Zhang, Dai‐Min

Rationale: 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. Methods and 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.

Rationale : The large conductance Ca 2+ -activated K + (BK) channel, a key determinant of vascular tone, is regulated by angiotensin II (Ang II) type 1 receptor signaling. Upregulation of Ang II functions and downregulation of BK channel activities have been reported in diabetic vessels. However, the molecular mechanisms underlying Ang II-mediated BK channel modulation, especially in diabetes mellitus, have not been thoroughly examined. Objectives : The aim in this study was to determine whether caveolae-targeting facilitates BK channel dysfunction in diabetic vessels. Methods and Results : Using patch clamp techniques and molecular biological approaches, we found that BK channels, Ang II type 1 receptor, G αq/11 (G protein q/11 α subunit), nonphagocytic NAD(P)H oxidases (NOX-1), and c-Src kinases (c-Src) were colocalized in the caveolae of rat arterial smooth muscle cells and the integrity of caveolae in smooth muscle cells was critical for Ang II-mediated BK channel regulation. Most importantly, membrane microdomain targeting of these proteins was upregulated in the caveolae of streptozotocin-induced rat diabetic vessels, leading to enhanced Ang II-induced redox-mediated BK channel modification and causing BK channel and coronary dysfunction. The absence of caveolae abolished the effects of Ang II on vascular BK channel activity and preserved BK channel function in diabetes. Conclusions : These results identified a molecular scheme of receptor/enzyme/channel/caveolae microdomain complex that facilitates the development of vascular BK channel dysfunction in diabetes.