SK Activation Research Articles

Role of Protein Kinase C in Metabolic Regulation of Coronary Endothelial Small Conductance Calcium-Activated Potassium Channels.

Small conductance calcium-activated potassium (SK) channels are largely responsible for endothelium-dependent coronary arteriolar relaxation. Endothelial SK channels are downregulated by the reduced form of nicotinamide adenine dinucleotide (NADH), which is increased in the setting of diabetes, yet the mechanisms of these changes are unclear. PKC (protein kinase C) is an important mediator of diabetes-induced coronary endothelial dysfunction. Thus, we aimed to determine whether NADH signaling downregulates endothelial SK channel function via PKC. SK channel currents of human coronary artery endothelial cells were measured by whole cell patch clamp method in the presence/absence of NADH, PKC activator phorbol 12-myristate 13-acetate, PKC inhibitors, or endothelial PKCα/PKCβ knockdown by using small interfering RNA. Human coronary arteriolar reactivity in response to the selective SK activator NS309 was measured by vessel myography in the presence of NADH and PKCβ inhibitor LY333531. NADH (30-300 μmol/L) or PKC activator phorbol 12-myristate 13-acetate (30-300 nmol/L) reduced endothelial SK current density, whereas the selective PKCᵦ inhibitor LY333531 significantly reversed the NADH-induced SK channel inhibition. PKCβ small interfering RNA, but not PKCα small interfering RNA, significantly prevented the NADH- and phorbol 12-myristate 13-acetate-induced SK inhibition. Incubation of human coronary artery endothelial cells with NADH significantly increased endothelial PKC activity and PKCβ expression and activation. Treating vessels with NADH decreased coronary arteriolar relaxation in response to the selective SK activator NS309, and this inhibitive effect was blocked by coadministration with PKCβ inhibitor LY333531. NADH-induced inhibition of endothelial SK channel function is mediated via PKCβ. These findings may provide insight into novel therapeutic strategies to preserve coronary microvascular function in patients with metabolic syndrome and coronary disease.

Activation of small conductance Ca2+ -activated K+ channels suppresses Ca2+ transient and action potential alternans in ventricular myocytes.

At the cellular level, cardiac alternans is observed as beat-to-beat alternations in contraction strength, action potential (AP) morphology and Ca2+ transient (CaT) amplitude, and is a risk factor for cardiac arrhythmia. The (patho)physiological roles of small conductance Ca2+ -activated K+ (SK) channels in ventricles are poorly understood. We tested the hypothesis that in single rabbit ventricular myocytes pharmacological modulation of SK channels plays a causative role for the development of pacing-induced CaT and AP duration (APD) alternans. SK channel blockers (apamin, UCL1684) had only a minor effect on AP repolarization. However, SK channel activation by NS309 resulted in significant APD shortening, demonstrating that functional SK channels are well expressed in ventricular myocytes. The effects of NS309 were prevented or reversed by apamin and UCL1684, indicating that NS309 acted on SK channels. SK channel activation abolished or reduced the degree of pacing-induced CaT and APD alternans. Inhibition of KV 7.1 (with HMR1556) and KV 11.1 (with E4031) channels was used to mimic conditions of long QT syndromes type-1 and type-2, respectively. Both HMR1556 and E4031 enhanced CaT alternans that was prevented by SK channel activation. In AP voltage-clamped cells the SK channel activator had no effect on CaT alternans, confirming that suppression of CaT alternans was caused by APD shortening. APD shortening contributed to protection from alternans by lowering sarcoplasmic reticulum Ca2+ content and curtailing Ca2+ release. The data suggest that SK activation could be a potential intervention to avert development of alternans with important ramifications for arrhythmia prevention and therapy for patients with long QT syndrome. KEY POINTS: At the cellular level, cardiac alternans is observed as beat-to-beat alternations in contraction strength, action potential (AP) morphology and intracellular Ca2+ release amplitude, and is a risk factor for cardiac arrhythmia. The (patho)physiological roles of small conductance Ca2+ -activated K+ (SK) channels in ventricles are poorly understood. We investigated whether pharmacological modulation of SK channels affects the development of cardiac alternans in normal ventricular cells and in cells with drug-induced long QT syndrome (LQTS). While SK channel blockers have only a minor effect on AP morphology, their activation leads to AP shortening and abolishes or reduces the degree of pacing-induced Ca2+ and AP alternans. AP shortening contributed to protection against alternans by lowering sarcoplasmic reticulum Ca2+ content and curtailing Ca2+ release. The data suggest SK activation as a potential intervention to avert the development of alternans with important ramifications for arrhythmia prevention for patients with LQTS.

