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PA-6 inhibits inward rectifier currents carried by V93I and D172N gain-of-function KIR2.1 channels, but increases channel protein expression
- Source :
- Journal of Biomedical Science, Vol 24, Iss 1, Pp 1-10 (2017), Journal of Biomedical Science, Journal of Biomedical Science, 24(1). BioMed Central
- Publication Year :
- 2017
-
Abstract
- 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 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). 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.
- Subjects :
- 0301 basic medicine
Endocrinology, Diabetes and Metabolism
Clinical Biochemistry
Action Potentials
lcsh:Medicine
030204 cardiovascular system & hematology
Pharmacology
Biochemistry
medical
Membrane Potentials
0302 clinical medicine
Endocrinology
Pharmacology (medical)
KvLQT1
Membrane potential
Trafficking
Inward-rectifier potassium ion channel
Kir2.1
K2.1
Drugs
Cardiac action potential
General Medicine
Molecular Docking Simulation
Diabetes and Metabolism
cardiovascular system
IK1
Short QT syndrome
Biology
03 medical and health sciences
PA-6
KCNJ5
Potassium Channel Blockers
Journal Article
Humans
Patch clamp
Potassium Channels, Inwardly Rectifying
Molecular Biology
Ion channel
Pentamidine
Biochemistry, medical
Research
Biochemistry (medical)
lcsh:R
Cell Biology
Atrial fibrillation
030104 developmental biology
HEK293 Cells
Gene Expression Regulation
biology.protein
Biophysics
KIR2.1
Subjects
Details
- Language :
- English
- ISSN :
- 10217770
- Volume :
- 24
- Issue :
- 1
- Database :
- OpenAIRE
- Journal :
- Journal of Biomedical Science
- Accession number :
- edsair.doi.dedup.....d78706751446ca0cd4e20d015ddfc35e