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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

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

Zhang Dai-Min DM He Tongrong T Katusic Zvonimir S ZS Lee Hon-Chi HC Lu Tong T

Circulation research 20101021 12

<h4>Rationale</h4>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.<h4>Objective</h4>we hypothesize that UPS facilitates vascular BK-β(1) degradation in diabetes.<h4>Methods and r ...[more]

PMID: 20966391

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Project description:Aims:Type 2 diabetes (T2D) is strongly associated with cardiovascular morbidity and mortality in patients. Vascular large conductance Ca2+-activated potassium (BK) channels, composed of four pore-forming ? subunits (BK-?), and four regulatory ?1 subunits (BK-?1), are densely expressed in coronary arterial smooth muscle cells (SMCs) and play an important role in regulating vascular tone and myocardial perfusion. However, the role of BK channels in coronary microvascular dysfunction of human subjects with diabetes is unclear. In this study, we examined BK channel function and protein expression, and BK channel-mediated vasodilation in freshly isolated coronary arterioles from T2D patients. Methods and results:Atrial tissues were obtained from 16 patients with T2D and 25 matched non-diabetic subjects during cardiopulmonary bypass procedure. Microvessel videomicroscopy and immunoblot analysis were performed in freshly dissected coronary arterioles and inside-out single BK channel currents was recorded in enzymatically isolated coronary arteriolar SMCs. We found that BK channel sensitivity to physiological Ca2+ concentration and voltage was downregulated in the coronary arteriolar SMCs of diabetic patients, compared with non-diabetic controls. BK channel kinetics analysis revealed that there was significant shortening of the mean open time and prolongation of the mean closed time in diabetic patients, resulting in a remarkable reduction of the channel open probability. Functional studies showed that BK channel activation by dehydrosoyasaponin-1 was diminished and that BK channel-mediated vasodilation in response to shear stress was impaired in diabetic coronary arterioles. Immunoblot experiments confirmed that the protein expressions of BK-? and BK-?1 subunits were significantly downregulated, but the ratio of BK-?/BK-?1 was unchanged in the coronary arterioles of T2D patients. Conclusions:Our results demonstrated for the first time that BK channel function and BK channel-mediated vasodilation were abnormal in the coronary microvasculature of diabetic patients, due to decreased protein expression and altered intrinsic properties of BK channels.

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

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

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