The Pore-Lipid Interface: Role of Amino-Acid Determinants of Lipophilic Access by Ivabradine to the hERG1 Pore Domain

Abstract

Abnormal cardiac electrical activity is a common side effect caused by unintended block of the promiscuous drug target human ether-à-go-go-related gene (hERG1), the pore-forming domain of the delayed rectifier K⁺ channel in the heart. hERG1 block leads to a prolongation of the QT interval, a phase of the cardiac cycle that underlies myocyte repolarization detectable on the electrocardiogram. Even newly released drugs such as heart-rate lowering agent ivabradine block the rapid delayed rectifier current I_Kr, prolong action potential duration, and induce potentially lethal arrhythmia known as torsades de pointes. In this study, we describe a critical drug-binding pocket located at the lateral pore surface facing the cellular membrane. Mutations of the conserved M651 residue alter ivabradine-induced block but not by the common hERG1 blocker dofetilide. As revealed by molecular dynamics simulations, binding of ivabradine to a lipophilic pore access site is coupled to a state-dependent reorientation of aromatic residues F557 and F656 in the S5 and S6 helices. We show that the M651 mutation impedes state-dependent dynamics of F557 and F656 aromatic cassettes at the protein-lipid interface, which has a potential to disrupt drug-induced block of the channel. This fundamentally new mechanism coupling the channel dynamics and small-molecule access from the membrane into the hERG1 intracavitary site provides a simple rationale for the well established state-dependence of drug blockade. SIGNIFICANCE STATEMENT: The drug interference with the function of the cardiac hERG channels represents one of the major sources of drug-induced heart disturbances. We found a novel and a critical drug-binding pocket adjacent to a lipid-facing surface of the hERG1 channel, which furthers our molecular understanding of drug-induced QT syndrome.

Graphical abstract

Fig. 1.

Structural organization of drug binding site in the intracellular cavity of hERG1 channel. (A) Organization of the hERG1 pore domain (two subunits are shown for clarity) in the open (top) and closed (bottom) states, respectively. The location of key residues involved in the state-dependent dynamics of membrane-facing side windows is shown in color-coded stick mode. (B) Access pathway to the intracellular cavity of hERG1 channel from the intracellular milieu. The differences in orientation of key hydrophobic residues involved to ivabradine-induced block are shown for the open (top) and closed (bottom) states, respectively. (C) The trajectory-averaged iso-surfaces available for the drug’s lipophilic access to the intracellular cavity. The residue-based color coding was used to highlight protein-membrane interfaces present in the open (top) and closed (bottom) states of the channel, respectively.

Fig. 2.

Effect of ivabradine on WT-hERG1 (A) and M651T-hERG1 currents (B), respectively. (C) The representative time-courses of the WT and M651T current in response to application of various concentrations of ivabradine. The original current traces (times indicated with arrows) were shown in (A) and (B). (D) The dose-response curves of dofetilide (triangles) and ivabradine (circles) blockade of M651T-hERG1 (open symbols) and WT-hERG1 currents (solid symbols). For all experiments with ivabradine blocking M651T-hERG1, n = 3, 6, 7 for concentrations of drugs 1, 10, and 100 μM, respectively. For ivabradine block of WT-hERG1, n = 5 was used for every concentrations. The dose-response curves of dofetilide block of M651T-hERG1 were obtained with n = 5, 7, 5, 10 with concentrations of 0.01, 0.03, 0.1, 1 μM. For dofetilide block of WT-hERG1, n = 1, 3, 2, 2, 5 in the concentrations of 0.002, 0.02, 0.1, 0.2, 2 μM. (E) Effect of ivabradine on M651T/T618I hERG1 currents. (F) The dose-response curves of ivabradine blockade of M651T/T618I hERG1 in comparison with blockade of M651T-hERG1. All experiments for M651T/T618I-hERG1 system were performed with n = 3.

Fig. 3.

