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

• Published in: Journal of biomedical science Vol. 24; no. 1; pp. 1 - 10
• Main Authors: Ji, Yuan, Veldhuis, Marlieke G, Zandvoort, Jantien, Romunde, Fee L, Houtman, Marien J. C, Duran, Karen, van Haaften, Gijs, Zangerl-Plessl, Eva-Maria, Takanari, Hiroki, Stary-Weinzinger, Anna, van der Heyden, Marcel A. G
• Format: Journal Article
• Language: English
• Published: Basel BioMed Central Ltd 15-07-2017; BioMed Central; BMC
• Subjects: Accumulation; Action potential; Atrial fibrillation; Binding sites; Calcium channels; Cardiac arrhythmia; Coding; Docking; Drugs; Electrocardiography; Electrophysiology; Fibrillation; Gene mutations; Heart diseases; Homology; Human performance; IK1; Immunofluorescence; Intracellular; Ion channels; KIR2.1; Kir2.1 protein; Localization; Membrane potential; Microscopy; Molecular modelling; Mutants; Mutation; PA-6; Pentamidine; Physiological aspects; Polyamines; Potassium; Potassium channels (inwardly-rectifying); Properties; Short QT syndrome
• ISSN: 1423-0127; 1021-7770; 1423-0127
• DOI: 10.1186/s12929-017-0352-x

Background The 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. Methods Molecular 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. Results PA-6 docking in the V93I/D172N double mutant homology model of KIR2.1 demonstrated that mutations and drug-binding site are >30 A 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). Conclusions 1) 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.