Age-dependent neuroprotective effect of an SK3 channel agonist on excitotoxity to dopaminergic neurons in organotypic culture.

BACKGROUND: Small conductance, calcium-activated (SK3) potassium channels control the intrinsic excitability of dopaminergic neurons (DN) in the midbrain and modulate their susceptibility to toxic insults during development. METHODS: We evaluated the age-dependency of the neuroprotective effect of an SK3 agonist, 1-Ethyl-1,3-dihydro-2H-benzimidazol-2-one (1-EBIO), on Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) excitotoxicity to DN in ventral mesencephalon (VM) organotypic cultures. RESULTS: Most tyrosine hydroxylase (TH)+ neurons were also SK3+; SK3+/TH- cells (DN+) were common at each developmental stage but more prominently at day in vitro (DIV) 8. Young DN+ neurons were small bipolar and fusiform, whereas mature ones were large and multipolar. Exposure of organotypic cultures to AMPA (100 µm, 16 h) had no effect on the survival of DN+ at DIV 8, but caused significant toxicity at DIV 15 (n = 15, p = 0.005) and DIV 22 (n = 15, p<0.001). These results indicate that susceptibility of DN to AMPA excitotoxicity is developmental stage-dependent in embryonic VM organotypic cultures. Immature DN+ (small, bipolar) were increased after AMPA (100 µm, 16 h) at DIV 8, at the expense of the number of differentiated (large, multipolar) DN+ (p = 0.039). This effect was larger at DIV 15 (p<<<0.0001) and at DIV 22 (p<<<0.0001). At DIV 8, 30 µM 1-EBIO resulted in a large increase in DN+. At DIV 15, AMPA toxicity was prevented by exposure to 30 µM, but not 100 µM 1-EBIO. At DIV 22, excitotoxicity was unaffected by 30 µM 1-EBIO, and partially reduced by 100 µM 1-EBIO. CONCLUSION: The effects of the SK3 channel agonist 1-EBIO on the survival of SK3-expressing dopaminergic neurons were concentration-dependent and influenced by neuronal developmental stage.

SK channel activation potentiates auranofin-induced cell death in glio- and neuroblastoma cells.

Brain tumours are among the deadliest tumours being highly resistant to currently available therapies. The proliferative behaviour of gliomas is strongly influenced by ion channel activity. Small-conductance calcium-activated potassium (SK/KCa) channels are a family of ion channels that are associated with cell proliferation and cell survival. A combined treatment of classical anti-cancer agents and pharmacological SK channel modulators has not been addressed yet. We used the gold-derivative auranofin to induce cancer cell death by targeting thioredoxin reductases in combination with CyPPA to activate SK channels in neuro- and glioblastoma cells. Combined treatment with auranofin and CyPPA induced massive mitochondrial damage and potentiated auranofin-induced toxicity in neuroblastoma cells in vitro. In particular, mitochondrial integrity, respiration and associated energy generation were impaired. These findings were recapitulated in patient-derived glioblastoma neurospheres yet not observed in non-cancerous HT22 cells. Taken together, integrating auranofin and SK channel openers to affect mitochondrial health was identified as a promising strategy to increase the effectiveness of anti-cancer agents and potentially overcome resistance.

Activation of small conductance calcium-activated potassium channels suppresses seizure susceptibility in the genetically epilepsy-prone rats.

Small conductance calcium-activated potassium (SK) channels dampen neuronal excitability by contributing to slow afterhyperpolarization (AHP) that follows a series of action potentials, and therefore may represent an intrinsic inhibitory mechanism to prevent seizures. We have previously reported that susceptibility to acoustically evoked seizures was associated with downregulation of SK1 and SK3 subtypes of SK channels in the inferior colliculus of the moderated seizure severity strain of the genetically epilepsy-prone rats (GEPR-3s). Here, we evaluated the effects of 1-ethyl-2-benzimidazolinone (1-EBIO), a potent activator of SK channels, on acoustically evoked seizures in both male and female adult GEPR-3s at various time points post-treatment. Systemic administration of 1-EBIO at various tested doses suppressed seizure susceptibility in both male and female GEPR-3s; however, the complete seizure suppression was only observed following administration of relatively higher doses of 1-EBIO in females. These findings indicate that activation of SK channels results in anticonvulsive action against generalized tonic-clonic seizures in both male and female GEPR-3s, with males exhibiting higher sensitivity than females.

