New potential binding determinant for hERG channel inhibitors

Abstract

Human ether-à-go-go related gene (hERG) 1 channels conduct the rapid delayed rectifier K(+) current (IKr) and are essential for the repolarization of the cardiac action potential. hERG1 inhibition by structurally diverse drugs may lead to life threatening arrhythmia. Putative binding determinants of hERG1 channel blockers include T623, S624 and V625 on the pore helix, and residues G648, Y652 and F656, located on segment S6. We and others have previously hypothesized that additional binding determinants may be located on helix S5, which is in close contact with the S6 segments. In order to test this hypothesis, we performed a detailed investigation combining ionic current measurements with two-microelectrode voltage clamp and molecular modeling techniques. We identified a novel aromatic high affinity binding determinant for blockers located in helix S5, F557, which is equally potent as Y652. Modeling supports a direct interaction with the outer pore helix.

Figure 1. Location of tested residues and known binding determinants T623, S624, Y652 and F656.

All residues are represented as sticks in one subunit of the hERG_KvAP-m6 homology model.

Figure 2. Mutation of F557L alters the sensitivity of hERG channel to drug block.

(A) Representative current traces of hERG and the indicated mutants in response to the pulse protocol illustrated. (B–G) Concentration-response relationships for block of WT, mutants F557L and Y652A channels by dofetilide (B) haloperidol (C) cisapride (D) astemizole (E) amiodarone (F) and terfenadine (G).

Figure 3. Effects of mutations surrounding F557 on channel inhibition by dofetilide, haloperidol and cisapride.

(A) Representative currents through the indicated hERG channel mutants in control (black traces) and after steady state block by 25 μM dofetelide was reached (red traces, same voltage protocol as shown in top panel. (B–D) Inhibition of currents through the indicated mutants by dofetilide (25 μM), haloperidol (10 μM) and cisapride (9 μM). Data are represented throughout the figure as mean ± SEM. The numbers of experiments (n) are indicated as small insets within the bars. ***P < 0.001, **P < 0.01, one-way ANOVA. The value of 1 indicates no detectable decrease in current by drugs.

Figure 4. Docking poses revealing aromatic interactions with residue F557.

Left side: side view of two neighboring subunits colored in yellow and blue. Right side: top view of hERG blockers and interacting residues, presented as sticks. π-π interactions between aromatic rings are indicated by blue dots (cutoff distances: sandwich-shaped = 5Å; t-shaped = 6.5 Å; parallel displaced = 5.5 Å).