hERG subunit composition determines differential drug sensitivity

Abstract

Background and purpose: The majority of human ether-a-go-go-related gene (hERG) screens aiming to minimize the risk of drug-induced long QT syndrome have been conducted using heterologous systems expressing the hERG 1a subunit, although both hERG 1a and 1b subunits contribute to the K+ channels producing the repolarizing current I(Kr) . We tested a range of compounds selected for their diversity to determine whether hERG 1a and 1a/1b channels exhibit different sensitivities that may influence safety margins or contribute to a stratified risk analysis.

Experimental approach: We used the IonWorks™ plate-based electrophysiology device to compare sensitivity of hERG 1a and 1a/1b channels stably expressed in HEK293 cells to 50 compounds previously shown to target hERG channels. Potency was determined as IC₅₀ values (µM) obtained from non-cumulative, eight-point concentration-effect curves of normalized data, fitted to the Hill equation. To minimize possible sources of variability, compound potency was assessed using test plates arranged in alternating columns of cells expressing hERG 1a and 1a/1b.

Key results: Although the potency of most compounds was similar for the two targets, some surprising differences were observed. Fluoxetine (Prozac) was more potent at blocking hERG 1a/1b than 1a channels, yielding a corresponding reduction in the safety margin. In contrast, E-4031 was a more potent blocker of hERG 1a compared with 1a/1b channels, as previously reported, as was dofetilide, another high-affinity blocker.

Conclusions and implications: The current assays may underestimate the risk of some drugs to cause torsades de pointes arrhythmia, and overestimate the risk of others.

Figure 1

Western blot showing hERG 1a and 1b protein stably expressed in HEK-293 cells and visualized with a C-terminal pan-hERG antibody (Novus Biologicals, Littleton, CO, USA). From the top, the signals represent mature (Golgi-glycosylated) hERG 1a at 155 kDa, immature (ER-resident) 1a at 135 kDa (Zhou et al., 1998), mature hERG 1b at 95 kDa, and immature 1b at 85 kDa (Jones et al., 2004).

Figure 2

Activation and deactivation properties of hERG 1a and 1a/1b channels measured using IonWorks™. (A and B) Typical traces of hERG 1a and 1a/1b currents elicited by the voltage protocol shown in the upper inset of (A). (C) Steady state activation plots. The V_1/2 and the slope factor for 1a channels are +8.4 ± 1.3 mV and +8.7 ± 1, respectively (n = 76); and for 1a/1b channels, +4.8 ± 1.1 mV and +8.9 ± 1, respectively (n = 108). (D) Time course of activation. Apparent activation is faster for hERG 1a/1b compared with hERG 1a currents. Time constants of activation were 1262 ± 134 ms and 1104 ± 82 ms for hERG 1a (n = 28–278) and 1a/1b (n = 41–748), respectively. (E) Deactivation is faster for hERG 1a/1b versus 1a currents. Scaled tail currents recorded at −105 mV are shown. (F) Time constants of fast and slow components from double exponential fits to deactivating tail currents are plotted for comparison between 1a and 1a/1b channels. **P < 0.001 versus hERG 1a values.

Figure 3

Inactivation properties of hERG 1a and 1a/1b channels measured using IonWorks™. (A and B) Typical traces of hERG currents elicited by the three-pulse protocol (upper inset of Figure 2A) to measure the time course of inactivation of 1a and 1a/1b channels. (C) There were no significant differences in the time constants of inactivation for hERG 1a (n = 20–24) and hERG 1a/1b (n = 17–24) channels (P > 0.05 versus hERG 1a values). (D) Plot quantifying data showing recovery from inactivation is faster in hERG 1a/1b (n = 29) compared with hERG 1a (n = 21) channels (P < 0.0001 vs. hERG 1a values). Tail currents were evoked by the protocol shown in the upper insert.

Figure 4

Concentration-response curves to encainide (A), E-4031 (B) and fluoxetine (C) against hERG 1a () or 1a/1b channels () measured using IonWorks™. The IC₅₀ values for these compounds are shown in Table 1. Although encainide exhibited similar potency for 1a and 1a/1b, E-4031 and fluoxetine exhibited greater potency for 1a (as previously reported by Sale et al., 2008) and 1a/1b respectively.

Figure 5

(A) The potency Bland-Altman plot to illustrate the difference in potency (pIC₅₀) as a function of the average potency of a compound for hERG 1a versus 1a/1b channels. The axis y = 0 represents no difference in the potency for the two hERG channels. Negative deviations represent greater potency for hERG 1a/1b versus 1a, whereas positive deviations represent greater potency for hERG 1a versus 1a/1b. 49 hERG blockers, previously selected for their diversity (Männikköet al., 2010), were tested. (B) Safety margin Bland-Altman plot illustrates the difference in margin as a function of the average margin of a compound for hERG 1a versus 1a/1b channels. The axis y = 0 represents no difference in the safety margin for the two hERG channels. Negative deviations represent lower margins for hERG 1a versus 1a/1b, whereas positive deviations represent lower margins for hERG 1a/1b versus 1a channels. The safety margin was defined as hERG IC₅₀ (µM)/EFTPC_max (µM).