State dependent dissociation of HERG channel inhibitors

Abstract

Background and purpose: Inhibition of HERG channels prolongs the ventricular action potential and the QT interval with the risk of torsade de pointes arrhythmias and sudden cardiac death. Many drugs induce greater inhibition of HERG channels when the cell membrane is depolarized frequently. The dependence of inhibition on the pulsing rate may yield different IC(50) values at different frequencies and thus affect the quantification of HERG channel block. We systematically compared the kinetics of HERG channel inhibition and recovery from block by 8 blockers at different frequencies.

Experimental approach: HERG channels were expressed heterologously in Xenopus oocytes and currents were measured with the two-electrode voltage clamp technique.

Key results: Frequency-dependent block was observed for amiodarone, cisapride, droperidol and haloperidol (group 1) whereas bepridil, domperidone, E-4031 and terfenadine (group 2) induced similar pulse-dependent block at all frequencies. With the group 1 compounds, HERG channels recovered from block in the presence of drug (recovery being voltage-dependent). No substantial recovery from block was observed with the second group of compounds. Washing out of bepridil, domperidone, E-4031 and terfenadine was substantially augmented by frequent pulsing. Mutation D540K in the HERG channel (which exhibits reopening at negative voltages) facilitated recovery from block by these compounds at -140 mV.

Conclusion and implications: Drug molecules dissociate at different rates from open and closed HERG channels ('use-dependent' dissociation). Our data suggest that apparently 'trapped' drugs (group 2) dissociated from the open channel state whereas group 1 compounds dissociated from open and resting states.

Figure 1

Different kinetics of human ether-a-go-go-related gene (HERG) channel inhibition. (a) The development of channel inhibition by haloperidol (3 μM), bepridil (3 μM), compound E-4031 (3 μM) and domperidone (3 μM) during repetitive stimulation at a frequency of 0.3 Hz. Pulse trains were applied after a 3 min equilibration period in drug without stimulation. HERG currents were evoked by a repolarizing step to −40 mV after a 300 ms conditioning pulse to 20 mV (see inset in b). Peak HERG tail currents are plotted vs pulse number during a train. Steady-state inhibition by E-4031 was achieved after 180 pulses (see interrupted x axis) whereas steady-state block by haloperidol occurred within five pulses. (b) Representative currents illustrating the difference on rates. Ultra-slow inhibition of HERG current by E-4031 is illustrated by plotting every 10th current.

Figure 2

Frequency-dependent human ether-a-go-go-related gene channel inhibition. (a) Representative current traces during pulse trains of indicated frequencies in the presence of amiodarone (10 μM). The voltage protocol is shown in the inset (top left). Normalized peak current values are plotted against pulse number for (b) amiodarone (10 μM) and (c) haloperidol (3 μM), cisapride (3 μM) and droperidol (2 μM). Lines represent fit to single exponential functions (I_peak=A exp(−N/N_const)+I_ss). The steepness parameters N_const and steady-state levels are given in Table 1.

Figure 3

Frequency-independent human ether-a-go-go-related gene channel inhibition. (a) Representative current traces during pulse trains of indicated frequencies in the presence of domperidone (3 μM). The voltage protocol is the same as in Figure 2. Normalized peak current values are plotted vs pulse number for (b) domperidone; (c) bepridil (3 μM), E-4031 (10 μM, every 10th pulse is shown) and terfenadine (1 μM, every 10th current is shown). Lines represent fits to single exponential functions (I_peak=A·exp(−N/N_const)+I_ss). The steepness parameters N_const and steady-state levels are given in Table 1.

Figure 4

Recovery of human ether-a-go-go-related gene channel from block at rest. Channel block was induced by 1 Hz pulse trains (see inset and Figure 1). Ten conditioning pulses were applied to reach steady-state inhibition by amiodarone, cisapride, haloperidol, droperidole, domperidone, bepridil. A total of 100 pulses were required for E-4031 and terfenadine. Single test pulses were applied after a 330 s rest at −80 mV. All experiments were performed in the continued presence of drug. (a) Recovery from block by 10 μM amiodarone. Superimposed current traces were elicited by the 10th pulse of the conditioning train (0 s) and a single test pulse after a 330 s rest at −80 mV. (b) Lack of recovery from block by 3 μM bepridil. Superimposed traces of the current during the 10th pulse and the test pulse after the 330 s rest period at −80 mV. (c) Recovery after 330 s from block by amiodarone (n=7), cisapride (n=9), haloperidol (n=7), droperidol (n=6), domperidone (n=3), bepridil (n=3), E-4031 (n=3) and terfenadine (n=3).

Figure 5

Recovery from human ether-a-go-go-related gene channel block at different holding potentials. Recovery (normalized to maximum current amplitude in drug, see a and b) plotted vs recovery time. Solid lines are mono-exponential fits for recovery (a) in 10 μM amiodarone, (b) in 3 μM cisapride, (c) in 2 μM droperidol and (d) in 3 μM haloperidol at −80, −100 and −120 mV. The time constants (τ_recovery) are given in Table 2. A rest period of 5 min was introduced between each recovery protocol.

Figure 6

Repetitive stimulation accelerates washout of group 2 compounds. (a–d) Washout at a holding potential of −80 mV during infrequent pulsing (test pulses applied with an interval of 10 min), during 0.03 Hz pulsing and frequent stimulation at 1 Hz. Peak currents were normalized to the control (the tail current amplitude before application of drug).

Figure 7

Onset and recovery from block of the human ether-a-go-go-related gene (HERG) channel mutant D540 K by 3 μM terfenadine (a) and 10 μM domperidone (b). Channel block induced by repetitive pulsing to 0 mV (20 pulses after 3 min of equilibration without pulsing) is recovered from repetitive pulsing to −140 mV. Top: voltage protocol (5 s pulses to 0 or −140 mV, applied at 15 s intervals). Middle: Representative current traces. Labels a, b: superimposed 1st and 20th traces during the train of depolarizing pulses to 0 mV. Labels c and d: 1st and 20th traces during the train of hyperpolarizing pulses to −140 mV. Label e: 1st trace after recovery. Bottom: Plot of peak outward HERG current against pulse number.

Figure 8

Concentration/inhibition curves for the frequency-dependent inhibitor amiodarone (a) and the frequency-independent inhibitor domperidone (b). Human ether-a-go-go-related gene currents were evoked by either 0.03 or 1 Hz trains. Steady-state values of peak tail current are plotted against drug concentration. The IC₅₀ values for amiodarone were 7.17±2.05 μM, n_H=1.4, (0.03 Hz) and 1.08±0.65 μM, n_H=1.43 (0.3 Hz). The corresponding values for domperidone were 4.6±1.4 μM, n_H=2.29 (0.03 Hz) and 5.6±0.4 μM, n_H=1.47 (0.3 Hz).