Modulation of Cardiovascular Function in Primary Hypertension in Rat by SKA-31, an Activator of KCa2.x and KCa3.1 Channels.

The aim of this study was to investigate the hemodynamic effects of SKA-31, an activator of the small (KCa2.x) and intermediate (KCa3.1) conductance calcium-activated potassium channels, and to evaluate its influence on endothelium-derived hyperpolarization (EDH)-KCa2.3/KCa3.1 type relaxation in isolated endothelium-intact small mesenteric arteries (sMAs) from spontaneously hypertensive rats (SHRs). Functional in vivo and in vitro experiments were performed on SHRs or their normotensive controls, Wistar-Kyoto rats (WKY). SKA-31 (1, 3 and 10 mg/kg) caused a brief decrease in blood pressure and bradycardia in both SHR and WKY rats. In phenylephrine-pre-constricted sMAs of SHRs, SKA-31 (0.01-10 µM)-mediated relaxation was reduced and SKA-31 potentiated acetylcholine-evoked endothelium-dependent relaxation. Endothelium denudation and inhibition of nitric oxide synthase (eNOS) and cyclooxygenase (COX) by the respective inhibitors l-NAME or indomethacin, attenuated SKA-31-mediated vasorelaxation. The inhibition of KCa3.1, KCa2.3, KIR and Na+/K+-ATPase by TRAM-34, UCL1684, Ba2+ and ouabain, respectively, reduced the potency and efficacy of the EDH-response evoked by SKA-31. The mRNA expression of eNOS, prostacyclin synthase, KCa2.3, KCa3.1 and KIR were decreased, while Na+/K+-ATPase expression was increased. Collectively, SKA-31 promoted hypotension and vasodilatation, potentiated agonist-stimulated vasodilation, and maintained KCa2.3/KCa3.1-EDH-response in sMAs of SHR with downstream signaling that involved KIR and Na+/K+-ATPase channels. In view of the importance of the dysfunction of endothelium-mediated vasodilatation in the mechanism of hypertension, application of activators of KCa2.3/KCa3.1 channels such as SKA-31 seem to be a promising avenue in pharmacotherapy of hypertension.

Voltage-Independent SK-Channel Dysfunction Causes Neuronal Hyperexcitability in the Hippocampus of Fmr1 Knock-Out Mice.

Neuronal hyperexcitability is one of the major characteristics of fragile X syndrome (FXS), yet the molecular mechanisms of this critical dysfunction remain poorly understood. Here we report a major role of voltage-independent potassium (K+)-channel dysfunction in hyperexcitability of CA3 pyramidal neurons in Fmr1 knock-out (KO) mice. We observed a reduction of voltage-independent small conductance calcium (Ca2+)-activated K+ (SK) currents in both male and female mice, leading to decreased action potential (AP) threshold and reduced medium afterhyperpolarization. These SK-channel-dependent deficits led to markedly increased AP firing and abnormal input-output signal transmission of CA3 pyramidal neurons. The SK-current defect was mediated, at least in part, by loss of FMRP interaction with the SK channels (specifically the SK2 isoform), without changes in channel expression. Intracellular application of selective SK-channel openers or a genetic reintroduction of an N-terminal FMRP fragment lacking the ability to associate with polyribosomes normalized all observed excitability defects in CA3 pyramidal neurons of Fmr1 KO mice. These results suggest that dysfunction of voltage-independent SK channels is the primary cause of CA3 neuronal hyperexcitability in Fmr1 KO mice and support the critical translation-independent role for the fragile X mental retardation protein as a regulator of neural excitability. Our findings may thus provide a new avenue to ameliorate hippocampal excitability defects in FXS.SIGNIFICANCE STATEMENT Despite two decades of research, no effective treatment is currently available for fragile X syndrome (FXS). Neuronal hyperexcitability is widely considered one of the hallmarks of FXS. Excitability research in the FXS field has thus far focused primarily on voltage-gated ion channels, while contributions from voltage-independent channels have been largely overlooked. Here we report that voltage-independent small conductance calcium-activated potassium (SK)-channel dysfunction causes hippocampal neuron hyperexcitability in the FXS mouse model. Our results support the idea that translation-independent function of fragile X mental retardation protein has a major role in regulating ion-channel activity, specifically the SK channels, in hyperexcitability defects in FXS. Our findings may thus open a new direction to ameliorate hippocampal excitability defects in FXS.