Electrophysiological Studies Support Utility of Positive Modulators of SK Channels for the Treatment of Spinocerebellar Ataxia Type 2.

Spinocerebellar ataxia type 2 (SCA2) is an incurable hereditary disorder accompanied by cerebellar degeneration following ataxic symptoms. The causative gene for SCA2 is ATXN2. The ataxin-2 protein is involved in RNA metabolism; the polyQ expansion may interrupt ataxin-2 interaction with its molecular targets, thus representing a loss-of-function mutation. However, mutant ataxin-2 protein also displays the features of gain-of-function mutation since it forms the aggregates in SCA2 cells and also enhances the IP3-induced calcium release in affected neurons. The cerebellar Purkinje cells (PCs) are primarily affected in SCA2. Their tonic pacemaker activity is crucial for the proper cerebellar functioning. Disturbances in PC pacemaking are observed in many ataxic disorders. The abnormal intrinsic pacemaking was reported in mouse models of episodic ataxia type 2 (EA2), SCA1, SCA2, SCA3, SCA6, Huntington's disease (HD), and in some other murine models of the disorders associated with the cerebellar degeneration. In our studies using SCA2-58Q transgenic mice via cerebellar slice recording and in vivo recording from urethane-anesthetized mice and awake head-fixed mice, we have demonstrated the impaired firing frequency and irregularity of PCs in these mice. PC pacemaker activity is regulated by SK channels. The pharmacological activation of SK channels has demonstrated some promising results in the electrophysiological experiments on EA2, SCA1, SCA2, SCA3, SCA6, HD mice, and also on mutant CACNA1A mice. In our studies, we have reported that the SK activators CyPPA and NS309 converted bursting activity into tonic, while oral treatment with CyPPA and NS13001 significantly improved motor performance and PC morphology in SCA2 mice. The i.p. injections of chlorzoxazone (CHZ) during in vivo recording sessions converted bursting cells into tonic in anesthetized SCA2 mice. And, finally, long-term injections of CHZ recovered the precision of PC pacemaking activity in awake SCA2 mice and alleviated their motor decline. Thus, the SK activation can be used as a potential way to treat SCA2 and other diseases accompanied by cerebellar degeneration.

Abstract 13450: Role of PKC in Metabolic Regulation of Endothelial SK Channels

Introduction: Diabetes is associated with increases in metabolites NADH, and PKC. NADH has been recently found to inactivate endothelial SK channels. Overexpressed and activated PKC has been shown to play an important role in diabetes-induced endothelial dysfunction. However, it is undefined if PKC is involved in the NADH dysregulation of endothelial SK channels. Hypothesis: We hypothesized that PKC in a signaling cascade whereby NADH dysregulates endothelial SK channels and coronary relaxation function. Methods: SK channel currents of human coronary artery endothelial cells were measured by whole-cell patch-clamp method in the presence or absence of NADH, and/or PKC activator PMA, or PKC inhibitors or endothelial PKC α /PKC β knock-down by using short interfering RNA. The in-vitro coronary arterioles (n = 5) from the harvested atrial tissue samples of patients undergoing cardiac surgery were perfused with NADH in the presence or absence of PKC β inhibitor LY333531. Vascular reactivity was measured by vessel myography in the presence of vasoconstrictor and vasodilators. Results: NADH (30-300μM, n=7-9) or PKC activator PMA (30-300μM, n=6-12) reduced endothelial SK current density (p<0.05 vs. control (n=15), Fig. A-D), whereas the selective PKC β inhibitor LY333531 (50nM, n=12) significantly reversed the NADH-induced SK channel inhibition (p>0.05 vs. control (n=15), Fig. E). PKC α knock-down failed to affect NADH (n=13) and PMA (n=10) inhibition of endothelial SK currents (Fig. F). In contrast, PKC β knock-down significantly prevented NADH (n=12) and PMA (n=6)-induced SK inhibition (p>0.05, vs. control, Fig. G). Incubation of vessels with NADH significantly reduced relaxation response to the SK activator NS309 and this effect was abolished in the presence of LY333531. Conclusions: The metabolite NADH dysregulation of endothelial SK channels and impairment of endothelium-dependent relaxation function was via the PKC β signaling pathway.

