Muscle-specific f-box only proteins facilitate bk channel ?(1) subunit downregulation in vascular smooth muscle cells of diabetes mellitus.

ABSTRACT: activity of the large conductance Ca(2+)-activated K(+) (BK) channels is profoundly modulated by its ?(1) subunit (BK-?(1)). However, BK-?(1) expression is downregulated in diabetic vessels. The ubiquitin-proteasome system (UPS) is a major mechanism of intracellular protein degradation. Whether UPS participates in BK-?(1) downregulation in diabetic vessels is unknown.we hypothesize that UPS facilitates vascular BK-?(1) degradation in diabetes.using patch clamp and molecular biological approaches, we found that BK-?(1)-mediated channel activation and BK-?(1) protein expression were reduced in aortas of streptozotocin-induced diabetic rats and in human coronary arterial smooth muscle cells (CASMCs) cultured in high glucose. This was accompanied by upregulation of F-box only protein (FBXO)-9 and FBXO-32 (atrogin-1), the key components of the Skp1-Cullin-F-box (SCF) type ubiquitin ligase complex. BK-?(1) expression was suppressed by the FBXO activator doxorubicin but enhanced by FBXO-9 small interfering RNA or by the proteasome inhibitor MG-132. Cotransfection of atrogin-1 in HEK293 cells significantly reduced Flag-hSlo-?(1) expression by 2.16-fold, compared with expression of Flag-hSlo-?(1)V146A (a mutant without the PDZ-binding motif). After cotransfection with atrogin-1, the ubiquitination of Flag-hSlo-?(1) was increased by 1.91-fold, compared with that of hSlo-?(1)V146A, whereas cotransfection with atrogin-1?F (a nonfunctional mutant without the F-box motif) had no effect. Moreover, inhibition of Akt signaling attenuated the phosphorylation of forkhead box O transcription factor (FOXO)-3a and enhanced atrogin-1 expression, which in turn suppressed BK-?(1) protein levels in human CASMCs.downregulation of vascular BK-?(1) expression in diabetes and in high-glucose culture conditions was associated with FOXO-3a/FBXO-dependent increase in BK-?(1) degradation.

SUBMITTER: Zhang DM 

PROVIDER: S-EPMC3076051 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature