Quantitative Analysis of the Cytoskeleton’s Role in Inward Rectifier KIR2.1 Forward and Backward Trafficking

• Published in: Frontiers in physiology 2022-01, Vol.12
• Main Authors: Li, Encan, Loen, Vera, van Ham, Willem B., Kool, Willy, van der Heyden, Marcel A. G., Takanari, Hiroki
• Format: Article
• Language: English
• Subjects: Chloroquine; Cytochalasin; cytoskeleton; ion channel; Nocodazole; Physiology; trafficking
• ISSN: 1664-042X; 1664-042X
• DOI: 10.3389/fphys.2021.812572

Alteration of the inward rectifier current I K 1 , carried by K IR 2.1 channels, affects action potential duration, impacts resting membrane stability and associates with cardiac arrhythmias. Congenital and acquired K IR 2.1 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 K IR 2.1 trafficking processes is unknown. We aimed to quantify the dependence of K IR 2.1 trafficking on cytoskeleton function. GFP or photoconvertible Dendra2 tagged K IR 2.1 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 K IR 2.1 at the plasma membrane. Red fluorescent decay (τr,s) represented internalization of photoconverted K IR 2.1. Patch clamp electrophysiology was used to quantify I KIR 2 . 1 . Biochemical methods were used for cytoskeleton isolation and detection of K IR 2.1-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 K IR 2.1 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 K IR 2.1 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 I KIR 2 . 1 . Chloroquine treatment (10 μM, 24 h) induced intracellular aggregation of K IR 2.1 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 K IR 2.1 channels, whereas initial backward trafficking relies on a functional dynamin system. Chronic disturbance of the actin system inhibits K IR 2.1 currents.