Muscle-Specific F-Box Only Proteins Facilitate BK Channel beta(1) Subunit Downregulation in Vascular Smooth Muscle Cells of Diabetes Mellitus

作者：Zhang Dai min; He Tongrong; Katusic Zvonimir S; Lee Hon Chi; Lu Tong^*

来源：Circulation Research, 2010, 107(12): 1454-+.

DOI：10.1161/CIRCRESAHA.110.228361

• ubiquitin-proteasome system
• BK channel
• beta(1) subunit
• protein degradation
• diabetes mellitus

Rationale: Activity of the large conductance Ca2+-activated K+ (BK) channels is profoundly modulated by its beta(1) subunit (BK-beta(1)). However, BK-beta(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-beta(1) downregulation in diabetic vessels is unknown.
Objective: We hypothesize that UPS facilitates vascular BK-beta(1) degradation in diabetes.
Methods and Results: Using patch clamp and molecular biological approaches, we found that BK-beta(1)-mediated channel activation and BK-beta(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-beta(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-beta(1) expression by 2.16-fold, compared with expression of Flag-hSlo-beta(1)V146A (a mutant without the PDZ-binding motif). After cotransfection with atrogin-1, the ubiquitination of Flag-hSlo-beta(1) was increased by 1.91-fold, compared with that of hSlo-beta(1)V146A, whereas cotransfection with atrogin-1 Delta 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-beta(1) protein levels in human CASMCs.
Conclusions: Downregulation of vascular BK-beta(1) expression in diabetes and in high-glucose culture conditions was associated with FOXO-3a/FBXO-dependent increase in BK-beta(1) degradation. (Circ Res. 2010; 107: 1454-1459.)

• 出版日期2010-12-10