SKA-31, an activator of endothelial Ca2+-activated K+ channels evokes robust vasodilation in rat mesenteric arteries.

It is now well recognized that endothelial KCa2.3 and KCa3.1 channel activities contribute to dilation of resistance arteries via endothelium-mediated hyperpolarization and vascular smooth muscle relaxation. In this study, we have investigated the functional effect of the KCa channel activator SKA-31 in third order rat mesenteric arteries using arterial pressure myography. Isolated arteries were cannulated, pressurized intraluminally to 70 mmHg at 36 °C and then constricted with 1 µM phenylephrine. Acute bath exposure to SKA-31 evoked a robust and reversible inhibition of developed tone (IC50 = 0.22 µM). The vasodilatory effects of SKA-31 and acetylcholine were blunted in the presence of KCa2.3 and KCa3.1 channel antagonists, and were largely prevented following endothelial denudation. Western blot and q-PCR analyses of isolated mesenteric arteries revealed KCa2.3 and KCa3.1 channel expression at the protein and mRNA levels, respectively. Penitrem-A, an inhibitor of KCa1.1 channels, decreased vasodilatory responses to acetylcholine, sodium nitroprusside and NS-1619, but had little effect on SKA-31. Similarly, bath exposure to the eNOS inhibitor L-NAME did not alter SKA-31 and acetylcholine-mediated vasodilation. Collectively, these data highlight the major cellular mechanisms by which the endothelial KCa channel activator SKA-31 inhibits agonist-evoked vasoconstriction in rat small mesenteric arteries.

Identity and function of a cardiac mitochondrial small conductance Ca2+-activated K+ channel splice variant.

We provide evidence for location and function of a small conductance, Ca2+-activated K+ (SKCa) channel isoform 3 (SK3) in mitochondria (m) of guinea pig, rat and human ventricular myocytes. SKCa agonists protected isolated hearts and mitochondria against ischemia/reperfusion (IR) injury; SKCa antagonists worsened IR injury. Intravenous infusion of a SKCa channel agonist/antagonist, respectively, in intact rats was effective in reducing/enhancing regional infarct size induced by coronary artery occlusion. Localization of SK3 in mitochondria was evidenced by Western blot of inner mitochondrial membrane, immunocytochemical staining of cardiomyocytes, and immunogold labeling of isolated mitochondria. We identified a SK3 splice variant in guinea pig (SK3.1, aka SK3a) and human ventricular cells (SK3.2) by amplifying mRNA, and show mitochondrial expression in mouse atrial tumor cells (HL-1) by transfection with full length and truncated SK3.1 protein. We found that the N-terminus is not required for mitochondrial trafficking but the C-terminus beyond the Ca2+ calmodulin binding domain is required for Ca2+ sensing to induce mK+ influx and/or promote mitochondrial localization. In isolated guinea pig mitochondria and in SK3 overexpressed HL-1 cells, mK+ influx was driven by adding CaCl2. Moreover, there was a greater fall in membrane potential (ΔΨm), and enhanced cell death with simulated cell injury after silencing SK3.1 with siRNA. Although SKCa channel opening protects the heart and mitochondria against IR injury, the mechanism for favorable bioenergetics effects resulting from SKCa channel opening remains unclear. SKCa channels could play an essential role in restraining cardiac mitochondria from inducing oxidative stress-induced injury resulting from mCa2+ overload.

SK2 channels regulate mitochondrial respiration and mitochondrial Ca2+ uptake.