Coronary endothelial dysfunction prevented by small-conductance calcium-activated potassium channel activator in mice and patients with diabetes

ObjectiveTo investigate coronary endothelial protection of a small-conductance calcium-activated potassium (SK) channel activator against a period of cardioplegic-hypoxia and reoxygenation (CP-H/R) injury in mice and patients with diabetes (DM) and those without diabetes (nondiabetic [ND]). MethodsMouse small coronary arteries/heart endothelial cells (MHECs) and human coronary arterial endothelial cells (HCAECs) were dissected from the harvested hearts of mice (n = 16/group) and from discarded right atrial tissue samples of patients with DM and without DM (n = 8/group). The SK current density of MHECs was measured. The in vitro small arteries/arterioles, MHECs, and HCAECs were subjected to 60 minutes of CP hypoxia, followed by 60 minutes of oxygenation. Vessels were treated with or without the selective SK activator NS309 for 5 minutes before and during CP hypoxia. ResultsDM and/or CP-H/R significantly inhibited the total SK currents of MHECs and HCAECs and significantly diminished the mouse coronary relaxation response to NS309. Administration of NS309 immediately before and during CP hypoxia significantly improved the recovery of coronary endothelial function, as demonstrated by increased relaxation responses to adenosine 5′-diphosphate and substance P compared with those seen in controls (P < .05). This protective effect was more pronounced in vessels from ND mice and patients compared with DM mice and patients (P < .05). Cell surface membrane SK3 expression was significantly reduced after hypoxia, whereas cytosolic SK3 expression was greater than that of the sham control group (P < .05). ConclusionsApplication of NS309 immediately before and during CP hypoxia protects mouse and human coronary microvasculature against CP-H/R injury, but this effect is diminished in the diabetic coronary microvasculature. SK inhibition/inactivation and/or internalization/redistribution may contribute to CP-H/R–induced coronary endothelial and vascular relaxation dysfunction.

Both Lobes of Calmodulin Bound to KCa2.2 Respond to Ca2+

An increase in intracellular Ca2+ opens KCa2.2 Ca2+-activated K+-channels (SK) to conduct K+ down its electrochemical gradient. SK activation requires bound calmodulin proteins to decode dynamic Ca2+ fluctuations in a cell. Bilobal calmodulin has four Ca2+ binding sites. A common model proposes that just the N-terminal domain (N-lobe) of calmodulin binds Ca2+ to open SK, while an immobile C-terminal domain (C-lobe) has constitutive, Ca2+-independent binding. Such a mechanism is compatible with x-ray and EM structures of intact SK channels and fragments. While molecular structures provide snapshots of the channels in different conformations with and without calcium, gating is a dynamic process involving several different conformations, calcium binding to calmodulin, interactions between calmodulin and the channel and channel opening. We measured SK activity using inside-out patch recordings from “calmodulin-disrupted” SK channels where calcium-dependent activation can be restored by exogenously applied full-length calmodulin. Isolated N and C lobes have little ability to activate SK. We measured calmodulin binding to a C-terminal SK2 peptide (SKp) using both composition-gradient multi-angle light scattering and tryptophan emission spectra. While they do not rescue activation, isolated lobes do bind to a SK cytoplasmic domain with high affinity. Consistent with earlier models, N-lobe binding to SKp is stronger in Ca2+, and C-lobe binding affinity is strong regardless of Ca2+. However, a native tryptophan in SKp is sensitive to Ca2+ binding to either the N- or C-lobe of calmodulin at Ca2+ concentrations that activate SK. Our results show that the calmodulin C-lobe bound to SK is Ca2+ sensitive and that both lobes of calmodulin bind SK in Ca2+-dependent states governed by the different properties of each lobe.

Loose coupling between SK and P/Q-type Ca2+ channels in cartwheel cells of the dorsal cochlear nucleus.

Small-conductance Ca2+-activated K+ (SK) and large-conductance voltage- and Ca2+-activated K+ (BK) channels are Ca2+-activated K+ channels that control action potential firing in diverse neurons in the brain. In cartwheel cells of the dorsal cochlear nucleus, blockade of either channel type leads to excessive production of spike bursts. In the same cells, P/Q-type Ca2+ channels in plasma membrane and ryanodine receptors in endoplasmic reticulum supply Ca2+ to BK channels through Ca2+ nanodomain signaling. In this study, voltage-clamp experiments were performed in cartwheel cells in mouse brain slices to examine the Ca2+ signaling pathways underlying activation of SK channels. As with BK channels, SK channels required the activity of P/Q-type Ca2+ channels. However, this signaling occurred across Ca2+ micro- rather than nanodomain distances and was independent of Ca2+ release from endoplasmic reticulum. These differential modes of activation may lead to distinct time courses of the two K+ currents and therefore control excitability of auditory neurons across different timescales.NEW & NOTEWORTHY This study has shown for the first time that in cartwheel cells of the dorsal cochlear nucleus, small-conductance Ca2+-activated K+ (SK) channels were triggered by the activation of P/Q-type Ca2+ channels in which SK-P/Q-type coupling is mediated within the Ca2+ microdomains (loose coupling). Although Ca2+-induced Ca2+ release is able to activate large-conductance voltage- and Ca2+-activated K+ (BK) channels in cartwheel cells, it did not contribute to SK activation.

