AUTHOR=Li Encan , Loen Vera , van Ham Willem B. , Kool Willy , van der Heyden Marcel A. G. , Takanari Hiroki 

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

JOURNAL=Frontiers in Physiology

VOLUME=12

YEAR=2022

URL=https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2021.812572

DOI=10.3389/fphys.2021.812572

ISSN=1664-042X

ABSTRACT=<p>Alteration of the inward rectifier current <italic>I</italic><sub><italic>K</italic>1</sub>, carried by K<sub>IR</sub>2.1 channels, affects action potential duration, impacts resting membrane stability and associates with cardiac arrhythmias. Congenital and acquired K<sub>IR</sub>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<sub>IR</sub>2.1 trafficking processes is unknown. We aimed to quantify the dependence of K<sub>IR</sub>2.1 trafficking on cytoskeleton function. GFP or photoconvertible Dendra2 tagged K<sub>IR</sub>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<sub>IR</sub>2.1 at the plasma membrane. Red fluorescent decay (τr,s) represented internalization of photoconverted K<sub>IR</sub>2.1. Patch clamp electrophysiology was used to quantify <italic>I</italic><sub><italic>KIR</italic>2</sub>.<sub>1</sub>. Biochemical methods were used for cytoskeleton isolation and detection of K<sub>IR</sub>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<sub>IR</sub>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<sub>IR</sub>2.1 forward trafficking [τg,s 13 ± 2 <italic>vs.</italic> 131 ± 31* and 160 ± 40* min, for control, Cytochalasin B and Nocodazole, respectively (*<italic>p</italic> &lt; 0.05 <italic>vs.</italic> 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 <italic>vs.</italic> 87 ± 14*, 60 ± 16 and 64 ± 20 min (*<italic>p</italic> &lt; 0.05 <italic>vs.</italic> control)]. Cytochalasin B treatment (20 μM, 24 h) inhibited <italic>I</italic><sub><italic>KIR</italic>2</sub>.<sub>1</sub>. Chloroquine treatment (10 μM, 24 h) induced intracellular aggregation of K<sub>IR</sub>2.1 channels and enhanced interaction with the actin/intermediate filament system (103 ± 90 fold; <italic>p</italic> &lt; 0.05 <italic>vs.</italic> control). Functional actin and tubulin cytoskeleton systems are essential for forward trafficking of K<sub>IR</sub>2.1 channels, whereas initial backward trafficking relies on a functional dynamin system. Chronic disturbance of the actin system inhibits K<sub>IR</sub>2.1 currents. Internalized K<sub>IR</sub>2.1 channels become recruited to the cytoskeleton, presumably in lysosomes.</p>