PA-6 inhibits inward rectifier currents carried by V93I and D172N gain-of-function KIR2.1 channels, but increases channel protein expression

Abstract

BackgroundThe inward rectifier potassium current IK1 contributes to a stable resting membrane potential and phase 3 repolarization of the cardiac action potential. KCNJ2 gain-of-function mutations V93I and D172N associate with increased IK1, short QT syndrome type 3 and congenital atrial fibrillation. Pentamidine-Analogue 6 (PA-6) is an efficient (IC50 = 14 nM with inside-out patch clamp methodology) and specific IK1 inhibitor that interacts with the cytoplasmic pore region of the KIR2.1 ion channel, encoded by KCNJ2. At 10 μM, PA-6 increases wild-type (WT) KIR2.1 expression in HEK293T cells upon chronic treatment. We hypothesized that PA-6 will interact with and inhibit V93I and D172N KIR2.1 channels, whereas impact on channel expression at the plasma membrane requires higher concentrations.MethodsMolecular modelling was performed with the human KIR2.1 closed state homology model using FlexX. WT and mutant KIR2.1 channels were expressed in HEK293 cells. Patch-clamp single cell electrophysiology measurements were performed in the whole cell and inside-out mode of the patch clamp method. KIR2.1 expression level and localization were determined by western blot analysis and immunofluorescence microscopy, respectively.ResultsPA-6 docking in the V93I/D172N double mutant homology model of KIR2.1 demonstrated that mutations and drug-binding site are >30 Å apart. PA-6 inhibited WT and V93I outward currents with similar potency (IC50 = 35.5 and 43.6 nM at +50 mV for WT and V93I), whereas D172N currents were less sensitive (IC50 = 128.9 nM at +50 mV) using inside-out patch-clamp electrophysiology. In whole cell mode, 1 μM of PA-6 inhibited outward IK1 at −50 mV by 28 ± 36%, 18 ± 20% and 10 ± 6%, for WT, V93I and D172N channels respectively. Western blot analysis demonstrated that PA-6 (5 μM, 24 h) increased KIR2.1 expression levels of WT (6.3 ± 1.5 fold), and V93I (3.9 ± 0.9) and D172N (4.8 ± 2.0) mutants. Immunofluorescent microscopy demonstrated dose-dependent intracellular KIR2.1 accumulation following chronic PA-6 application (24 h, 1 and 5 μM).Conclusions1) KCNJ2 gain-of-function mutations V93I and D172N in the KIR2.1 ion channel do not impair PA-6 mediated inhibition of IK1, 2) PA-6 elevates KIR2.1 protein expression and induces intracellular KIR2.1 accumulation, 3) PA-6 is a strong candidate for further preclinical evaluation in treatment of congenital SQT3 and AF.

Emerging therapeutic targets in the short QT syndrome

“…Recently, a pentamadine analogue, PA-6, has been identified that inhibits Kir2.1 selectively at submicromolar concentrations [119]. It has been shown also to be able to inhibit D172N Kir2.1, albeit with a modest reduction in potency [120]. PA-6 exerts an agonist effect on Kir2.1 protein expression, but this action is likely to be outweighed by its acute channel blocking effect [120].…”

“…It has been shown also to be able to inhibit D172N Kir2.1, albeit with a modest reduction in potency [120]. PA-6 exerts an agonist effect on Kir2.1 protein expression, but this action is likely to be outweighed by its acute channel blocking effect [120]. The encouraging in vitro results with PA-6 point towards the potential for selective I K1 inhibitors in SQT3.…”

Electrophysiological and Pharmacological Characterization of Human Inwardly Rectifying Kir2.1 Channels on an Automated Patch-Clamp Platform

“…31 These inhibitory activities are consistent with published data obtained using traditional manual patch-clamp. Thus, whole-cell K ir 2.1 currents elicited at −110 mV in KCNJ2 -tranfected HEK cells were inhibited by 68% in the presence of 3 μM external PA-6, 48 whereas ML133 showed an IC 50 value of 1.8 μM at −100 mV. 31 Similarly, in feline ventricular myocytes, 3 μM chloroquine decreased I K1 currents at −100 mV by 67%.…”

Learning from studying very rare cardiac conditions: the example of short QT syndrome

“…The E299 residue lies in the binding site for chloroquine [143] so it is quite likely that the drug would not be effective against the E299V SQT3 variant, whilst mutation of M301 did not impair chloroquine block [143] and so it may be effective against the M301K mutation. Recent work has discovered a pentamidine analogue PA-6 that shows inhibitory selectivity for I K1 [144] and reduces D172N-Kir2.1 current [145]. Our recent simulation work has shown in respect of atrial electrophysiology that 50% inhibition of I K1 alone was sufficient to prevent re-entry in E299V but not D17N mutant conditions; on the other hand, simulated combined I K1 + I Kr inhibition was effective at terminating reentry for both mutations [95].…”