Mitochondrial calcium ([Ca2+]m) overload and changes in mitochondrial metabolism are key players in neuronal death. Small conductance calcium-activated potassium (SK) channels provide protection in different paradigms of neuronal cell death. Recently, SK channels were identified at the inner mitochondrial membrane, however, their particular role in the observed neuroprotection remains unclear. Here, we show a potential neuroprotective mechanism that involves attenuation of [Ca2+]m uptake upon SK channel activation as detected by time lapse mitochondrial Ca2+ measurements with the Ca2+-binding mitochondria-targeted aequorin and FRET-based [Ca2+]m probes. High-resolution respirometry revealed a reduction in mitochondrial respiration and complex I activity upon pharmacological activation and overexpression of mitochondrial SK2 channels resulting in reduced mitochondrial ROS formation. Overexpression of mitochondria-targeted SK2 channels enhanced mitochondrial resilience against neuronal death, and this effect was inhibited by overexpression of a mitochondria-targeted dominant-negative SK2 channel. These findings suggest that SK channels provide neuroprotection by reducing [Ca2+]m uptake and mitochondrial respiration in conditions, where sustained mitochondrial damage determines progressive neuronal death.

SK channel enhancers attenuate Ca2+-dependent arrhythmia in hypertrophic hearts by regulating mito-ROS-dependent oxidation and activity of RyR.

Aims: Plasmamembrane small conductance Ca2+-activated K+ (SK) channels were implicated in ventricular arrhythmias in infarcted and failing hearts. Recently, SK channels were detected in the inner mitochondria membrane (IMM) (mSK), and their activation protected from acute ischaemia-reperfusion injury by reducing intracellular levels of reactive oxygen species (ROS). We hypothesized that mSK play an important role in regulating mitochondrial function in chronic cardiac diseases. We investigated the role of mSK channels in Ca2+-dependent ventricular arrhythmia using rat model of cardiac hypertrophy induced by banding of the ascending aorta thoracic aortic banding (TAB). Methods and results: Dual Ca2+ and membrane potential optical mapping of whole hearts derived from TAB rats revealed that membrane-permeable SK enhancer NS309 (2 µM) improved aberrant Ca2+ homeostasis and abolished VT/VF induced by ß-adrenergic stimulation. Using whole cell patch-clamp and confocal Ca2+ imaging of cardiomyocytes derived from TAB hearts (TCMs) we found that membrane-permeable SK enhancers NS309 and CyPPA (10 µM) attenuated frequency of spontaneous Ca2+ waves and delayed afterdepolarizations. Furthermore, mSK inhibition enhanced (UCL-1684, 1 µM); while activation reduced mitochondrial ROS production in TCMs measured with MitoSOX. Protein oxidation assays demonstrated that increased oxidation of ryanodine receptors (RyRs) in TCMs was reversed by SK enhancers. Experiments in permeabilized TCMs showed that SK enhancers restored SR Ca2+ content, suggestive of substantial improvement in RyR function. Conclusion: These data suggest that enhancement of mSK channels in hypertrophic rat hearts protects from Ca2+-dependent arrhythmia and suggest that the 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.

Temporal decorrelation by SK channels enables efficient neural coding and perception of natural stimuli.

It is commonly assumed that neural systems efficiently process natural sensory input. However, the mechanisms by which such efficient processing is achieved, and the consequences for perception and behaviour remain poorly understood. Here we show that small conductance calcium-activated potassium (SK) channels enable efficient neural processing and perception of natural stimuli. Specifically, these channels allow for the high-pass filtering of sensory input, thereby removing temporal correlations or, equivalently, whitening frequency response power. Varying the degree of adaptation through pharmacological manipulation of SK channels reduced efficiency of coding of natural stimuli, which in turn gave rise to predictable changes in behavioural responses that were no longer matched to natural stimulus statistics. Our results thus demonstrate a novel mechanism by which the nervous system can implement efficient processing and perception of natural sensory input that is likely to be shared across systems and species.

Regulation of excitability in tonic firing substantia gelatinosa neurons of the spinal cord by small-conductance Ca(2+)-activated K(+) channels.