Abstract 297: Therapeutic Effects of SK Channel Activator on Cardioprotection in the Setting of Cardioplegic Ischemia-Reperfusion and Cardiopulmonary Bypass

Background: Inactivation of small-conductance calcium-activated potassium (SK) channels contributes to coronary microvascular dysfunction in patients after cardioplegic arrest, cardiopulmonary bypass (CP/CPB) and cardiac surgery. SK-channel activation is cardioprotective in rodent isolated heart model of myocardial infarction and ischemia/reperfusion. In the current study, we investigated the impact of SK activation on myocardial protection in a clinically relevant large animal model of pigs subjected to cardioplegic ischemia/reperfusion and CPB. Methods and Results: Ten Yorkshire pigs were subjected to 1 hour of CP/CPB followed by 1 hour of reperfusion. Pigs received either cold blood hyperkalemic CP alone (CP control) or CP containing the selective SK channel activator NS309 (10 -6 M, n = 5/group). Left ventricular (LV) function was assessed by using pressure-volume loop analysis. Coronary arteriolar relaxation response (in-vitro) to the endothelium-dependent vasodilators adenosine diphosphate (ADP, 10 -9 -10 -4 M) and substance P (10 -12 -10 -7 M) and independent vasodilator sodium nitroprusside (SNP) was evaluated with video-microscopy. Inclusion of NS309 in cold blood CP tends to improve the recovery of LV function compared with the CP controls. CP/CPB and reperfusion significantly reduced endothelium-dependent relaxation response to ADP, 10 -5 M) and substance P (10 -8 M) as compared to control (p&lt;0.05). In contrast, CP-containing NS309 significant improved the recovery of endothelium-dependent relaxation response to ADP and substance P as compared with the control group (p&lt;0.05, respectively). Conclusion: In pig model of CP/CPB, inclusion of the selective SK channel activator in the cold blood cardioplegia improves recovery of coronary arteriolar endothelial function and microvascular relaxation, which may contribute to increased myocardial perfusion and improved LV function.

213 Nrf2 activation improves both hyperpolarization- and NO-mediated responses in penile vasculature from patients with erectile dysfunction

Oxidative stress has been proposed to be related to erectile dysfunction (ED) pathophysiology. Nrf2 is a master regulator of antioxidant defense. We have previously reported an improvement of endothelium-dependent relaxation by Nrf2 activator, sulforaphane, in rat corpus cavernosum (RCC) from aged rats and in penile resistance arteries (HPRA) and corpus cavernosum (HCC) from patients with ED. The aim of this work was to characterize the effects of Nrf2 activation in human penile tissue function. HPRA and HCC were dissected from cavernosal specimens from 17 patients (64.3±2.4 years old) undergoing penile prosthesis insertion, who gave informed consent, and functionally evaluated in wire myographs and organ chambers, respectively. Six patients had diabetes, 11 hypertension, 7 dyslipidemia, 2 obesity and 4 established cardiovascular disease. All patients had one or more cardiovascular risk factors (CVRFs). Two different Nrf2 activators, sulforaphane (10 μM) and oltipraz (30 μM) were able to improve endothelial relaxation in HCC, contributed by NO, and in HPRA, contributed by NO and endothelium-derived hyperpolarizing factor (EDHF). In fact, sulforaphane markedly improved EDHF-mediated vasodilation (after NO synthesis and cyclooxygenase inhibition) in HPRA (Emax 47.0±10.7% vs. 77.1±3.9%, p < 0.05). Small- and intermediate-conductance calcium-activated potassium channels (SK and IK) are involved in EDHF-mediated vasodilation. In this sense, sulforaphane enhanced the relaxation induced by the SK and IK activator, NS309, in HPRA. However, the very limited endothelial vasodilation (10.2±4.9%) remaining after blocking hyperpolarization with KCl in HPRA was also significantly potentiated by sulforaphane (24.7±4.7%, p < 0.05), suggesting that Nrf2 activation potentiates NO-dependent vasodilation. In this sense, treatment of HPRA from ED patients with sulforaphane resulted in an improvement of vasodilation driven by the PDE5 inhibitor, tadalafil (pD2 6.24±0.51 vs. 7.55±0.55, p < 0.05).

Confirming a Role for α9nAChRs and SK Potassium Channels in Type II Hair Cells of the Turtle Posterior Crista.

