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Original Article

Effects of Na+ Channel Blocker, Pilsicainide, on HERG Current Expressed in HEK-293 Cells

Wu, Long-Mei MD; Orikabe, Minako MD; Hirano, Yuji MD, PhD; Kawano, Seiko MD, PhD; Hiraoka, Masayasu MD, PhD

Author Information
Journal of Cardiovascular Pharmacology: September 2003 - Volume 42 - Issue 3 - p 410-418
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Abstract

Several potassium currents are involved in action potential repolarization of cardiac cells. 1,2 The rapidly activating component of delayed rectifier K+ current, Ikr, is a main component to determine the final repolarization (phase 3) of action potentials for most species with a few exceptions. Ikr is encoded by HERG, the human ether-a-go-go-related gene. 3,4 Abnormalities of HERG genes having a loss of channel function cause one form of congenital long QT syndrome (LQT2). 4,5 In addition, HERG current in heterologous expression system and Ikr in native cardiac myocytes are inhibited by various drugs including the class III and several class I antiarrhythmic agents. 6–17 Block of Ikr is implicated as a main mechanism of antiarrhythmic actions for the class III drugs. In certain settings, however, the block may be arrhythmogenic due to excessive action potential lengthening at the cellular levels and prolongation of QT intervals on surface electrocardiogram leading to the development of torsades de pointes and polymorphic ventricular tachycardias.

The antiarrhythmic drug, pilsicainide (PIL), has a main effect of inhibiting voltage-dependent Na+ channel, and is claimed to exhibit its least effect on K+ and Ca2+ currents at therapeutic concentrations. 18–21 Thus, the drug is interpreted to exert its antiarrhythmic action clinically mainly through a pure Na+ channel blockade and does not cause prolongation of action potential duration (APD). Actually, PIL has been shown to exert antiarrhythmic efficacy on various experimental as well as clinical arrhythmias. 22–24 There have been, however, case reports describing QT prolongation by PIL in patients with renal failure or concomitant use of other QT-prolonging drugs. 25,26 This may indicate that PIL has effects on K+ currents with a relatively weak potency and the effects may become manifest at increased plasma drug concentrations or during concomitant use of QT-prolonging drugs. Since actions of PIL on Ikr in native cardiac myocytes or HERG current in heterologous expression system have not been reported, we studied its effects on the channel current of HERG stably expressed in HEK293 cells.

METHODS

Stable Expression System in HEK 293 Cells

Stably transfected HEK 293 cells by HERG were a gift from Dr. Craig T January (University of Wisconsin). The transfection techniques have been published elsewhere. 27 Briefly, HERG cDNA was sub cloned into Bam HI/ Eco RI sites of the pCDNA3 vector (Invitrogen). HEK 293 cells were transfected with this construct using the lipofectamine method (GIBCO), and the stably transfected cells were sub cloned to achieve a uniform HERG expression level. The cells were cultured in MEM supplemented with 10% FBS and 0.8% geneticin. The cells were harvested from the culture dish by trypsinization, washed twice with standard MEM, and stored in this medium at room temperature for electrophysiological study.

Method for Patch-Clamp Recording

Cells were allowed to settle on the bottom of a bath (< 0.5mL) mounted on an inverted microscope (Diaphoto TMD, Nikon, Tokyo). Cells were superfused with Tyrode's solution containing (in mmol/L) 137 NaCl, 4 KCl, 1.8 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES (pH = 7.4 adjusted with NaOH). Solution exchanges of the bath were completed within 40 seconds. Currents were recorded by the whole-cell patch-clamp configuration. The glass pipettes had inner diameter of ˜1.0 to 1.5 μm and resistance of 2 to 3 MΩ when filled with the internal solution. After forming a whole cell configuration, cell membrane capacitance was estimated by analyzing capacity transient elicited by 5-mV hyperpolarizing pulses. Series resistance compensation was done by 50 to 70% using the circuit built into the amplifier (Axopatch 200B, Axon Instruments, Foster City, CA). The internal solution contained (in mmol/L) 110 K-gluconate, 20 KCl, 1 MgCl2, 5 EGTA 5 MgATP, and 10 HEPES (pH = 7.2 adjusted with KOH). A patch-clamp amplifier (Axopatch 200B, Axon Instruments) was used to record membrane currents. Software (pCLAMP 8.0, Axon Instruments) was used to generate voltage protocols, to acquire data, and to analyze current signals. All experiments were done at temperature of 34 ± 1.0°C except for analysis of inactivation properties as indicated in the text. Pilsicainide (a gift from Suntory Pharmaceut. Co., Osaka) was prepared from the stock solution of 5 mmol/L in distilled water and diluted into Tyrode's solution as the final concentrations indicated in the text before every experiment.

pCLAMP software was used to analyze HERG current and fit current tracings to exponential functions. The voltage dependence of HERG current activation was determined by fitting peak values of tail current (Itail) versus test potential (Vt) to a Boltzmann function:

where Itail-max is maximum tail current. The voltage at which the current is half maximal activated (V1/2) and the slope factor (k) were calculated from these data.

