Regulation of Cardiac ATP-sensitive Potassium Channel Surface Expression by Calcium/Calmodulin-dependent Protein Kinase II

Denice M. Hodgson-Zingman,Asipu Sivaprasadarao,Ana Sierra,Ekaterina Subbotina,Nicolas Sapay,Michel Vivaudou,Peter J. Mohler,Crystal F. Kline,Peter M. Snyder,Elizabeth D. Luczak,Mark E. Anderson,Vikas Sharotri,Zhiyong Zhu,Leonid V. Zingman

doi:10.1074/jbc.m112.429548

Abstract

Cardiac ATP-sensitive potassium (K(ATP)) channels are key sensors and effectors of the metabolic status of cardiomyocytes. Alteration in their expression impacts their effectiveness in maintaining cellular energy homeostasis and resistance to injury. We sought to determine how activation of calcium/calmodulin-dependent protein kinase II (CaMKII), a central regulator of calcium signaling, translates into reduced membrane expression and current capacity of cardiac K(ATP) channels. We used real-time monitoring of K(ATP) channel current density, immunohistochemistry, and biotinylation studies in isolated hearts and cardiomyocytes from wild-type and transgenic mice as well as HEK cells expressing wild-type and mutant K(ATP) channel subunits to track the dynamics of K(ATP) channel surface expression. Results showed that activation of CaMKII triggered dynamin-dependent internalization of K(ATP) channels. This process required phosphorylation of threonine at 180 and 224 and an intact (330)YSKF(333) endocytosis motif of the K(ATP) channel Kir6.2 pore-forming subunit. A molecular model of the μ2 subunit of the endocytosis adaptor protein, AP2, complexed with Kir6.2 predicted that μ2 docks by interaction with (330)YSKF(333) and Thr-180 on one and Thr-224 on the adjacent Kir6.2 subunit. Phosphorylation of Thr-180 and Thr-224 would favor interactions with the corresponding arginine- and lysine-rich loops on μ2. We concluded that calcium-dependent activation of CaMKII results in phosphorylation of Kir6.2, which promotes endocytosis of cardiac K(ATP) channel subunits. This mechanism couples the surface expression of cardiac K(ATP) channels with calcium signaling and reveals new targets to improve cardiac energy efficiency and stress resistance.

Highlights

Surface expression of cardiac ATP-sensitive potassium (KATP) channels impacts cellular energy homeostasis
Using real-time patch clamp monitoring of the KATP channel, whole cell current induced by the opener pinacidil, and metabolic inhibition by DNP, we found that application of isoproterenol significantly reduces KATP current in isolated ventricular cardiomyocytes within 2–3 min (Fig. 1, A and D, fractional reduction ϭ 0.66 Ϯ 0.1, p Ͻ 0.05)
Very little KATP channel current reduction was observed in cardiomyocytes isolated from transgenic mice expressing the calmodulin-dependent protein kinase II (CaMKII) inhibitory peptide, AC3-I (Fig. 1, C and D, fractional reduction ϭ 0.14 Ϯ 0.06, p Ͻ 0.05 versus WT), supporting the hypothesis that the isoproterenol effect on reducing whole cell KATP channel current in the WT cardiomyocytes occurred through CaMKII activation

Summary

Background

Surface expression of cardiac ATP-sensitive potassium (KATP) channels impacts cellular energy homeostasis. We concluded that calcium-dependent activation of CaMKII results in phosphorylation of Kir6.2, which promotes endocytosis of cardiac KATP channel subunits This mechanism couples the surface expression of cardiac KATP channels with calcium signaling and reveals new targets to improve cardiac energy efficiency and stress resistance. A molecular model of Kir6.2 predicts that the ␮2 subunit of the endocytosis adaptor protein, AP2, docks with the KATP channel by interacting with 330YSKF333 and Thr180 on one and Thr-224 on an adjacent Kir6.2 subunit These findings provide new insight into the regulation of cardiac KATP channels and provide valuable new targets for the promotion of cardiac energy efficiency and stress resistance

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION