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 8.03 μM; peak IhERG during ventricular action potential clamp was inhibited ~ 62% at 10 μM. 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.

hERG Potassium Channel Blockade by the HCN Channel Inhibitor Bradycardic Agent Ivabradine

“…Inactivation‐dependence of ivabradine inhibition of I hERG was probed further using the N588K attenuated‐inactivation mutant. 16,30–31 Figure 6A shows representative traces of N588K I hERG before and during exposure to ivabradine, elicited using the same experimental protocol employed to study WT I hERG , whereas Figure 6B shows the concentration dependence of ivabradine inhibition of N588K I hERG , superimposed on that for the WT channel. The IC 50 for N588K inhibition was 10.29 μmol/L (CI: 8.73 to 12.15); n H 0.68 (CI: 0.57 to 0.79), which was ≈5‐fold that for WT I HERG .…”

“…16,19 Cells were plated on small sterilized glass shards in 40‐mm Petri dishes after at least 6 hours of incubation at 37°C (5% CO 2 ). At least 48 hours after plating, cells were transfected with Lipofectamine ™ LTX (Invitrogen) following the manufacturer's instructions.…”

“…16,19 Patch‐pipettes (Schott #8250 glass; A‐M Systems Inc, USA) were pulled (Narishige, PP 830) and polished (Narishige, MF 83) to obtain a final resistance between 2 and 4 MΩ. The intracellular solution used to fill the patch‐pipettes contained (in mmol/L): 130 KCl, 1 MgCl 2 , 5 EGTA, 5 MgATP, and 10 HEPES (titrated to pH 7.2 with KOH).…”

“…The intracellular solution used to fill the patch‐pipettes contained (in mmol/L): 130 KCl, 1 MgCl 2 , 5 EGTA, 5 MgATP, and 10 HEPES (titrated to pH 7.2 with KOH). 16,19 All hERG currents were recorded with an Axopatch 200B amplifier (Axon Instruments, now Molecular Devices) and a CV‐4/100 or CV203BU head‐stage. Pipette resistance compensation was between 70% and 80%.…”

Molecular Insights into hERG Potassium Channel Blockade by Lubeluzole

“…Due to the known low expression and altered kinetic properties of some hERG mutants (in particular T623A and F656A clones), we measured the inward current of the T623A and F656A mutants in high (94 mM) external K + solution, since the external K + concentration increase is aimed to maximize the inward tail currents [25, 26, 30]. Consequently, we tested lubeluzole block of wild-type I hERG under similar experimental conditions, to examine whether a change in the ionic conditions affects hERG affinity toward lubeluzole.…”