In turtle posterior cristae, cholinergic vestibular efferent neurons (VENs) synapse on type II hair cells, bouton afferents innervating type II hair cells, and afferent calyces innervating type I hair cells. Electrical stimulation of VENs releases acetylcholine (ACh) at these synapses to exert diverse effects on afferent background discharge including rapid inhibition of bouton afferents and excitation of calyx-bearing afferents. Efferent-mediated inhibition is most pronounced in bouton afferents innervating type II hair cells near the torus, but becomes progressively smaller and briefer when moving longitudinally through the crista toward afferents innervating the planum. Sharp-electrode recordings have inferred that efferent-mediated inhibition of bouton afferents requires the sequential activation of alpha9-containing nicotinic ACh receptors (α9*nAChRs) and small-conductance, calcium-dependent potassium channels (SK) in type II hair cells. Gradations in the strength of efferent-mediated inhibition across the crista likely reflect variations in α9*nAChRs and/or SK activation in type II hair cells from those different regions. However, in turtle cristae, neither inference has been confirmed with direct recordings from type II hair cells. To address these gaps, we performed whole-cell, patch-clamp recordings from type II hair cells within a split-epithelial preparation of the turtle posterior crista. Here, we can easily visualize and record hair cells while maintaining their native location within the neuroepithelium. Consistent with α9*nAChR/SK activation, ACh-sensitive currents in type II hair cells were inward at hyperpolarizing potentials but reversed near −90 mV to produce outward currents that typically peaked around −20 mV. ACh-sensitive currents were largest in torus hair cells but absent from hair cells near the planum. In current clamp recordings under zero-current conditions, ACh robustly hyperpolarized type II hair cells. ACh-sensitive responses were reversibly blocked by the α9nAChR antagonists ICS, strychnine, and methyllycaconitine as well as the SK antagonists apamin and UCL1684. Intact efferent terminals in the split-epithelial preparation spontaneously released ACh that also activated α9*nAChRs/SK in type II hair cells. These release events were accelerated with high-potassium external solution and all events were blocked by strychnine, ICS, methyllycaconitine, and apamin. These findings provide direct evidence that activation of α9*nAChR/SK in turtle type II hair cells underlies efferent-mediated inhibition of bouton afferents.

Endothelial Caveolae-TRPV4-SK Channel Interactions in Shear Stress-Mediated Coronary Arteriole Dilation

We have previously reported that shear stress-mediated dilation (SSD) of coronary arterioles is caveolae-dependent. We found that both TRPV4 and SK channels are targeted to the low-buoyant density caveolin-rich membrane fraction in freshly isolated bovine coronary endothelial cells (BCEC) and TRPV4 and SK3 channels are co-immunoprecipitated by anti-caveolin-1 antibody. Isoproterenol (1 µM) enhanced the whole-cell K+ currents in BCEC by 5-fold that are inhibited by apamin (200 nM), an SK channel-specific inhibitor, but not by iberiotoxin (100 nM) or Tram34 (200 nM). Exposure of the BCEC to the TRPV4 activator, GSK1016790A (150 nM) resulted in a 3.5-fold activation of SK currents. Acute exposure of BCEC seeded in a capillary tube to 10 dynes/cm2 of shear stress (SS) resulted in a 4-fold increase in TRPV4 currents that are inhibited by HC067047 (200 nM), a TRPV4-specific inhibitor, and a 3.5-fold increase in SK currents that are inhibited by apamin (200 nM). However, after incubation with HC067047, a TRPV4 specific inhibitor, SK currents could no longer be activated by SS, suggesting SK activation by SS was mediated through TRPV4. In addition, pre-incubation of isolated coronary arterioles with apamin (200 nM) resulted in a significant diminution of SSD while pre-incubation with HC067047 (150 nM) results in vasoconstriction by SS. Exposure of BECE to SS (10 dynes/cm2 12 h) enhanced the production of endothelium-derived vasodilators, NO and PGI2, and facilitated the translocation of TRPV4 to the caveolae. Inhibition of TRPV4 abolished the SS-mediated [Ca2+]i increase in BECE. These novel results indicate that there is a dynamic interaction in the vascular endothelium between caveolae, TRPV4 and SK channels in the modulation of SSD.

