreversible block or slow recovery from block.

Block of KCB-328 was more pronounced at more positive potential and IC₅₀ of KCB-328 in blocking HERG was significantly different at all potentials examined (Fig. 3). These results suggest that the block of HERG by KCB-328 is voltage-dependent. However, KCB-328 (IC₅₀ of 2.1 μM) was less potent than dofetilide at 0 mV (IC₅₀ of 0.3–0.6 μM; Kiehn et al., 1996; Ficker et al., 1998) in blocking HERG expressed in Xenopus oocyte. These results indicate that the blockade produced by KCB-328 is similar to that previously reported with dofetilide except the potency (Kiehn et al., 1996; Snyders and Chaudhary, 1996; Ficker et al., 1998). This may be due to the structural similarity between KCB-328 and dofetilide.

An ideal class III antiarrhythmic agent would selectively prolong the ventricular action potential at increased heart rates. Currently available class III antiarrhythmic agents do not possess this desired rate dependence. Therapeutic potentials of class III antiarrhythmic drugs are limited by their tendency to cause LQT and cardiac sudden death. Methanesulfonanilides of class III antiarrhythmic drugs such as dofetilide, E-4031, ibutilide, and MK-499 selectively act on I_Kr and prolong APD (Jurkiewicz and Sanguinetti, 1993; Krafte and Volberg, 1994; Yang et al., 1995). KCB-328 also prolonged APD and inhibited I_K (Lee et al., 1998). Furthermore, it was shown that KCB-328 suppressed ventricular fibrillation induced by coronary artery ligation and reperfusion, and programmed electrical stimulation-induced ventricular arrhythmias with old myocardial infarction in an intravenous infusion of 0.5 mg/kg/30 min (Xue et al., 1998a). However, KCB-328 in a similar condition (0.3 mg/kg/30 min) was also reported to induce arrhythmias and intensify proarrhythmic effects of adrenaline, although KCB-328 is weaker than MS-551 in prolonging QT intervals at the same doses (Xue et al., 1998b). In view of the above, with our result of KCB-328 at a micromolar concentration, we expect that KCB-328 might produce antiarrhythmic and proarrythmic effects through blockade of HERG. The difference in effective concentrations of KCB-328 in our present study from the previous reports (Xue et al., 1998a,b) may have originated from the different experimental condition.

In the present study, we first reported that KCB-328 blocks HERG current in a time-, voltage-, and concentration-dependent manner, which may account for antiarrhythmic effects of KCB-328 (Xue et al., 1998a) as well as for its proarrhythmic effect previously reported (Xue et al., 1998b). However, there are several limitations in our study. First, we are unable to provide information regarding the detailed structural determinants of drug binding and blocking. Extensive mutational study is required to address this question. Second, our data do not exclude the possibility of closed state-dependent block by KCB-328. Third, the effect of KCB-328 on the channels formed by coexpression of KCNE2 (encoding MiRP1) and HERG should be addressed because HERG channels coassembled with MiRP1 resemble native cardiac I_Kr channels (Abbott et al., 1999). Furthermore, it would be greatly helpful for understanding the mechanisms of the drug actions and developing class III antiarrhythmic drugs if the effect of KCB-328 on action potential and K⁺ current in human cardiac myocytes were examined.