Small conductance Ca²⁺-activated K⁺ channels (K_Ca2 or SK channels) have been reported in excitable cells, where they aid in integrating changes in intracellular Ca²⁺ (⁠⁠) with membrane potentials. We have recently reported the functional expression of SK channels in human and mouse cardiac myocytes. Additionally, we have found that the channel is highly expressed in atria compared with the ventricular myocytes. We demonstrated that human cardiac myocytes expressed all three members of SK channels (SK1, 2, and 3); moreover, the different members are capable of forming heteromultimers. Here, we directly tested the contribution of SK3 to the overall repolarization of atrial action potentials. We took advantage of a mouse model with site-specific insertion of a tetracycline-based genetic switch in the 5′ untranslated region of the KCNN3 (SK3 channel) gene (SK3^T/T). The gene-targeted animals overexpress the SK3 channel without interfering with the normal profile of SK3 expression. Whole-cell, patch-clamp techniques show a significant shortening of the action potential duration mainly at 90% repolarization (APD₉₀) in atrial myocytes from the homozygous SK3^T/T animals. Conversely, treatment with dietary doxycycline results in a significant prolongation of APD₉₀ in atrial myocytes from SK3^T/T animals. We further demonstrate that the shortening of APDs in SK3 overexpression mice predisposes the animals to inducible atrial arrhythmias. SK3 channel contributes importantly towards atrial action potential repolarization. Our data suggest the important role of the SK3 isoform in atrial myocytes.

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.

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.

Cyclic 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 (K_ATP) 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. Single-channel recordings of cardiac K_ATP 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 K_ATP) 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 K_ATP 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. The present study provides novel evidence that PKG exerts dual regulation of cardiac K_ATP 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 K_ATP channels and contribute to cardiac protection against ischemia-reperfusion injury.

Life-threatening malignant arrhythmias in pathophysiological conditions can increase the mortality and morbidity of patients with cardiovascular diseases. Cardiac electrical activity depends on the coordinated propagation of excitatory stimuli and the generation of action potentials in cardiomyocytes. Action potential formation results from the opening and closing of ion channels. Recent studies have indicated that small-conductance calcium-activated potassium (SK) channels play a critical role in cardiac repolarization in pathophysiological but not normal physiological conditions. The aim of this review is to describe the role of SK channels in healthy and diseased hearts, to suggest cardiovascular pathophysiologic targets for intervention, and to discuss studies of agents that target SK channels for the treatment of cardiovascular diseases.