Mitochondrial SK Channels Attenuate Ca-Dependent Arrhythmia in Hypertrophic Hearts by Regulating Mito-ROS-Dependent Oxidation and Activity of RyR

Rationale: Small conductance Ca2+-activated K+ (SK) channels are implicated in ventricular arrhythmias in the context of heart failure and myocardial infarct. In addition to their plasmalemmal localization, SK channels were recently discovered in mitochondria inner membrane (mSK). Activation of these channels is protective against ischemia-reperfusion injury by reducing intracellular levels of reactive oxygen species (ROS). We hypothesized that mSK may also play an important role in regulating mitochondrial function in cardiac disease as well.Objective: We investigated the role of mSK channels in Ca2+-dependent ventricular arrhythmia using a rat model of hypertrophy induced by banding of the ascending aorta (TAB).Methods and Results: Dual Ca2+ and membrane potential optical mapping of whole hearts from TAB rats revealed that membrane-permeable SK activator improved aberrant Ca2+ homeostasis and abolished VT/VF induced by b-adrenergic stimulation. Using whole cell patch-clamp and confocal Ca2+ imaging we found that membrane-permeable SK enhancers attenuated frequency of spontaneous Ca2+ waves and delayed afterdepolarizations in hypertrophic cardiomyocytes. Furthermore, mSK inhibition enhanced; and activation reduced mitochondrial ROS production in TAB myocytes measured with MitoSOX. Monobromobimane assay demonstrated that increased oxidation of ryanodine receptors (RyRs) in TAB cells was reversed by SK enhancers. Experiments in permeabilized TAB myocytes showed that SK enhancers restored SR Ca2+ content, suggestive of substantial improvement in RyR function.Conclusions: These data suggest that enhancement of mSK channels in hypertrophic rat hearts protects from Ca2+-dependent arrhythmia. This protection is mediated via decreased mitochondrial ROS and subsequent decreased oxidation of reactive cysteines in RyR, which ultimately leads to stabilization of RyR-mediated Ca2+ release.

Abstract 17442: Effects of Small Conductance Ca2+-activated K+ Channels on Cardiogenesis in Human Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (hiPSCs) with the ability to form all somatic cells can generate pacemaking cardiomyocytes (CMs) for engineering of biological pacemakers. However, differentiation efficiency of hiPSCs into pacemaking cardiomyocytes is merely at 5% of the total CMs. Membrane potential of a cell can alter cell fate. Activation of small conductance Ca2+-activated K+ channels (SKs) has been shown to induce membrane hyperpolarization. In this study, we demonstrated that SKs are present and functional in hiPSCs and their differentiation progeny. Addition of SK activator (EBIO or CyPPA) hyperpolarized hiPSCs and differentiating hiPSCs 2 and 5 days post-differentiation by -2.5±0.97, -22.7±1.8 and -13.6±3.3 mV, respectively. The magnitude of hyperpolarization exhibited dose-dependency to SK activator in differentiating hiPSCs 2 days post-differentiation. Activation of SK channels by EBIO or CyPPA on day 2-3 from the onset of differentiation significantly upregulated the expression of Islet (Isl)1, a marker for a subset of cardiac progenitor cells that are known to form the sinoatrial node where the pacemaking cardiomyocyte reside. The number of Isl1-positive cells in differentiating hiPSCs with SK activation on day 2-3 has also increased relative to control. Our findings indicate that the activation of SK channels at an appropriate treatment time and length results in a facilitated increase in the differentiation of hiPSCs to pacemaking cardiomyocyte precursors.

Activation of Mitochondrial SK Channels in Cardiomyocytes Derived from Hypertrophic Hearts Attenuates Ca2+-Dependent Arrhythmia by Reducing Mitochondrial ROS Production Thereby Stabilizing RyRs

Small conductance Ca2+-activated K+ (SK) channels were recently discovered in mitochondria inner membrane. Activation of these voltage-independent channels was found to be protective against ischemia-reperfusion injury by reducing intracellular levels of reactive oxygen species (ROS). Considering submicromolar [Ca2+] sensitivity of SKs we hypothesized that these channels may play an important role in cardiac hypertrophy. To investigate the role of mSK channels in cardiac hypertrophy we used a rat model of pressure-overload induced by ligation of ascending aorta (TAB). Using confocal Ca2+ imaging and patch-clamp we found that inhibition of SK channels with cell-permeable inhibitor UCL-1468 (1 uM) increased frequency of spontaneous Ca2+ waves (SCWs) and delayed afterdepolarizations (DADs) in TAB ventricular myocytes (TCMs). Conversely, membrane-permeable SK enhancer CyPPA (10 uM) attenuated pro-arrhythmic SCWs and DADs in TCMs. Furthermore, inhibition of mSKs enhanced; and activation reduced ROS production by mitochondria in TCMs measured with MitoSox. Monobromobimane assay demonstrated that increased oxidation of ryanodine receptors (RyRs) in TCMs was reversed by CyPPA. Experiments in permeabilized myocytes showed that CyPPA was unable to completely reverse increase in spark frequency in TCMs. However, incubation of TCMs with CyPPA restored SR Ca2+ content, suggestive of substantial improvement in RyR function. Optical mapping experiments of TAB hearts using dual Ca2+ and membrane potential imaging revealed that incubation with membrane-permeable SK activator NS309 (2 uM) improved aberrant Ca2+ homeostasis and abolished DADs and VT/VF induced by beta-adrenergic stimulation. These data suggest that pharmacological activation of mSK channels in hypertrophy protects from Ca2+-dependent arrhythmia via reduction of mitochondrial ROS, and thereby reduction of oxidized RyR leading to its stabilization.

