Alteration of the inward rectifier current IK1, carried by KIR2.1 channels, affects action potential duration, impacts resting membrane stability and associates with cardiac arrhythmias. Congenital and acquired KIR2.1 malfunction frequently associates with aberrant ion channel trafficking. Cellular processes underlying trafficking are intertwined with cytoskeletal function. The extent to which the cytoskeleton is involved in KIR2.1 trafficking processes is unknown. We aimed to quantify the dependence of KIR2.1 trafficking on cytoskeleton function. GFP or photoconvertible Dendra2 tagged KIR2.1 constructs were transfected in HEK293 or HeLa cells. Photoconversion of the Dendra2 probe at the plasma membrane and subsequent live imaging of trafficking processes was performed by confocal laser-scanning microscopy. Time constant of green fluorescent recovery (τg,s) represented recruitment of new KIR2.1 at the plasma membrane. Red fluorescent decay (τr,s) represented internalization of photoconverted KIR2.1. Patch clamp electrophysiology was used to quantify IKIR2.1. Biochemical methods were used for cytoskeleton isolation and detection of KIR2.1-cytoskeleton interactions. Cytochalasin B (20 μM), Nocodazole (30 μM) and Dyngo-4a (10 nM) were used to modify the cytoskeleton. Chloroquine (10 μM, 24 h) was used to impair KIR2.1 breakdown. Cytochalasin B and Nocodazole, inhibitors of actin and tubulin filament formation respectively, strongly inhibited the recovery of green fluorescence at the plasma membrane suggestive for inhibition of KIR2.1 forward trafficking [τg,s 13 ± 2 vs. 131 ± 31* and 160 ± 40* min, for control, Cytochalasin B and Nocodazole, respectively (*p < 0.05 vs. control)]. Dyngo-4a, an inhibitor of dynamin motor proteins, strongly slowed the rate of photoconverted channel internalization, whereas Nocodazole and Cytochalasin B had less effect [τr,s 20 ± 2 vs. 87 ± 14*, 60 ± 16 and 64 ± 20 min (*p < 0.05 vs. control)]. Cytochalasin B treatment (20 μM, 24 h) inhibited IKIR2.1. Chloroquine treatment (10 μM, 24 h) induced intracellular aggregation of KIR2.1 channels and enhanced interaction with the actin/intermediate filament system (103 ± 90 fold; p < 0.05 vs. control). Functional actin and tubulin cytoskeleton systems are essential for forward trafficking of KIR2.1 channels, whereas initial backward trafficking relies on a functional dynamin system. Chronic disturbance of the actin system inhibits KIR2.1 currents. Internalized KIR2.1 channels become recruited to the cytoskeleton, presumably in lysosomes.
Expression and activity of inwardly rectifying potassium (KIR) channels within the heart are strictly regulated. KIR channels have an important role in shaping cardiac action potentials, having a limited conductance at depolarized potentials but contributing to the final stage of repolarization and resting membrane stability. Impaired KIR2.1 function causes Andersen-Tawil Syndrome (ATS) and is associated with heart failure. Restoring KIR2.1 function by agonists of KIR2.1 (AgoKirs) would be beneficial. The class 1c antiarrhythmic drug propafenone is identified as an AgoKir; however, its long-term effects on KIR2.1 protein expression, subcellular localization, and function are unknown. Propafenone’s long-term effect on KIR2.1 expression and its underlying mechanisms in vitro were investigated. KIR2.1-carried currents were measured by single-cell patch-clamp electrophysiology. KIR2.1 protein expression levels were determined by Western blot analysis, whereas conventional immunofluorescence and advanced live-imaging microscopy were used to assess the subcellular localization of KIR2.1 proteins. Acute propafenone treatment at low concentrations supports the ability of propafenone to function as an AgoKir without disturbing KIR2.1 protein handling. Chronic propafenone treatment (at 25–100 times higher concentrations than in the acute treatment) increases KIR2.1 protein expression and KIR2.1 current densities in vitro, which are potentially associated with pre-lysosomal trafficking inhibition.
Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Chinese Scholarship Council Background Inward rectifying potassium (Kir) channel expression and activity are tightly regulated within the heart. Kir channels play key roles in shaping cardiac action potentials, having a reduced conductance at depolarized potentials but contributing to the final stage of repolarization and resting membrane stability. The Kir2.1 channel protein has polyamine binding residues in both the transmembrane (D172) and the cytoplasmic domains (E224,E299), responsible for the process of inward rectification. A reduced functioning of Kir2.1 causes 1) Andersen-Tawil Syndrom and 2) is present in a subset of heart failure patients. Restoration of normal Kir2.1 function by agonists of Kir2.1 (AgoKirs) would be beneficial. The drug propafenone is identified as an AgoKir, but its long-term effects on Kir2.1 protein expression and subcellular localisation is unknown. Purpose To investigate propafenone's long-term effect on Kir2.1 expression and its underlying mechanisms in cell systems. Methods GFP, Dendra2 or non-tagged wildtype (WT) and mutant Kir2.1 expression constructs were transiently or stably (HEK-KWGF; CHO-KD cell lines) expressed in Human Embryonic Kidney and Chinese Hamster Ovary cells. Kir2.1 carried currents were measured by single cell patch clamp electrophysiology. Kir2.1 proteins expression levels were determined by Western blot analysis, whereas conventional immunofluorescence and advanced live-imaging microscopy were used to assess the subcellular localisation of Kir2.1 proteins. Propafenone was dissolved in DMSO,BaCl2 was used as Kir2.1 channel inhibitor. Results Acute administration of 0.1 and 0.5 µM propafenone increased Kir2.1 carried outward current confirming the drug's Agokir status. Propafenone dose-dependently increased WT Kir2.1 expression levels (2.63±0.40 fold increase at 25 µM and 2.75±0.56 fold increase at 50 µM, 24h) in a process that is independent of the E224, E299 and D172 polyamine binding sites, and the R312 residue which is adjacent to the proposed propafenone binding site. Propafenone (25, 50 µM, 24h) induced intracellular accumulation of WT and mutant Kir2.1 proteins in the late endosome/lysosome compartment (Figure). Channel inhibition by BaCl2 did not affect the propafenone responses on Kir2.1 expression levels or subcellular localisation. Conclusion Acute administration of propafenone at low concentrations increases Kir2.1 currents. Chronic propafenone treatment at only 25-100 times higher concentrations results in increased Kir2.1 protein expression levels and intracellular accumulation in late endosomes and/or lysosomes. Our data support the ability of propafenone at low concentrations to function as AgoKirs without disturbing Kir2.1 protein handing.
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