TY - JOUR
T1 - Muscle-specific F-Box only proteins facilitate BK channel β1 subunit downregulation in vascular smooth muscle cells of diabetes mellitus
AU - Zhang, Dai Min
AU - He, Tongrong
AU - Katusic, Zvonimir S.
AU - Lee, Hon Chi
AU - Lu, Tong
PY - 2010/12/10
Y1 - 2010/12/10
N2 - Rationale: Activity of the large conductance Ca-activated K (BK) channels is profoundly modulated by its β1 subunit (BK-β1). However, BK-β1 expression is downregulated in diabetic vessels. The ubiquitin-proteasome system (UPS) is a major mechanism of intracellular protein degradation. Whether UPS participates in BK-β1 downregulation in diabetic vessels is unknown. Objective: We hypothesize that UPS facilitates vascular BK-β1 degradation in diabetes. Methods and results: Using patch clamp and molecular biological approaches, we found that BK-β1-mediated channel activation and BK-β1 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-β1 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-β1 expression by 2.16-fold, compared with expression of Flag-hSlo-β1V146A (a mutant without the PDZ-binding motif). After cotransfection with atrogin-1, the ubiquitination of Flag-hSlo- β1 was increased by 1.91-fold, compared with that of hSlo-β1V146A, 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-β1 protein levels in human CASMCs. Conclusions: Downregulation of vascular BK-β1 expression in diabetes and in high-glucose culture conditions was associated with FOXO-3a/FBXO-dependent increase in BK-β1 degradation.
AB - Rationale: Activity of the large conductance Ca-activated K (BK) channels is profoundly modulated by its β1 subunit (BK-β1). However, BK-β1 expression is downregulated in diabetic vessels. The ubiquitin-proteasome system (UPS) is a major mechanism of intracellular protein degradation. Whether UPS participates in BK-β1 downregulation in diabetic vessels is unknown. Objective: We hypothesize that UPS facilitates vascular BK-β1 degradation in diabetes. Methods and results: Using patch clamp and molecular biological approaches, we found that BK-β1-mediated channel activation and BK-β1 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-β1 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-β1 expression by 2.16-fold, compared with expression of Flag-hSlo-β1V146A (a mutant without the PDZ-binding motif). After cotransfection with atrogin-1, the ubiquitination of Flag-hSlo- β1 was increased by 1.91-fold, compared with that of hSlo-β1V146A, 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-β1 protein levels in human CASMCs. Conclusions: Downregulation of vascular BK-β1 expression in diabetes and in high-glucose culture conditions was associated with FOXO-3a/FBXO-dependent increase in BK-β1 degradation.
KW - BK channelβ subunit
KW - diabetes mellitus
KW - protein degradation
KW - ubiquitin-proteasome system
UR - http://www.scopus.com/inward/record.url?scp=78650705169&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=78650705169&partnerID=8YFLogxK
U2 - 10.1161/CIRCRESAHA.110.228361
DO - 10.1161/CIRCRESAHA.110.228361
M3 - Article
C2 - 20966391
AN - SCOPUS:78650705169
SN - 0009-7330
VL - 107
SP - 1454
EP - 1459
JO - Circulation research
JF - Circulation research
IS - 12
ER -