Ankyrin-B Regulates Kir6.2 Membrane Expression and Function in Heart

Jingdong Li,Crystal F. Kline,Mark E. Anderson,Thomas J. Hund,Peter J. Mohler

doi:10.1074/jbc.m110.147868

Abstract

Ankyrin polypeptides are critical for normal membrane protein expression in diverse cell types, including neurons, myocytes, epithelia, and erythrocytes. Ankyrin dysfunction results in defects in membrane expression of ankyrin-binding partners (including ion channels, transporters, and cell adhesion molecules), resulting in aberrant cellular function and disease. Here, we identify a new role for ankyrin-B in cardiac cell biology. We demonstrate that cardiac sarcolemmal K(ATP) channels directly associate with ankyrin-B in heart via the K(ATP) channel alpha-subunit Kir6.2. We demonstrate that primary myocytes lacking ankyrin-B display defects in Kir6.2 protein expression, membrane expression, and function. Moreover, we demonstrate a secondary role for ankyrin-B in regulating K(ATP) channel gating. Finally, we demonstrate that ankyrin-B forms a membrane complex with K(ATP) channels and the cardiac Na/K-ATPase, a second key membrane transporter involved in the cardiac ischemia response. Collectively, our new findings define a new role for cardiac ankyrin polypeptides in regulation of ion channel membrane expression in heart.

Highlights

Ankyrins are multivalent adapter proteins required for the proper membrane expression of ion channels, transporters, cell adhesion molecules, and structural and signaling molecules in excitable and non-excitable cells [1]
We observed a parallel decrease in the expression of both KATP channel ␤-subunits, SUR1 and SUR2A
Our new findings demonstrate a role for ankyrin-B in Kir6.2 membrane expression and regulation in heart

Summary

EXPERIMENTAL PROCEDURES

Electrophysiology—Mice were killed after deep anesthesia with 2.5% Avertin at a dose of 0.2 ml/10 g (10 g of tribromoethanol alcohol ϩ 10 ml of tert-amyl alcohol with the addition of 1 mg/ml heparin (187 USP units/mg). 100 ␮g of wild-type heart lysate or 200 ␮g of ankyrin-Bϩ/Ϫ heart lysate was added to the washed beads, along with protease inhibitor mixture and co-immunoprecipitation binding buffer, and incubated for 12 h at 4 °C. The reactions were incubated overnight at 4 °C and centrifuged and washed three times with binding buffer prior to elution, SDS-PAGE, and immunoblotting with anti-ankyrin-B Ig. Cell Culture—HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% FBS (HyClone) and 0.1% penicillin/streptomycin. 100 ␮g of wild-type heart lysate was added to the beads, along with 500 ␮l of binding buffer and protease inhibitor mixture, and the reactions were incubated for 12 h at 4 °C. The null hypothesis was rejected for p Յ 0.05

RESULTS

The abbreviations used are

DISCUSSION