Quantitative Analysis of the Cytoskeleton's Role in Inward Rectifier K(IR)2.1 Forward and Backward Trafficking

Alteration of the inward rectifier current I-K1, 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 (tau g,s) represented recruitment of new K(IR)2.1 at the plasma membrane. Red fluorescent decay (tau r,s) represented internalization of photoconverted K(IR)2.1. Patch clamp electrophysiology was used to quantify I-KIR2.(1). Biochemical methods were used for cytoskeleton isolation and detection of K(IR)2.1-cytoskeleton interactions. Cytochalasin B (20 mu M), Nocodazole (30 mu M) and Dyngo-4a (10 nM) were used to modify the cytoskeleton. Chloroquine (10 mu M, 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 [tau g,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 [tau r,s 20 +/- 2 vs. 87 +/- 14*, 60 +/- 16 and 64 +/- 20 min (*p < 0.05 vs. control)]. Cytochalasin B treatment (20 mu M, 24 h) inhibited I-KIR2.(1). Chloroquine treatment (10 mu M, 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. Internalized K(IR)2.1 channels become recruited to the cytoskeleton, presumably in lysosomes.