Deactivating currents were fitted to a double-exponential function. Inactivating currents and currents recovering from inactivation were fitted to a single- or a double-exponential function using a least squares algorithm. Steady-state inactivation was analyzed as described previously. 28 At negative potential, the current decline due to significant closing of channels occurred through deactivation. This was corrected for by extrapolating the exponential falling phase back to the start of the negative conditioning pulses. The steady-state inactivation curves were fitted with a Boltzmann function in the following form:

Where I is the amplitude of inactivating current corrected for deactivation, Imax is the maximum of I, Imin is the minimum of I, Vt is the prepulse of test potential, V1/2 is the voltage at which I is half of Imax and k is the slope factor.

The percent block of HERG tail current by different concentrations of PIL relative to the control was a fit with a Hill equation: relative current = 1/{([drug]/IC50)n + 1} to determine the concentration required for half block (IC50) and Hill coefficient (n). IC50 values were determined from tail current measurements after test depolarization to + 50 mV for 4 seconds followed by repolarization to –50 mV for 6 seconds.

Statistical Analysis

All data are expressed as means ± SE. Student t tests for paired and unpaired groups, and one-way ANOVA with multiple comparison method were performed for statistical evaluation. P < 0.05 was considered to indicate a significant difference.

RESULTS

Effects of Pilsicainide on HERG Current Stably Expressed in HEK293 Cells

Figure 1 illustrates the effect of pilsicainide (PIL) on HERG current during equilibration with 10 and 20 μM PIL (pulses applied every 15 seconds). Currents during depolarizing pulses (peak current) as well as tail currents upon repolarization were gradually suppressed. Suppression of HERG tail current by 10 μM PIL developed with an approximate half time of 2 minutes reaching a steady state after ˜5 minutes. Addition of 20 μM PIL further suppressed the current. During the application of 10 and 20 μM PIL, the peak currents were suppressed at test voltages positive to –10 mV, and the maximum current decreased to 71.6% of the control by 10 μM and 45.0% by 20 μM PIL. The maximum tail currents upon repolarization to –50 mV were also reduced by the drugs to 61.8% and to 48.2% of the control, respectively (n = 7) (Fig. 2 A,B). The current recovered to 70.0% of the control (n = 6) after 10 to 15 minutes of the drug (20 μM PIL) washout, leaving an incomplete recovery (Fig. 1A).

F1-13
FIGURE 1.:
Effects of 10 and 20 μM pilsicainide on HERG current expressed in a HEK 293 cell. A, Time course of the HERG tail current block by 10 and 20 μM pilsicainide (PIL). Pulse protocol indicated in the inset. Test depolarization to +30 mV for 4 seconds from a holding potential of -60 mV was followed by repolarization to -50 mV for 6 seconds applied every 15 seconds. Relative tail current was plotted against time. Application and washout of PIL were indicated on the top. Con, control. B, Superimposed current traces elicited by test depolarization from a holding potential of -60 mV to -40 mV cc+50 mV by 10-mV increment steps for 4 seconds followed by repolarization to -50 mV for 2 seconds. Test pulses were applied every 15 seconds. The current traces (a), (b), and (c) were recorded at indicated time in (A).
F2-13
FIGURE 2.:
Effects of 10 and 20 μM PIL on I-V relationships of HERG current and concentration-response curve. A, Peak currents during depolarizing test pulses. B, Tail currents upon repolarization to -50 mV. Summarized data from 7 cells. Data were obtained by a pulse protocol shown in Fig.1 (B). * P < 0.05 versus control. ** P < 0.01 versus control. C, Dose-response curve of the HERG tail current block by PIL. Dose-response curve was fitted to the Hill equation expressed by a solid line with the data at each concentration of PIL with percent tail current block. Each data point represents a mean of 5 to 9 measurements and SE (error bars). Pulse protocol was the same as the one shown in the inset of Figure 1 (A).

Concentration-dependent block of HERG current was constructed by measuring percentage block of the maximum tail currents with various concentrations of PIL and the dose-response curve was a fit to a Hill equation with an IC50 of 20.4 ± 1.1 μM and Hill coefficient of 0.98 ± 0.04 (Fig. 2C).

