Rationale: 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.Objective: We hypothesize that UPS facilitates vascular BK-␤ 1 degradation in diabetes. Methods and Results: 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. Key Words: ubiquitin-proteasome system Ⅲ BK channel ␤ 1 subunit Ⅲ protein degradation Ⅲ diabetes mellitus T he large conductance Ca 2ϩ -activated K ϩ (BK) channels play an important role in the regulation of vascular physiology. Functional BK channels in coronary arterial smooth muscle cells (CASMCs) are composed of the poreforming ␣ subunits (BK-␣, encoded by the Slo gene) and the regulatory ␤ 1 subunits (BK-␤ 1 ) in 4:4 stoichiometry. However, BK channel function is impaired in diabetes, 1,2 which is associated with microvessel complications. Recently, we and other investigators have reported that impaired BK channel activation was attributable to reduced BK-␤ 1 expression in diabetic vessels. 3,4 However, the underlying molecular mechanisms is unknown. Conclusions:The ubiquitin-proteasome system (UPS) accounts for 80% to 90% of intracellular protein turnover. 5 UPS-mediated protein degradation involves 3 enzyme systems: ubiquitinactivation enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. 6 There are 1 E1, Ͼ25 E2, and Ͼ1000 E3 enzymes. Each E3 recognizes a specific motif on substrate proteins.F-box only proteins (FBXOs) are key components of the Skp1-Cullin-F-box (SCF) type ubiquitin ligase complex, functioning as sites for enzyme-substrate interaction. 7 FBXO expression is ...

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SK3 channel contributes importantly towards atrial action potential repolarization. Our data suggest the important role of the SK3 isoform in atrial myocytes.

Key pointsr Both the ATP-sensitive potassium (K ATP ) channel and the gaseous messenger nitric oxide (NO) play fundamental roles in protecting the heart from injuries related to ischaemia. r NO has previously been suggested to modulate cardiac K ATP channels; however, the underlying mechanism remains largely unknown.r In this study, by performing electrophysiological and biochemical assays, we demonstrate that NO potentiation of K ATP channel activity in ventricular cardiomyocytes is prevented by pharmacological inhibition of soluble guanylyl cyclase (sGC), cGMP-dependent protein kinase (PKG), Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) and extracellular signal-regulated protein kinase 1/2 (ERK1/2), by removal of reactive oxygen species and by genetic disruption of CaMKIIδ.r These results suggest that NO modulates cardiac K ATP channels via a novel cGMP-sGC-cGMP-PKG-ROS-ERK1/2-calmodulin-CaMKII (δ isoform in particular) signalling cascade.r This novel intracellular signalling pathway may regulate the excitability of heart cells and provide protection against ischaemic or hypoxic injury, by opening the cardioprotective K ATP channels.Abstract The ATP-sensitive potassium (K ATP ) channels are crucial for stress adaptation in the heart. It has previously been suggested that the function of K ATP channels is modulated by nitric oxide (NO), a gaseous messenger known to be cytoprotective; however, the underlying mechanism remains poorly understood. Here we sought to delineate the intracellular signalling mechanism responsible for NO modulation of sarcolemmal K ATP (sarcK ATP ) channels in ventricular cardiomyocytes. Cell-attached patch recordings were performed in transfected human embryonic kidney (HEK) 293 cells and ventricular cardiomyocytes freshly isolated from adult rabbits or genetically modified mice, in combination with pharmacological and biochemical approaches. Bath application of the NO donor NOC-18 increased the single-channel activity of Kir6.2/SUR2A (i.e. the principal ventricular-type K ATP ) channels in HEK293 cells, whereas the increase was abated by KT5823 [a selective cGMP-dependent protein kinase (PKG) inhibitor], mercaptopropionyl glycine [MPG; a reactive oxygen species (ROS) scavenger], catalase (an H 2 O 2 -degrading enzyme), myristoylated autocamtide-2 related inhibitory peptide (mAIP) selective for Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) and U0126 [an extracellular signal-regulated protein kinase 1/2 (ERK1/2) inhibitor], respectively. The NO donors (glycol-SNAP-2) were also capable of stimulating native sarcK ATP channels preactivated by the channel opener pinacidil in rabbit ventricular myocytes, through reducing the occurrence and the dwelling time of the long closed states whilst increasing the frequency of channel opening; in contrast, all these changes were reversed in the presence of Abbreviations APD 90 , action potential duration at 90% repolarization; CaMKII, calcium/calmodulin-dependent protein kinase II; E K , equilibrium potential for potassium; ERK, ...

BackgroundCyclic GMP (cGMP)-dependent protein kinase (PKG) is recognized as an important signaling component in diverse cell types. PKG may influence the function of cardiac ATP-sensitive potassium (KATP) channels, an ion channel critical for stress adaptation in the heart; however, the underlying mechanism remains largely unknown. The present study was designed to address this issue.Methods and FindingsSingle-channel recordings of cardiac KATP channels were performed in both cell-attached and inside-out patch configurations using transfected human embryonic kidney (HEK)293 cells and rabbit ventricular cardiomyocytes. We found that Kir6.2/SUR2A (the cardiac-type KATP) channels were activated by cGMP-selective phosphodiesterase inhibitor zaprinast in a concentration-dependent manner in cell-attached patches obtained from HEK293 cells, an effect mimicked by the membrane-permeable cGMP analog 8-bromo-cGMP whereas abolished by selective PKG inhibitors. Intriguingly, direct application of PKG moderately reduced rather than augmented Kir6.2/SUR2A single-channel currents in excised, inside-out patches. Moreover, PKG stimulation of Kir6.2/SUR2A channels in intact cells was abrogated by ROS/H2O2 scavenging, antagonism of calmodulin, and blockade of calcium/calmodulin-dependent protein kinase II (CaMKII), respectively. Exogenous H2O2 also concentration-dependently stimulated Kir6.2/SUR2A channels in intact cells, and its effect was prevented by inhibition of calmodulin or CaMKII. PKG stimulation of KATP channels was confirmed in intact ventricular cardiomyocytes, which was ROS- and CaMKII-dependent. Kinetically, PKG appeared to stimulate these channels by destabilizing the longest closed state while stabilizing the long open state and facilitating opening transitions.ConclusionThe present study provides novel evidence that PKG exerts dual regulation of cardiac KATP channels, including marked stimulation resulting from intracellular signaling mediated by ROS (H2O2 in particular), calmodulin and CaMKII, alongside of moderate channel suppression likely mediated by direct PKG phosphorylation of the channel or some closely associated proteins. The novel cGMP/PKG/ROS/calmodulin/CaMKII signaling pathway may regulate cardiomyocyte excitability by opening KATP channels and contribute to cardiac protection against ischemia-reperfusion injury.

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Our data indicate that H S is a novel regulator of FoxO1 in cardiac cells and provide evidence supporting the potential of H S in inhibiting the progression of DCM.