B-AB18-02 KIR2.1 CHANNELS IN A NOVEL SARCOPLASMIC RETICULUM MICRODOMAIN CONTROL INTRACELLULAR CA2+ DYNAMICS

Abstract

Background: The strong inward rectifier K+ channel, Kir2.1, is known to localize at the sarcolemma to control the resting potential and the final phase of ventricular repolarization. K+ channels have been suggested to contribute countercurrent to calcium flux across the sarcoplasmic reticulum (SR) membrane, but their identity and function remain controversial. Objective: To test whether a fraction of Kir2.1 channels that cluster within a novel SR membrane microdomain function to provide essential countercurrent to balance Ca2+ reuptake, helping to control intracellular calcium dynamics and excitationcontraction coupling. Methods: Using confocal microscopy we analyzed the ultrastructure of mouse and rat skeletal muscle slices, cardiomyocytes, and isolated mouse cardiac SR vesicles. Immunolocalization of target proteins was analyzed in intact and detubulated cardiomyocytes treated with formamide by immunofluorescence and biochemically by western-blotting after membrane fractionation. Functional assays included patchclamping and calcium transient dynamics. Results: Cardiomyocytes and skeletal muscle slices revealed two distinct microdomain bands of Kir2.1 immunostaining, one colocalizing with NaV1.5 near the Z disk, the other colocalizing with Ankyrin-B in the M line. The latter is a previously unknown Kir2.1 channel microdomain localized at the SR membrane. Its ionic current was sensitive to spermine and caffeine, and modified by asymmetrical potassium concentrations. Finally, chloroquinemediated inhibition of the SR Kir2.1 current resulted in a larger but slower calcium SR reuptake. Hence, we revealed a previously unknown physiological role for Kir2.1 channels at the SR membrane in the control of intracellular Ca2+ dynamics, conducting K+ as a potential countercurrent ion to calcium reuptake. Conclusion: Altogether, the data provide original structural and functional demonstration of a major K+ channel localizing at the SR and contributing to the control of intracellular calcium homeostasis. Aberrant Kir2.1 localization at the SR could underly cardiac arrhythmogenesis and periodic skeletal muscle paralysis in several inheritable ion channel diseases.