Voltage- and Frequency-dependent Block of HERG Current by Pilsicainide

Voltage dependence of activation was examined by plotting the relative tail current to the maximum at each test voltages from the data shown in Fig. 2B (Fig. 3A). In control, a sigmoid activation was achieved with a half activation voltage (V1/2) = –16.3 ± 0.3 mV and slope factor = –7.8 ± 0.3 mV. After equilibration of 10 μM PIL, the voltage-dependent activation shifted in a negative direction (V1/2 = –24.9 ± 0.4 mV) without a change in slope factor (–8.0 ± 0.4 mV). An increase in the concentration of PIL to 20 μM did not further shift activation voltage (V1/2 = –25.4 ± 0.7 mV and slope factor = –8.3 ± 0.7 mV; n = 7). To confirm whether shift in activation curve was caused by real drug effect or by other non-specific factors such as cell swelling due to patch break, another set of experiments were performed. In 5 control cells, activation curve was measured twice, the first time after 2 to 3 minutes of patch break and the second time after 15 minutes of the first measurement, in the absence of the drug. The 2 curves were super-imposable and no time-dependent shift in activation curve was seen (Fig. 3A, inset). Therefore, we believe shift in activation curve after PIL representing drug-induced changes.

F3-13
FIGURE 3.:
Effects of PIL on voltage dependence of activation and voltage-dependent block. A, Activation curves of HERG current was constructed by normalization of tail currents at each test voltages to the maximum. The average value of tail currents in Figure 2 (B) was fitted to a Boltzmann function. Activation curve was shifted in a negative direction by about 10 mV in the presence of 10 μM PIL and no further shift was seen in 20 μM PIL. The inset shows activation curves recorded from control cells (n = 5) in the absence of PIL after 2 to 3 minutes of patch break (open triangle) and after 15 minutes (closed triangle). B, Voltage-dependent block by PIL was obtained by expressing percent block of tail currents (1 - Idrug / Icontrol) at each test voltage from the experiments shown in Fig. 2 (B) (n = 7).

Voltage-dependent block was examined with calculating relative tail current amplitudes at each test voltage during the drug application to the control (Fig. 3B). At test voltage of –40 and –30 mV, no block of tail currents by 10 μM PIL was observed. At test voltages between –20 and 0 mV, block increased with increased depolarization, while further increase in test depolarization positive to 0 mV did not further augment the degree of block. In the presence of 20 μM PIL, a similar voltage dependence was observed at negative voltages with increased block by test depolarization up to 0 mV and the block saturated at positive voltages without further increase. The degree of block (1 – Idrug / Icontrol) was 0.38 ± 0.13 (n = 7) by10 μM and it was 0.53 ± 0.09 by 20 μM PIL at +50 mV (n = 7).

Frequency-dependent block of HERG current was examined by applying 3 different frequencies of pulsation (1.33, 0.5, and 0.2 Hz) after the wash-in PIL for 10 minutes while the holding potential was held constant at –100 mV. Twenty (or 10 at 0.2 Hz) test pulses with depolarization to +30 mV for 200 milliseconds followed by repolarization to –50 mV for 200 milliseconds were applied with 3 different frequencies. Tail current amplitudes upon repolarization to –50 mV were measured at each pulse. The tail current evoked by the first pulse after 10 minute-perfusion of 20 μM PIL was already substantially decreased compared with the control, and no further decrease in current amplitudes with successive pulses was noted, while a stable current amplitude in successive pulses was seen in the absence of PIL (control) (Fig. 4A). In addition, the degrees of block at the 20th (or 10th at 0.2 Hz) pulse by 3 different frequencies of pulsation were almost equal and no frequency-dependent block was apparent. The relative tail current values of the last test pulse were 0.48 ± 0.08 at 1.35 Hz, 0.52 ± 0.10 at 0.5 Hz and 0.54 ± 0.07 at 0.2 Hz (n = 6, NS by ANOVA).

F4-13
FIGURE 4.:
Frequency-dependent block of HERG current by PIL. A, Development of block with 20 successive numbers of pulses (10 pulses at 0.2 Hz) from a holding potential of -100 mV to +30 mV for 200 milliseconds and repolarization to -50 mV for 200 milliseconds at 3 different frequencies. Tail current amplitudes of each pulse were measured at -50 mV and plotted versus pulse numbers (n = 6). B, Superimposed current traces in control and in the presence of the drug elicited by the first pulse after 10-minute application, while a holding potential was kept constant at -100 mV. Note that the initial portion of the current during the first depolarizing test pulse in the drug was not different from the current level in control, and block developed thereafter to decrease the peak as well as the tail current.