The excitability of substantia gelatinosa (SG) neurons in the spinal dorsal horn determines the processing of nociceptive information from the periphery to the central nervous system. Small conductance Ca(2+)-activated K(+) (SK) channels on neurons supply strong negative feedback control on neuronal excitability by affecting afterhyperpolarization (AHP). However, the role of SK channels in regulating tonic-firing SG neuron excitability remains elusive. In the present study, whole-cell recordings were conducted in SG neurons from acute spinal cord slices of adult rats. The SK channel opener 1-ethyl-2-benzimidazolinone (1-EBIO) attenuated spike discharges and increased AHP amplitudes; this effect was mimicked by a high Ca(2+) external solution. Systemic administration of 1-EBIO attenuated the thermal-induced nociception behavior. Conversely, the inhibition of SK channels with apamin, a specific SK channel inhibitor, increased neuronal excitability and decreased the AHP amplitudes; this effect was mimicked by a Ca(2+)-free external solution. Apamin increased excitatory synaptic transmission by increasing the amplitudes of evoked excitatory postsynaptic potentials (eEPSPs). This facilitation depended on N-methyl-d-aspartate (NMDA) receptors, extracellular Mg(2+) and intracellular Ca(2+). Voltage-gated Ca(2+) channels (VGCCs) were also involved in the apamin-induced effects. Strikingly, 1-EBIO action on decreasing excitability persisted in the presence of apamin, indicating that 1-EBIO manipulates SK channels via a pathway rather than via apamin-sensitive SK channels. The data reveal a previously uncharacterized mechanism for manipulating SG neuronal excitability by Ca(2+) conductances via both apamin-sensitive and apamin-insensitive pathways. Because SG neurons in the dorsal horn are involved in regulating nociception, manipulating neuronal excitability via SK channels indicates a potential therapeutic target.

A pharmacologic activator of endothelial KCa channels increases systemic conductance and reduces arterial pressure in an anesthetized pig model.

SKA-31, an activator of endothelial KCa2.3 and KCa3.1 channels, reduces systemic blood pressure in mice and dogs, however, its effects in larger mammals are not well known. We therefore examined the hemodynamic effects of SKA-31, along with sodium nitroprusside (SNP), in anesthetized, juvenile male domestic pigs. Experimentally, continuous measurements of left ventricular (LV), aortic and inferior vena cava (IVC) pressures, along with flows in the ascending aorta, carotid artery, left anterior descending coronary artery and renal artery, were performed during acute administration of SKA-31 (0.1, 0.3, 1.0, 3.0 and 5.0mg/ml/kg) and a single dose of SNP (5.0 µg/ml/kg). SKA-31 dose-dependently reduced mean aortic pressure (mPAO), with the highest dose decreasing mPAO to a similar extent as SNP (-23 ± 3 and -28 ± 4 mmHg, respectively). IVC pressure did not change. Systemic conductance and conductance in coronary and carotid arteries increased in response to SKA-31 and SNP, but renal artery conductance was unaffected. There was no change in either LV stroke volume (SV) or heart rate (versus the preceding control) for any infusion. With no change in SV, drug-evoked decreases in LV stroke work (SW) were attributed to reductions in mPAO (SW vs. mPAO, r(2)=0.82, P<0.001). In summary, SKA-31 dose-dependently reduced mPAO by increasing systemic and arterial conductances. Primary reductions in mPAO by SKA-31 largely account for associated decreases in SW, implying that SKA-31 does not directly impair cardiac contractility.

Orai1 forms a signal complex with SK3 channel in gallbladder smooth muscle.

Orai1 is one of the key components of store-operated Ca(2+) entry (SOCE) involved in diverse physiological functions. Orai1 may associate with other proteins to form a signaling complex. In the present study, we investigated the interaction between Orai1 and small conductance Ca(2+)-activated potassium channel 3 (SK3). With the use of RNA interference technique, we found that the SOCE and its associated membrane hyperpolarization were reduced while Orai1 was knocked down by a specific Orai1 siRNA in guinea pig gallbladder smooth muscle. However, with the use of isometric tension measurements, our results revealed that agonist-induced muscle contractility was significantly enhanced after Orai1 protein was knocked down or the tissue was treated by SK3 inhibitor apamin, but not affected by larger conductance Ca(2+)-activated potassium channel inhibitor iberiotoxin or intermediate conductance Ca(2+)-activated potassium channel inhibitor TRAM-34. In addition, in the presence of apamin, Orai1 siRNA had no additional effect on agonist-induced contraction. In coimmunoprecipitation experiment, SK3 and Orai1 pulled down each other. These data suggest that, Orai1 physically associated with SK3 to form a signaling complex in gallbladder smooth muscle. Ca(2+) entry via Orai1 activates SK3, resulting in membrane hyperpolarization in gallbladder smooth muscle. This hyperpolarizing effect of Orai1-SK3 coupling could serve to prevent excessive contraction of gallbladder smooth muscle in response to contractile agonists.

