Abstract TP434: Regulation of BK Channel ß1 Subunit Expression by Muscle Ring Finger-1 Protein in Diabetic Vessels
BK channels, abundantly expressed in vascular smooth muscle cells (VSMCs), are the key determinant of vascular physiology. BK channel activity is tightly regulated by its accessory β1 (BK-β1) subunits. BK-β1 protein expression is down-regulated in diabetic vessels, contributing to diabetic vascular complications including hypertension, stroke and coronary heart disease. However, BK-β1 mRNA is not reduced in diabetic vessels suggesting increased BK-β1 turnover. Since ubiquitin-proteasome system (UPS) accounts for 80~90% of intracellular protein degradations in mammalian cells, we hypothesize that muscle ring finger-1 (MuRF-1) protein, the muscle specific E3 ligase, accelerates vascular BK-β1 degradation in diabetes mellitus. In this study, we found that BK-β1 protein level was decreased by 1.95±0.14 fold, accompanied by a 2.83±0.16 fold increase of MuRF-1 expression in streptozotocin-induced diabetic mouse vessels (n=3, p<0.05 vs. control). Moreover, response of vascular BK channel activation by DHS-1 (a specific BK-β1 activator) was absent in diabetic mouse vascular smooth muscle cells (VSMCs). Similar results were observed in human VSMCs cultured with 22 mM glucose (HG) compared to those cultured with 5 mM glucose. Knockdown of MuRF-1 by siRNA (100 nM) produced a 4.2-fold decrease of MuRF-1 protein level and a 1.8-fold increase in BK-β1 expression in HG-cultured VSMCs. In addition, the total BK-β1 expression was 2.58±0.04 fold lower but the ubiquitinated BK-β1 protein was 5.18±0.04 fold higher in HEK293 cells 48-h after transfection with MuRF-1, compared to those with MuRF-1ΔR (an inactive mutant with the Ring structure deletion) transfection (n=3, p<0.05). Immunoprecipitation experiments confirmed that the N-terminus of BK-β1 was physically associated with MuRF-1. Inhibition of UPS activity by 10 μM MG-132 (a proteasome inhibitor) enhanced BK-β1 expression by 1.70±0.01 fold (n=3, p<0.05 vs. control). Our results indicate that MuRF-1 participated in ubiquitin-mediated vascular BK-β1 protein proteasomal degradation and delineated a fundamental mechanism underlying vasculopathy in diabetes.
- © 2012 by American Heart Association, Inc.