These results may indicate that PIL has a tonic block with an affinity to the rested channels. However, a close examination of the first current during the test depolarization in the drug showed no inhibition at the initial 20˜30 milliseconds of the depolarizing pulse, and the block developed in a time-dependent fashion thereafter, excluding a possibility of tonic block. The tail current upon repolarization was substantially suppressed (Fig. 4B) and no further increase in inhibition of the tail current was observed up to 20th pulse. Similar results were confirmed in all 6 examined preparations. It was also noted the decay of the tail current in 20 μM PIL was slower than that in control (B).

Effects of Pilsicainide on Time Course of the HERG Current Activation

Effects of PIL on HERG current activation were examined using the envelope of the tail test in the absence and presence of 20 μM PIL after stable current inhibition was achieved (Fig. 5A). The time course of activation was fitted to a single exponential function in the presence and absence of drug (B). At test voltage between –10 and +30 mV, time course of current activation became slow with longer time constants in the presence of PIL than in its absence at all examined test voltages (C).

F5-13
FIGURE 5.:
Effects of PIL on time course of HERG current activation. A, Time course of HERG current activation was measured by envelope of tail test in the absence and presence of 20 μM PIL. Voltage protocol is indicated in the inset. Test depolarization to +30 mV with various durations (20 3400 milliseconds) followed by repolarization to -50 mV for 400 milliseconds was applied from a holding potential of -80 mV every 15 seconds. Superimposed current records in control (A-1) and in the presence of 20 μM PIL (A-2) are shown. B, Activation time constants were measured by fitting tail current amplitudes versus pulse durations with a single-exponential function from the experiments shown in (A). C, Time constants of activation at different test potentials. Voltage protocol was similar to the one shown in A with varying test potential levels. Time constants of activation became longer at voltages between -10 and + 30 mV after PIL (n = 7).

Deactivation was analyzed by applying long hyperpolarizing test pulses after a conditioning pulse to + 50 mV. Deactivating currents during test pulses could be fitted to a double-exponential function. In the presence of 20 μM PIL, the time constants of the slow component (τs) became longer than that in control, while the time constant of the fast component (τf) was unchanged (n = 7) (Fig. 6 B & C). Because of the slowed deactivation in the presence of PIL, the decay of the tail current showed “crossover” (not shown).

F6-13
FIGURE 6.:
Effects of PIL on HERG current deactivation. A, Superimposed tail currents in control (A-1) and in the presence of 20 μM PIL (A-2). Pulse protocol is indicated in the inset. A conditioning pulse to +50 mV for 2 seconds from a holding potential of -80 mV followed by repolarizing test pulses of 8 seconds to potentials from -80 to -40 mV by 10-mV increment steps was applied every 15 seconds. Current records represent responses to the final portion of conditioning pulse to +50 mV and the initial 1500 milliseconds of repolarizing test pulses. B, Time course of deactivation was fitted to a double-exponential function. Time constants of fast component (τf) versus repolarizing test potentials were plotted. C, Time constants of slow component (τs) versus repolarizing test potentials were plotted (n = 7). ** P < 0.01.

Effects of Pilsicainide on HERG Current Inactivation

To obtain better resolution of HERG current inactivation, experiments to analyze fast inactivation and steady state inactivation were performed at room temperature of 23 ± 1°C. The fast inactivation time course of HERG current was measured by applying a conditioning pulse to +50 mV for 1 second followed by a brief hyperpolarizing pulse (–120 mV for 5 milliseconds) to allow the HERG channel to recover from inactivation. Depolarizing test pulses were applied to record inactivating currents. The time course of fast inactivating currents could be fitted to a single-exponential function. PIL slightly but significantly shortened the time constants of the fast inactivation of HERG channel at all examined potentials (Fig. 7A).

F7-13
FIGURE 7.:
Effects of PIL on HERG current inactivation. A, Time constants of fast inactivation against test voltages. Pulse protocol is indicated in the inset. A conditioning pulse to +50 mV for 1000 milliseconds from a holding potential of -80 mV was followed by a hyperpolarizing pulse to -120 mV for 5 milliseconds, and subsequent depolarizing test pulses for 500 milliseconds to potentials between -40 and +30 mV in 10-mV steps were applied every 10 seconds (left inset). Inactivation time constants were measured by fitting inactivating currents during test pulse potentials with a single-exponential function (n = 4). The experiments were done at room temperature (23°C). * P < 0.05, ** P < 0.01 B, Normalized steady-state inactivation curves. Pulse protocol is indicated in the inset. A conditioning pulse to +30 mV for 1000 milliseconds was followed by short (3 milliseconds) test pulses between -160 mV and +20 mV in 10-mV increment steps, and a fixed depolarizing pulse to +30 mV for 1000 milliseconds was then applied every 10 seconds. Normalized inactivation curves as a function of test pulse potential were fitted with a Boltzmann function (n = 4). The experiments were performed at room temperature.