Inactivation of Endothelial Small/Intermediate Conductance of Calcium-Activated Potassium Channels Contributes to Coronary Arteriolar Dysfunction in Diabetic Patients.

BACKGROUND: Diabetes is associated with coronary arteriolar endothelial dysfunction. We investigated the role of the small/intermediate (SK(Ca)/IK(Ca)) conductance of calcium-activated potassium channels in diabetes-related endothelial dysfunction. METHODS AND RESULTS: Coronary arterioles (80 to 150 µm in diameter) were dissected from discarded right atrial tissues of diabetic (glycosylated hemoglobin = 9.6±0.25) and nondiabetic patients (glycosylated hemoglobin 5.4±0.12) during coronary artery bypass graft surgery (n=8/group). In-vitro relaxation response of precontracted arterioles was examined in the presence of the selective SK(Ca)/IK(Ca) activator NS309 and other vasodilatory agents. The channel density and membrane potential of diabetic and nondiabetic endothelial cells was measured by using the whole cell patch-clamp technique. The protein expression and distribution of the SK(Ca)/IK(Ca) in the human myocardium and coronary arterioles was examined by Western blotting and immunohistochemistry. Our results indicate that diabetes significantly reduced the coronary arteriolar response to the SK(Ca)/IK(Ca) activator NS309 compared to the respective responses of nondiabetic vessels (P<0.05 versus nondiabetes). The relaxation response of diabetic arterioles to NS309 was prevented by denudation of endothelium (P=0.001 versus endothelium-intact). Diabetes significantly decreased endothelial SK(Ca)/IK(Ca) currents and hyperpolarization induced by the SK(Ca)/IK(Ca) activator NS309 as compared with that of nondiabetics. There were no significant differences in the expression and distribution of SK(Ca)/IK(Ca) proteins in the coronary microvessels. CONCLUSIONS: Diabetes is associated with inactivation of endothelial SK(Ca)/IK(Ca) channels, which may contribute to endothelial dysfunction in diabetic patients.

UTP activates small-conductance Ca2+-activated K+ channels in murine detrusor PDGFRα+ cells.

Purines induce transient contraction and prolonged relaxation of detrusor muscles. Transient contraction is likely due to activation of inward currents in smooth muscle cells, and prolonged relaxation may be due to activation of small-conductance Ca(2+)-activated K(+) (SK) channels via P2Y1 receptors expressed by detrusor PDGF receptor (PDGFR)α(+) cells. We investigated whether other subtypes of P2Y receptors are involved in the activation of SK channels in PDGFRα(+) cells of detrusor muscles. Quantitative analysis of transcripts revealed that P2ry2, P2ry4, and P2ry14 are expressed in PDGFRα(+) cells of P2ry1-deficient/enhanced green fluorescent protein (P2ry1(-/-)/eGFP) mice at similar levels as in wild-type mice. UTP, a P2Y2/P2Y4 agonist, activated large outward currents in detrusor PDGFRα(+) cells. SK channel blockers and an inhibitor of phospholipase C completely abolished currents activated by UTP. In contrast, UTP activated nonselective cation currents in smooth muscle cells. Under current-clamp (current = 0), UTP induced significant hyperpolarization of PDGFRα(+) cells. MRS2500, a selective P2Y1 antagonist, did not affect UTP-activated outward currents in PDGFRα(+) cells from wild-type mice, and activation of outward currents by UTP was retained in P2ry1(-/-)/eGFP mice. As a negative control, we tested the effect of MRS2693, a selective P2Y6 agonist. This compound did not activate outward currents in PDGFRα(+) cells, and currents activated by UTP were unaffected by MRS2578, a selective P2Y6 antagonist. The nonselective P2Y receptor blocker suramin inhibited UTP-activated outward currents in PDGFRα(+) cells. Our data demonstrate that P2Y2 and/or P2Y4 receptors function, in addition to P2Y1 receptors, in activating SK currents in PDGFRα(+) cells and possibly in mediating purinergic relaxation responses in detrusor muscles.

