Muscle-specific f-box only proteins facilitate bk channel β(1) subunit downregulation in vascular smooth muscle cells of diabetes mellitus.
BACKGROUND
activity of the large conductance Ca(2+)-activated K(+) (BK) channels is profoundly modulated by its β(1) subunit (BK-β(1)). However, BK-β(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-β(1) downregulation in diabetic vessels is unknown.


OBJECTIVE
we hypothesize that UPS facilitates vascular BK-β(1) degradation in diabetes.


RESULTS
using patch clamp and molecular biological approaches, we found that BK-β(1)-mediated channel activation and BK-β(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-β(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-β(1) expression by 2.16-fold, compared with expression of Flag-hSlo-β(1)V146A (a mutant without the PDZ-binding motif). After cotransfection with atrogin-1, the ubiquitination of Flag-hSlo-β(1) was increased by 1.91-fold, compared with that of hSlo-β(1)V146A, whereas cotransfection with atrogin-1ΔF (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-β(1) protein levels in human CASMCs.


CONCLUSIONS
downregulation of vascular BK-β(1) expression in diabetes and in high-glucose culture conditions was associated with FOXO-3a/FBXO-dependent increase in BK-β(1) degradation.
Zhang
Dai-Min
DM
Division of Cardiovascular Diseases, Department of Internal Medicine, Mayo Clinic, 200 First St SW, Rochester, MN 55905, USA.
He
Tongrong
T
Katusic
Zvonimir S
ZS
Lee
Hon-Chi
HC
Lu
Tong
T
D002478:Cells, Cultured; D003920:Diabetes Mellitus / Q000378:metabolism*; D015536:Down-Regulation; D044783:F-Box Proteins / Q000502:physiology*; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000502:physiology; D005947:Glucose; D006801:Humans; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000473:pathology*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D055550:Protein Stability; D021122:Protein Subunits / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism
D044783:F-Box Proteins; C499517:FOXO3 protein, human; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; D051036:Large-Conductance Calcium-Activated Potassium Channels; D021122:Protein Subunits; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex; D005947:Glucose