The steady-state inactivation curves of HERG channel were determined by fitting the initial current measured on return to +50 mV against test potentials between –160 and +30 mV by 10 mV step to a Boltzmann function. Steadystate inactivation curve had a tendency to shift in a negative direction after PIL, but this shift was not significant (Fig. 7B). In the control, V1/2 was -81.7 ± 0.7 mV and the slope factor was 22.7 ± 0.6 mV. In the presence of 20 μM PIL V1/2 was –83.5 ± 1.0 mV and the slope factor was 23.6 ± 0.9 mV (n = 4).

DISCUSSION

The present study demonstrated that PIL, representing a relatively pure Na+ channel blocker (IC50 = 10–20 μM), inhibited HERG current expressed in HEK 293 cells with the IC50 value of 20 μM. PIL also shifted the voltage dependence of activation, and delayed the time course of activation. The inactivation properties of HERG current were mildly affected. PIL seemed to have an affinity, at least, to the open state of the channels. The drug had a fast access to the HERG channels upon depolarization, but the dissociation from the drug-bound channels appeared to be very slow.

Pilsicainide, a lidocaine derivative, has as its main action to suppress the maximum upstroke velocity of action potential and the Na+ current of native cardiac cells. 18,19 The drug has been shown to affect action potential duration (APD) minimally and to exhibit almost no effects on K+ and Ca2+ currents at therapeutic drug concentrations. 18,20,21 Therefore, PIL is generally regarded as a pure Na+ channel blocker at therapeutic concentrations. As for the action on Ik, there have been no reports describing the effect of PIL on this current, or actions on Ikr, Iks, and HERG current, separately. Hence, this is the first report to describe the action of PIL on HERG channel current at comparable or slightly higher therapeutic concentrations.

Block of the HERG current by PIL increased with depolarization and reached a maximum level at depolarizing voltage levels with full channel activation. Prolongation of depolarizing pulse rapidly increased the channel inhibition reaching a plateau around ˜200 milliseconds, and the suppression of the tail current was maximal after 200-millisecond depolarization in the first pulse. PIL shifted activation curve of HERG channel, and delayed the time course of activation. Furthermore, the decay of tail current upon repolarization became slower with drug than in control, exhibiting “crossover”. All of these features are consistent with PIL as an open channel blocker of HERG channels. 9–14 However, the drug did not show frequency-dependent block at stimulation frequencies of 0.2, 0.5, and 1.33 Hz. The reason for frequency-independent inhibition can be explained by a fast access of the drug to open channels during depolarization faster than ˜200 milliseconds (Fig. 4B), but dissociation from drug-bound channels was slower than 5 seconds, since there was no recovery of the current at pulsing frequency of 0.2 Hz. The blocking properties of an open channel affinity and frequency-independent inhibition resembled those of other Na+ channel blockers including quinidine and flecainide. 7,9,13,14 These drugs were also considered to be open channel blockers with a fast access to the channels but with a slow dissociation from the blocked channels. Similar action to PIL was shared by vesnarinone, which is an open channel blocker of HERG channel with the IC50 value of 18 μM and shows a fast access and a slow dissociation from the blocked channels. 29 Methanesulfonanilides including dofetilide and E-4031 are also regarded as open channel blockers but they show frequency-dependent block, requiring several pulses to develop a full inhibition due to a relatively slow access to the channel. 10–12,14 However, azimilide, a methanesulfonanilide compound, has reverse frequency-dependent blocking properties. This may reflect that the binding sites of different drugs of diverse chemical structures differ in various locations of HERG channel 30, and/or allosteric interactions influence the mode of action of individual drugs.

A therapeutic blood concentration of PIL in the clinical setting has been reported to be around 1 to 3 μM. 22,23,31 The IC50 value of PIL for Na+ current is 10 ˜ 20 μM 18, and the one for HERG current is somewhat higher than the therapeutic level. Therefore, PIL may not induce the side effects of QT prolongation at therapeutic drug concentrations. Caution must be paid to avoid proarrhythmic effects, when the plasma concentration is expected to increase (e.g., in the settings of renal failure because of excretion of the drug from the kidney). 32

ACKNOWLEDGMENT

We acknowledge A. Mikawa for technical assistance and N. Fujita for secretarial assistance.

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Keywords:

activation of the HERG channel; Ikr; open channel block; QT prolongation; repolarization of action potential

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