Inhibition of Myogenic Tone in Rat Cremaster and Cerebral Arteries by SKA-31, an Activator of Endothelial KCa2.3 and KCa3.1 Channels.

Endothelial KCa2.3 and KCa3.1 channels contribute to the regulation of myogenic tone in resistance arteries by Ca(2+)-mobilizing vasodilatory hormones. To define further the functional role of these channels in distinct vascular beds, we have examined the vasodilatory actions of the KCa channel activator SKA-31 in myogenically active rat cremaster and middle cerebral arteries. Vessels pressurized to 70 mm Hg constricted by 80-100 µm (ie, 25%-45% of maximal diameter). SKA-31 (10 µM) inhibited myogenic tone by 80% in cremaster and ∼65% in middle cerebral arteries, with IC50 values of ∼2 µM in both vessels. These vasodilatory effects were largely prevented by the KCa2.3 blocker UCL1684 and the KCa3.1 blocker TRAM-34 and abolished by endothelial denudation. Preincubation with N(G) nitro L-arginine methyl ester (L-NAME, 0.1 mM) did not affect the inhibitory response to SKA-31, but attenuated the ACh-evoked dilation by ∼45%. Penitrem-A, a blocker of BK(Ca) channels, did not alter SKA-31 evoked vasodilation but did reduce the inhibition of myogenic tone by ACh, the BKCa channel activator NS1619, and sodium nitroprusside. Collectively, these data demonstrate that SKA-31 produces robust inhibition of myogenic tone in resistance arteries isolated from distinct vascular beds in an endothelium-dependent manner.

[The effect of modulators of SK channels on simple spike firing frequency in the discharge of the cerebellar Purkinje cells in laboratory mice].

The effect of CyPPA, a positive modulator of small conductance calcium-activated potassium channels of type 3 and 2 (SK3/SK2), and of NS309, an activator of intermediate and small conductance calcium-activated potassium channels (IK/SK), on the activity of cerebellar Purkinje cells was studied in 2-month-old male mice. The use of 1 mM of CyPPA has led to a decrease of simple spike firing frequency in the discharge of Purkinje cells by 25%, on average, during 1 h after application. At the same time, application of 100 µM of NS309 has promoted a decrease in simple spike firing frequency by 47 %, on average, during 1 h after the beginning of the action. The obtained results confirm the hypothesis that SK channels participate in regulation of simple spike firing frequency in the discharge of Purkinje cells and are responsible for restriction of signal frequency. The effect of NS309 on simple spike firing frequency was more pronounced; therefore, the IK/SK channels may be suggested to play the cardinal role in regulation of spike activity of Purkinje cells. Since increasing simple spike frequency in the discharge of Purkinje cells is observed at many disturbances of motor activity, in particular, at spinocerebellar ataxia, it can be suggested that the studied compounds or substances of similar action are of interest as potential medicinal agents.

Upregulation of SK3 and IK1 channels contributes to the enhanced endothelial calcium signaling and the preserved coronary relaxation in obese Zucker rats.

BACKGROUND AND AIMS: Endothelial small- and intermediate-conductance KCa channels, SK3 and IK1, are key mediators in the endothelium-derived hyperpolarization and relaxation of vascular smooth muscle and also in the modulation of endothelial Ca2+ signaling and nitric oxide (NO) release. Obesity is associated with endothelial dysfunction and impaired relaxation, although how obesity influences endothelial SK3/IK1 function is unclear. Therefore we assessed whether the role of these channels in the coronary circulation is altered in obese animals. METHODS AND RESULTS: In coronary arteries mounted in microvascular myographs, selective blockade of SK3/IK1 channels unmasked an increased contribution of these channels to the ACh- and to the exogenous NO- induced relaxations in arteries of Obese Zucker Rats (OZR) compared to Lean Zucker Rats (LZR). Relaxant responses induced by the SK3/IK1 channel activator NS309 were enhanced in OZR and NO- endothelium-dependent in LZR, whereas an additional endothelium-independent relaxant component was found in OZR. Fura2-AM fluorescence revealed a larger ACh-induced intracellular Ca2+ mobilization in the endothelium of coronary arteries from OZR, which was inhibited by blockade of SK3/IK1 channels in both LZR and OZR. Western blot analysis showed an increased expression of SK3/IK1 channels in coronary arteries of OZR and immunohistochemistry suggested that it takes place predominantly in the endothelial layer. CONCLUSIONS: Obesity may induce activation of adaptive vascular mechanisms to preserve the dilator function in coronary arteries. Increased function and expression of SK3/IK1 channels by influencing endothelial Ca2+ dynamics might contribute to the unaltered endothelium-dependent coronary relaxation in the early stages of obesity.