The temperature dependence of the concentration-response curves for ivabradine blockade of WT-hERG1 (panel A) and M651T-hERG1 (panel B) currents at T = 22°C and 37°C shown as closed and open circles, respectively. In the WT-hERG1 experiments at T = 22°C, n were 6, 5, 5, 5, 5 for concentrations of 1, 3, 10, 20, 100 μM of ivabradine. T = 37°C, n = 8, 7, 3, 4 for drug concentrations of 1, 3, 10, 100 μM, respectively. In the M651T construct at T = 22°C, n = 3, 6, 7 for experiments performed with 1, 10, 100 μM, respectively. At T = 37°C n = 4, 3, 5 for 10, 30, 100 μM, respectively.

Fig. 4.

Analysis of concomitant interactions of double mutations in blocking effects of saturating concentration of ivabradine (100 μM). Blocking effect of (A) M651T/F656C, (B) M651T/Y652A, (C) M651T/S620T, (D) M651T/F557L, (E) F656C/Y652A, (F) S620T/F557L, and their corresponding single mutations are shown. The following number of experiments was used: N = 5, 7, 5, 4 in order of in (A); 5, 7, 6, 3 in (B); 5, 7, 5, 5 in (C); 5, 7, 5, 5 in (D); 5, 5, 6, 5 in (E); 5, 5, 5, 4 in (F). *P < 0.05; **P < 0.01. One way ANOVA analysis was used. The precise statistic P values are provided in the Supplemental Table 1. WT was used as reference and was not included in the statistical analysis.

Fig. 5.

Current traces (A) and time-course (C) of ivabradine on M651T/F557L and their single mutations. (C) The concentration-response curves. The smooth curve was fitted to the Hill equation. Fitting of M651T and F557L was not possible. (E) The following numbers of experiments were performed: N = 3, 4, and 5 in F557L; n = 3, 6, and 7 in M651T; n = 4, 5, and 5 in M651T/F557L in concentrations of 1, 10, and 100 mM, the legend is shown on the figure. (D) Patch-clamp protocol.

Fig. 6.

Impact of the M651T mutation on the conformational dynamics of aromatic cassette in WT-hERG1 and M657T-hERG1 systems. The dihedral distributions and accessibility mapping were obtained from the last 750 nanoseconds of equilibrium all-atom MD simulations. (A) Side view of two subunits and top view of the pore domain showing relevant access pathways mapped by the MOLEonline tool (see Supplemental Information for details). Selected residues are shown: F557 (green), F656 (magenta), M651 (orange), and Y652 (yellow). (B) Key distances and torsional angles involving F557 (green sticks) and F656 (magenta sticks). Position of residues M651 in the S6 helix are colored in orange. (C) Top and side views of the superimposed positions of F557 (green) and F656 (magenta) for WT-hERG1 (left) and M651T-hERG1 (right). (D and E) one-dimensional and two-dimensional distribution maps for torsional angles in each subunit of WT-hERG1 (D) and M651T (E). One-dimensional distributions are shown for F557 (top panel) and F656 (side panel), respectively.

Fig. 7.

Molecular docking of ivabradine to different sites of the pore domain (PD) and the transmembrane domain (TMD). (A) Induced-Fit Docking (IFD) of ivabradine to PD access site (top), lipophilic site (middle), and intracellular cavity site (bottom). Ivabradine is shown in magenta and relevant interacting residues are labeled. The arrows indicate the potential access route that the drug might follow to access the cavity and produce the block. (B) Ensemble of binding poses mapped from the IFD docking showing the exploration of the different binding sites for WT (top), M651T (middle), and F557LM651T (bottom) in the PD of hERG1. No poses were found in the access path site for M651T. (C) Group of poses found by MD-ensemble docking showing the exploration of the different binding sites for WT (top), M651T (middle), and F557LM651T (bottom) TM hERG1. Results involving only one of the four subunits are shown for clarity. Results for all subunits are displayed in the corresponding pie charts beside each system and in Table 3. In agreement with IDF docking performed with cryo-EM structure, almost no poses were found on the access path site for M651T mutant.