The Effect of a Synthetic Estrogen, Ethinylestradiol, on the hERG Block by E-4031

Abstract

Inhibition of K⁺-conductance through the human ether-a-go-go related gene (hERG) channel leads to QT prolongation and is associated with cardiac arrhythmias. We previously reported that physiological concentrations of some estrogens partially suppress the hERG channel currents by interacting with the S6 residue F656 and increase the sensitivity of hERG blockade by E-4031. Although these studies suggested that clinically used synthetic estrogens with similar structures have the marked potential to alter hERG functions, the hERG interactions with synthetic estrogens have not been assessed. We therefore examined whether ethinylestradiol (EE2), a synthetic estrogen used in oral contraceptives, affects hERG function and blockade by drugs. Supratherapeutic concentrations of EE2 did not alter amplitudes or kinetics of the hERG currents elicited by train pulses at 20 mV (0.1 Hz). On the other hand, EE2 at therapeutic concentrations reduced the degree of hERG current suppression by E-4031. The administration of EE2 followed by E-4031 blockade reversed the current suppression, suggesting that the interaction of EE2 and E-4031 alters hERG at the drug-binding site. The effects of EE2 on hERG blockade raised the possibility that other estrogens, including synthetic estrogens, can alter hERG blockade by drugs that cause QT prolongation and ventricular arrhythmias.

Keywords: QT intervals; cardiac potassium channel; drug interaction; hERG blocker; synthetic estrogen.

Figure 1

Effects of the external application of ethinylestradiol, EE2, on hERG channel currents. Membrane currents were recorded from HEK293 cells stably expressing hERG. The hERG channels were sequentially activated at 0.1 Hz by 2-s test pulses from a holding potential (V_h) at −80 mV. (A), Chemical structures of E2 (left) and EE2 (right). (B), Representative traces before and after the application of EE2 (upper; 1 nM, lower; 10 nM) for 4 min. (C), Time courses of the effects of EE2 at 0.1 nM (open triangles, n = 5), 1 nM (closed triangles, n = 8), and 10 nM (n = 7), and E2 at 3 nM (closed triangles, n = 5). After stabilizing the tail amplitudes for 1 min, currents were recorded in the presence of each concentration of EE2 in the bath solution. Plots (means ± S.E.M.) are shown as ratios of the peak tail amplitudes just before the application of estrogens (control at time zero). (D–G), No effect on current-voltage relationships of the hERG channel elicited by a series of 2-s test pulses from −50 to 60 mV (10-mV increments, 0.1 Hz). (D), Representative superimposed traces at step pulses (2-s test pulses, −40 mV return, V_h = −80 mV) from a single cell before (left) and after a 5-min cumulative application of EE2 from 1 nM (middle) to 10 nM (right). (E), Current–voltage relationship at the end of test pulses. (F), Current–voltage relationship of tail peak currents recorded at −40 mV. (G), Normalized peaks of tail currents were plotted as a function of the hERG activation (Boltzmann fitting). Eighteen cells were used. Data are summarized in Table 1.

Figure 2

Blockade of hERG currents by E-4031 with or without EE2. hERG currents were recorded from HEK293 cells using the scheme as that described in Figure 1D. The hERG channels were sequentially activated at 0.1 Hz by 20-mV test pulses for 2 s from a holding potential (V_h) at −80 mV, whereas tail currents were recorded with a repolarizing step to −40 mV. EE2 at 1 nM was administered 5 min prior to the cumulative application of E-4031. (A). Representative traces in the presence of EtOH (left) and EE2 (right) are shown by superimposing the traces before (control) and after the addition of E-4031 (3, 30 and 300 nM). Scale, 10 pA/pF, 1 s. (B). Time course of hERG inhibition by cumulative application of E-4031 in the presence of 0.0001% EtOH (left) and 1 nM EE2 (right). After the current amplitudes were stabilized for 1 min, E-4031 was added in the presence of EtOH or EE2. The timing of E-4031 applications (3, 30, and 300 nM) indicated by the arrowheads above the plot. (C). Concentration-dependent inhibition of E-4031 was plotted as relative values of the tail amplitudes compared to the right before the application of E-4031. Colored lines indicate the presence of EE2. Control (no EE2); n = 8, EE2 at 1 nM; n = 8, EE2 at 10 nM; n = 8.

Figure 3

Effects of EE2 on blockade of hERG currents by E-4031. HERG currents were recorded from HEK293 cells stably expressing hERG as described in Figure 1. (A). Time course of hERG tail current blockade by E-4031 at 30 nM and the following recovery by the addition of EE2 at 1 nM. Plots from a representative experiment are normalized by the tail amplitude before E-4031 application (time 0). The current-voltage (I–V) relationships were tested before drug application (control, black), after E-4031 block (blue) and after the addition of EE2 (E-4031 + EE2, green). (B). Representative traces elicited by 20-mV test pulse (closed circles in I–V relationships) are shown by superimposing the traces before (control, black) and after the application of E-4031 only (blue) and E-4031 plus EE2 (green). Scale, 5 pA/pF, 1 s.

Figure 4

Effects of EE2 on I–V relationships of hERG currents by E-4031. I–V relationships of the hERG channel were activated by the same voltage protocol described in Figure 1D, and compared before drug application (control, black), after 30 nM E-4031 block (blue), and after the addition of EE2 (30 nM E-4031 + 1 nM EE2, green). (A), Representative traces elicited by step pulses (−40 mV to 60 mV). Tail peaks are indicated by arrows. Scale, 20 pA/pF, 1 s. (B), Tail I-V curves before (control), after the application of E-4031, and after the addition of EE2 (E-4031 + EE2). p < 0.05 ANOVA with repeated measures. * p < 0.05 vs. control. (C), Respective tail peak amplitudes were normalized to the tail amplitude preceding 60 mV before drug applications (control). p < 0.05 ANOVA with repeated measures. * p < 0.05, ** p < 0.01, vs. control, # p < 0.05 vs. E-4031. (D), Comparison of channel availability curves obtained from tail I-Vs. Smooth lines are Boltzmann fits, as described in the Methods, that generated V0.5 of activation in Control (black line), in E-4031 (blue line), and in E-4031/EE2 (green line); ns, ANOVA. Seven experiments were performed. Significance was evaluated using Student’s t-tests after repeated measures one-way ANOVA.