Ginsenoside Re enhances small-conductance Ca(2+)-activated K(+) current in human coronary artery endothelial cells.

AIMS: Ginsenosides, active components in ginseng, have been shown to increase nitric oxide (NO) production in aortic endothelial cells. This effect was reversed by tetraethylammonium (TEA) inhibition of endothelial Ca(2+)-activated K(+) (KCa) channels. The objectives of this study, therefore, were to test 1) whether vasorelaxing ginsenoside Re could affect KCa current, an important regulator of NO production, in human coronary artery endothelial cells (HCAECs); and 2) whether small-conductance KCa (SKCa) channel was the channel subtype involved. MAIN METHODS: Ionic currents of cultured HCAECs were studied using whole-cell patch clamp technique. KEY FINDINGS: Ginsenoside Re dose-dependently increased endothelial outward currents, with an EC50 of 408.90±1.59nM, and a maximum increase of 36.20±5.62% (mean±SEM; p<0.05). Apamin, an SKCa channel inhibitor, could block this effect, while La(3+), a nonselective cation channel (NSC) blocker, could not. When NSC channel, inward-rectifier K(+) channel, intermediate-, and large-conductance KCa channels were simultaneously blocked, ginsenoside Re could still increase outward currents significantly (35.49±4.22%; p<0.05); this effect was again abolished by apamin. Repeating the experiments when Cl(-) channel was additionally blocked gave similar results. Finally, we demonstrated that ginsenoside Re could hyperpolarize HCAECs; this effect was reversed by apamin. These data clearly indicate that ginsenoside Re increased HCAEC outward current via SKCa channel activation, and NSC channel was not involved. SIGNIFICANCE: This is the first report to demonstrate that ginsenoside Re could increase SKCa channel activity in HCAECs. This can be a mechanism mediating ginseng's beneficial actions on coronary vessels.

Platelet-derived growth factor receptor-α-positive cells and not smooth muscle cells mediate purinergic hyperpolarization in murine colonic muscles.

Enteric inhibitory neurotransmission is an important feature of the neural regulation of gastrointestinal motility. Purinergic neurotransmission, via P2Y1 receptors, mediates one phase of inhibitory neural control. For decades, ATP has been assumed to be the purinergic neurotransmitter and smooth muscle cells (SMCs) have been considered the primary targets for inhibitory neurotransmission. Recent experiments have cast doubt on both of these assumptions and suggested that another cell type, platelet-derived growth factor receptor-α-positive (PDGFRα(+)) cells, is the target for purinergic neurotransmission. We compared responses of PDGFRα(+) cells and SMCs to several purine compounds to determine if these cells responded in a manner consistent with enteric inhibitory neurotransmission. ATP hyperpolarized PDGFRα(+) cells but depolarized SMCs. Only part of the ATP response in PDGFRα(+) cells was blocked by MRS 2500, a P2Y1 antagonist. ADP, MRS 2365, ß-NAD, and adenosine 5-diphosphate-ribose, P2Y1 agonists, hyperpolarized PDGFRα(+) cells, and these responses were blocked by MRS 2500. Adenosine 5-diphosphate-ribose was more potent in eliciting hyperpolarization responses than ß-NAD. P2Y1 agonists failed to elicit responses in SMCs. Small hyperpolarization responses were elicited in SMCs by a small-conductance Ca(2+)-activated K(+) channel agonist, cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine, consistent with the low expression and current density of small-conductance Ca(2+)-activated K(+) channels in these cells. Large-amplitude hyperpolarization responses, elicited in PDGFRα(+) cells, but not SMCs, by P2Y1 agonists are consistent with the generation of inhibitory junction potentials in intact muscles in response to purinergic neurotransmission. The responses of PDGFRα(+) cells and SMCs to purines suggest that SMCs are unlikely targets for purinergic neurotransmission in colonic muscles.