CaV1.3-Driven SK Channel Activation Regulates Pacemaking and Spike Frequency Adaptation in Mouse Chromaffin Cells

Mouse chromaffin cells (MCCs) fire spontaneous action potentials (APs) at rest. Ca(v)1.3 L-type calcium channels sustain the pacemaker current, and their loss results in depolarized resting potentials (V(rest)), spike broadening, and remarkable switches into depolarization block after BayK 8644 application. A functional coupling between Ca(v)1.3 and BK channels has been reported but cannot fully account for the aforementioned observations. Here, using Ca(v)1.3(-/-) mice, we investigated the role of Ca(v)1.3 on SK channel activation and how this functional coupling affects the firing patterns induced by sustained current injections. MCCs express SK1-3 channels whose tonic currents are responsible for the slow irregular firing observed at rest. Percentage of frequency increase induced by apamin was found inversely correlated to basal firing frequency. Upon stimulation, MCCs build-up Ca(v)1.3-dependent SK currents during the interspike intervals that lead to a notable degree of spike frequency adaptation (SFA). The major contribution of Ca(v)1.3 to the subthreshold Ca(2+) charge during an AP-train rather than a specific molecular coupling to SK channels accounts for the reduced SFA of Ca(v)1.3(-/-) MCCs. Low adaptation ratios due to reduced SK activation associated with Ca(v)1.3 deficiency prevent the efficient recovery of Na(V) channels from inactivation. This promotes a rapid decline of AP amplitudes and facilitates early onset of depolarization block following prolonged stimulation. Thus, besides serving as pacemaker, Ca(v)1.3 slows down MCC firing by activating SK channels that maintain Na(V) channel availability high enough to preserve stable AP waveforms, even upon high-frequency stimulation of chromaffin cells during stress responses.

Chlorzoxazone, an SK-Type Potassium Channel Activator Used in Humans, Reduces Excessive Alcohol Intake in Rats

Alcoholism imposes a tremendous social and economic burden. There are relatively few pharmacological treatments for alcoholism, with only moderate efficacy, and there is considerable interest in identifying additional therapeutic options. Alcohol exposure alters SK-type potassium channel (SK) function in limbic brain regions. Thus, positive SK modulators such as chlorzoxazone (CZX), a US Food and Drug Administration-approved centrally acting myorelaxant, might enhance SK function and decrease neuronal activity, resulting in reduced alcohol intake. We examined whether CZX reduced alcohol consumption under two-bottle choice (20% alcohol and water) in rats with intermittent access to alcohol (IAA) or continuous access to alcohol (CAA). In addition, we used ex vivo electrophysiology to determine whether SK inhibition and activation can alter firing of nucleus accumbens (NAcb) core medium spiny neurons. Chlorzoxazone significantly and dose-dependently decreased alcohol but not water intake in IAA rats, with no effects in CAA rats. Chlorzoxazone also reduced alcohol preference in IAA but not CAA rats and reduced the tendency for rapid initial alcohol consumption in IAA rats. Chlorzoxazone reduction of IAA drinking was not explained by locomotor effects. Finally, NAcb core neurons ex vivo showed enhanced firing, reduced SK regulation of firing, and greater CZX inhibition of firing in IAA versus CAA rats. The potent CZX-induced reduction of excessive IAA alcohol intake, with no effect on the more moderate intake in CAA rats, might reflect the greater CZX reduction in IAA NAcb core firing observed ex vivo. Thus, CZX could represent a novel and immediately accessible pharmacotherapeutic intervention for human alcoholism.

