Ranolazine inhibition of hERG potassium channels: drug-pore interactions and reduced potency against inactivation mutants

Abstract

The antianginal drug ranolazine, which combines inhibitory actions on rapid and sustained sodium currents with inhibition of the hERG/IKr potassium channel, shows promise as an antiarrhythmic agent. This study investigated the structural basis of hERG block by ranolazine, with lidocaine used as a low potency, structurally similar comparator. Recordings of hERG current (IhERG) were made from cell lines expressing wild-type (WT) or mutant hERG channels. Docking simulations were performed using homology models built on MthK and KvAP templates. In conventional voltage clamp, ranolazine inhibited IhERG with an IC50 of 8.03μM; peak IhERG during ventricular action potential clamp was inhibited ~62% at 10μM. The IC50 values for ranolazine inhibition of the S620T inactivation deficient and N588K attenuated inactivation mutants were respectively ~73-fold and ~15-fold that for WT IhERG. Mutations near the bottom of the selectivity filter (V625A, S624A, T623A) exhibited IC50s between ~8 and 19-fold that for WT IhERG, whilst the Y652A and F656A S6 mutations had IC50s ~22-fold and 53-fold WT controls. Low potency lidocaine was comparatively insensitive to both pore helix and S6 mutations, but was sensitive to direction of K(+) flux and particularly to loss of inactivation, with an IC50 for S620T-hERG ~49-fold that for WT IhERG. Docking simulations indicated that the larger size of ranolazine gives it potential for a greater range of interactions with hERG pore side chains compared to lidocaine, in particular enabling interaction of its two aromatic groups with side chains of both Y652 and F656. The N588K mutation is responsible for the SQT1 variant of short QT syndrome and our data suggest that ranolazine is unlikely to be effective against IKr/hERG in SQT1 patients.

Keywords: Antiarrhythmic; Docking; Lidocaine; QT interval; Ranolazine; hERG.

Fig. 1

Wild-type I_hERG block by ranolazine and lidocaine. Ai. Representative current traces in control (normal Tyrode's) solution and in 10 μM ranolazine, overlying the applied AP voltage command. Aii. Time-course of inhibition of peak I_hERG during the repolarization phase of the AP. Peak I_hERG in control was normalized to a value of 1 and then current magnitudes in ranolazine were expressed as a proportion of this (I/I_max; n = 6 cells). B. Upper panel shows representative I_hERG traces in the absence (black line) and presence (grey line) of 10 μM ranolazine in normal Tyrode's solution (4 mM [K⁺]_e), elicited by the protocol shown in the lower panel. Horizontal arrows denote zero current. C. Upper panel shows inward I_hERG tail records in the absence (black line) and presence (grey line) of 10 μM ranolazine (~ 58% inhibition) in high K⁺ Tyrode's solution (94 mM [K⁺]_e). The current was evoked by the protocol shown in the lower panel and is shown on an expanded time-scale (denoted by the boxed area in the voltage protocol). D. Concentration–response relations for inhibition of I_hERG by ranolazine in normal K⁺ (filled square) and high K⁺ (open circle) Tyrode's solution. Data were fitted with a Hill-equation. Note that error bars for some points are small and are obscured by the symbols (n ≥ 5 cells per data-point). The data-points for 4 mM [K⁺]_e and 94 mM [K⁺]_e are closely overlain at 1 and 10 μM ranolazine.

Fig. 2

Ranolazine block of S620T and N588K I_hERG. Ai, Bi. Representative S620T (Ai) and N588K (Bi) I_hERG traces in the absence (black line) and presence (grey line) of 10 μM ranolazine, elicited by the protocol shown in the lower panels. Aii, Bii. Concentration–response plots (mean ± SEM) for S620T I_hERG block by ranolazine (Aii) and N588K I_hERG block by ranolazine (Bii). In each case the relation for WT I_hERG during the same protocol is reproduced for comparison. Data were fitted with a Hill-equation. Error bars for some points are small and are obscured by the symbols. (n ≥ 5 cells per data-point.)

Fig. 3

Effect of S6 mutations on ranolazine and lidocaine block of I_hERG. A. Expanded representative I_hERG traces of WT hERG and F656A hERG in the absence (black line) and presence (grey line) of either 10 μM ranolazine (Ai) or 300 μM lidocaine (Aii). The current was evoked by the same protocol shown in Fig. 1C and was recorded in high K⁺ Tyrode's solution (94 mM [K⁺]_e). Horizontal arrows denote zero current. B. Concentration–response plots (mean ± SEM) for both WT (black line) and F656A (grey line) I_hERG block by ranolazine (Bi) and lidocaine (Bii). Data were fitted with a Hill-equation. Error bars for some points are small and are obscured by the symbols. (n ≥ 5 cells per data-point.) C. Expanded example I_hERG traces of WT hERG and Y652A hERG in the absence (black line) and presence (grey line) of either 10 μM ranolazine (Ci) or 300 μM lidocaine (Cii). The current was evoked by the same protocol shown in Fig. 1B and was recorded in normal Tyrode's solution (4 mM [K⁺]_e). Horizontal arrows denote zero current. D. Concentration–response plots (mean ± SEM) for both WT (black line) and Y652A (grey line) I_hERG block by ranolazine (Di) and lidocaine (Dii). Data were fitted with a Hill-equation. Error bars for some points are small and are obscured by the symbols. (n ≥ 5 cells per data-point.)

Fig. 4

Effect of pore helix mutations on ranolazine and lidocaine block of I_hERG. A. Fitted concentration–response relations for WT hERG (black line) and T623A hERG (grey line) inhibition by ranolazine (Ai) and lidocaine (Aii). Panels B, and C show equivalent data for T624A, and V625A, respectively. Error bars for some points are small and are obscured by the symbols. For all plots n ≥ 5 cells per data-point.

Fig. 5

Docking analysis of ranolazine and lidocaine action. A, B. Cut-away view of hERG pore homology models. (A) MthK-based model with a representative lidocaine binding pose; (B) the equivalent residues of the Farid model with a representative ranolazine binding pose. The pale blue ribbon defines the pore helices and K⁺ ions in the S1 and S3 positions of the selectivity filter are purple spheres. Drug molecules are displayed as yellow sticks. Side chains of Y652 (salmon) and F656 (blue) are shown for three channel subunits. The side chain of one S620 residue (green) is shown for both models. C–F. Representative low energy score poses for lidocaine (C, D) and ranolazine (E, F) bound to the MthK-based model (C, E) or Farid model (D, F). The side chains of the aromatic side chains that make π–π or cation–π interactions with the drugs are shown in salmon (Y652) and light blue (F656). Side chains of T623 and S624 (green) and V625 (dark blue) are also shown as sticks. The K⁺ ion in the S3 site of the selectivity filter is represented as a sphere. The blue star in each panel identifies the positively-charged tertiary alkyl amino groups of lidocaine and ranolazine.