Endothelium-Dependent Vasodilation in Human Mesenteric Artery Is Primarily Mediated by Myoendothelial Gap Junctions Intermediate Conductance Calcium-Activated K+ Channel and Nitric Oxide

Myoendothelial microdomain signaling via localized calcium-activated potassium channel (K(Ca)) and gap junction connexins (Cx) is critical for endothelium-dependent vasodilation in rat mesenteric artery. The present study determines the relative contribution of NO and gap junction-K(Ca) mediated microdomain signaling to endothelium-dependent vasodilation in human mesenteric artery. The hypothesis tested was that such activity is due to NO and localized K(Ca) and Cx activity. In mesenteric arteries from intestinal surgery patients, endothelium-dependent vasodilation was characterized using pressure myography with pharmacological intervention. Vessel morphology was examined using immunohistochemical and ultrastructural techniques. In vessel segments at 80 mm Hg, the intermediate (I)K(Ca) blocker 1-[(2-chlorophenyl)diphenyl-methyl]-1H-pyrazole (TRAM-34; 1 μM) inhibited bradykinin (0.1 nM-3 μM)-induced vasodilation, whereas the small (S) K(Ca) blocker apamin (50 and 100 nM) had no effect. Direct IK(Ca) activation with 1-ethyl-2-benzimidazolinone (1-EBIO; 10-300 μM) induced vasodilation, whereas cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (1-30 μM), the SK(Ca) activator, failed to dilate arteries, whereas dilation induced by 1-EBIO (10-100 μM) was blocked by TRAM-34. Bradykinin-mediated vasodilation was attenuated by putative gap junction block with carbenoxolone (100 μM), with remaining dilation blocked by N-nitro l-arginine methyl ester (100 μM) and [1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one] (10 μM), NO synthase and soluble guanylate cyclase blockers, respectively. In human mesenteric artery, myoendothelial gap junction and IK(Ca) activity are consistent with Cx37 and IK(Ca) microdomain expression and distribution. Data suggest that endothelium-dependent vasodilation is primarily mediated by NO, IK(Ca), and gap junction Cx37 in this vessel. Myoendothelial microdomain signaling sites are present in human mesenteric artery and are likely to contribute to endothelium-dependent vasodilation via a mechanism that is conserved between species.

The small‐conductance calcium‐activated potassium channel is a key modulator of firing and long‐term depression in the dorsal striatum

The striatum is considered to be critical for the control of goal-directed action, with the lateral dorsal striatum (latDS) being implicated in modulation of habits and the nucleus accumbens thought to represent a limbic-motor interface. Although medium spiny neurons from different striatal subregions exhibit many similar properties, differential firing and synaptic plasticity could contribute to the varied behavioral roles across subregions. Here, we examined the contribution of small-conductance calcium-activated potassium channels (SKs) to action potential generation and synaptic plasticity in adult rat latDS and nucleus accumbens shell (NAS) projection neurons in vitro. The SK-selective antagonist apamin exerted a prominent effect on latDS firing, significantly decreasing the interspike interval. Furthermore, prolonged latDS depolarization increased the interspike interval and reduced firing, and this enhancement was reversed by apamin. In contrast, NAS neurons exhibited greater basal firing rates and less regulation of firing by SK inhibition and prolonged depolarization. LatDS neurons also had greater SK currents than NAS neurons under voltage-clamp. Importantly, SK inhibition with apamin facilitated long-term depression (LTD) induction in the latDS but not the NAS, without alterations in glutamate release. In addition, SK activation in the latDS prevented LTD induction. Greater SK function in the latDS than in the NAS was not secondary to differences in sodium or inwardly rectifying potassium channel function, and apamin enhancement of firing did not reflect indirect action through cholinergic interneurons. Thus, these data demonstrate that SKs are potent modulators of action potential generation and LTD in the dorsal striatum, and could represent a fundamental cellular mechanism through which habits are regulated.

Reduced Nucleus Accumbens SK Channel Activity Enhances Alcohol Seeking during Abstinence

The cellular mechanisms underlying pathological alcohol seeking remain poorly understood. Here, we show an enhancement of nucleus accumbens (NAcb) core action potential firing ex vivo after protracted abstinence from alcohol but not sucrose self-administration. Increased firing is associated with reduced small-conductance calcium-activated potassium channel (SK) currents and decreased SK3 but not SK2 subunit protein expression. Furthermore, SK activation ex vivo produces greater firing suppression in NAcb core neurons from alcohol- versus sucrose-abstinent rats. Accordingly, SK activation in the NAcb core significantly reduces alcohol but not sucrose seeking after abstinence. In contrast, NAcb shell and lateral dorsal striatal firing ex vivo are not altered after abstinence from alcohol, and SK activation in these regions has little effect on alcohol seeking. Thus, decreased NAcb core SK currents and increased excitability represents a critical mechanism that facilitates motivation to seek alcohol after abstinence.

SK channel activation for alcoholism

Ernest Gallo Center researchers have reported that activating SK channels in the nucleus accumbens region of the brain could help treat alcohol addiction. The team is planning human studies using chlorzoxazone, a marketed generic SK activator, but the researchers will have to address liver toxicity issues.