Small conductance calcium-activated potassium channel contributes to stress induced endothelial dysfunctions.

Patients with Takotsubo syndrome displayed endothelial dysfunction, but underlying mechanisms have not been fully clarified. This study aimed to explore molecular signalling responsible for catecholamine excess induced endothelial dysfunction. Human cardiac microvascular endothelial cells were challenged by epinephrine to mimic catecholamine excess. Patch clamp, FACS, ELISA, PCR, and immunostaining were employed for the study. Epinephrine (Epi) enhanced small conductance calcium-activated potassium channel current (ISK1-3) through activating α1 adrenoceptor. Phenylephrine enhanced edothelin-1 (ET-1) and reactive oxygen species (ROS) production, and the effects involved contribution of ISK1-3. H2O2 enhanced ISK1-3 and ET-1 production. Enhancing ISK1-3 caused a hyperpolarization, which increases ROS and ET-1 production. BAPTA partially reduced phenylephrine-induced enhancement of ET-1 and ROS, suggesting that α1 receptor activation can enhance ROS/ET-1 generation in both calcium-dependent and calcium-independent ways. The study demonstrates that high concentration catecholamine can activate SK1-3 channels through α1 receptor-ROS signalling and increase ET-1 production, facilitating vasoconstriction.

Inhibition of SK Channels in VTA Affects Dopaminergic Neurons to Improve the Depression-Like Behaviors of Post-Stroke Depression Rats.

Purpose: This study aimed to investigate the effect of small-conductance calcium-activated potassium channels (SK channels) on the dopaminergic (DA) neuron pathways in the ventral tegmental area (VTA) during the pathogenesis of post-stroke depression (PSD) and explore the improvement of PSD by inhibiting the SK channels. Patients and Methods: Four groups of Sprague-Dawley rats were randomly divided: Control, PSD, SK channel inhibitor (apamin) and SK channel activator (CyPPA) groups. In both control and CyPPA groups, sham surgery was performed. In the other two groups, middle cerebral arteries were occluded. The behavioral indicators related to depression in different groups were compared. Immunofluorescence was used to measure the activity of DA neurons in the VTA, while qRT-PCR was used to assess the expression of SK channel genes. Results: The results showed that apamin treatment improved behavioral indicators related to depression compared to the PSD group. Furthermore, the qRT-PCR analysis revealed differential expression of the KCNN1 and KCNN3 subgenes of the SK channels in each group. Immunofluorescence analysis revealed an increase in the expression of DA neurons in the VTA of the PSD group, which was subsequently reduced upon apamin intervention. Conclusion: This study suggests that SK channel activation following stroke contributes to depression-related behaviors in PSD rats through increased expression of DA neurons in the VTA. And depression-related behavior is improved in PSD rats by inhibiting the SK channels. The results of this study provide a new understanding of PSD pathogenesis and the possibility of developing new strategies to prevent PSD by targeting SK channels.

Insights into the mechanisms governing P01 scorpion toxin effect against U87 glioblastoma cells oncogenesis.

The emerging concept of small conductance Ca2+-activated potassium channels (SKCa) as pharmacological target for cancer treatment has significantly increased in recent years. In this study, we isolated the P01 toxin from Androctonus australis (Aa) scorpion venom and investigated its effect on biological properties of glioblastoma U87, breast MDA-MB231 and colon adenocarcinoma LS174 cancer cell lines. Our results showed that P01 was active only on U87 glioblastoma cells. It inhibited their proliferation, adhesion and migration with IC50 values in the micromolar range. We have also shown that P01 reduced the amplitude of the currents recorded in HEK293 cells expressing SK2 channels with an IC50 value of 3 pM, while it had no effect on those expressing SK3 channels. The investigation of the SKCa channels expression pattern showed that SK2 transcripts were expressed differently in the three cancer cell lines. Particularly, we highlighted the presence of SK2 isoforms in U87 cells, which could explain and rely on the specific activity of P01 on this cell line. These experimental data highlighted the usefulness of scorpion peptides to decipher the role of SKCa channels in the tumorigenesis process, and develop potential therapeutic molecules targeting glioblastoma with high selectivity.

A subfraction obtained from the venom of the tarantula Poecilotheria regalis contains inhibitor cystine knot peptides and induces relaxation of rat aorta by inhibiting L-type voltage-gated calcium channels.

Venoms from tarantulas contain low molecular weight vasodilatory compounds whose biological action is conceived as part of the envenomation strategy due to its propagative effects. However, some properties of venom-induced vasodilation do not match those described by such compounds, suggesting that other toxins may cooperate with these ones to produce the observed biological effect. Owing to the distribution and function of voltage-gated ion channels in blood vessels, disulfide-rich peptides isolated from venoms of tarantulas could be conceived into potential vasodilatory compounds. However, only two peptides isolated from spider venoms have been investigated so far. This study describes for the first time a subfraction containing inhibitor cystine knot peptides, PrFr-I, obtained from the venom of the tarantula Poecilotheria regalis. This subfraction induced sustained vasodilation in rat aortic rings independent of vascular endothelium and endothelial ion channels. Furthermore, PrFr-I decreased calcium-induced contraction of rat aortic segments and reduced extracellular calcium influx to chromaffin cells by the blockade of L-type voltage-gated calcium channels. This mechanism was unrelated to the activation of potassium channels from vascular smooth muscle, since vasodilation was not affected in the presence of TEA, and PrFr-I did not modify the conductance of the voltage-gated potassium channel Kv10.1. This work proposes a new envenomating function of peptides from venoms of tarantulas, and establishes a new mechanism for venom-induced vasodilation.

Dapagliflozin regulates myocardial SK2 protein expression in diabetic rats / 中国药理学通报

Aim To investigate the effect of dapagliflozin on the small conductance calcium-activated potassium channel 2 (SK2 channel) protein in the myocardium of diabetic rats and its possible mechanism of action. Methods In vivo: type 2 diabetes model was established by high-glucose and high-fat diet combined with intraperitoneal injection of low-dose streptozotocin (35 mg

Effect of protease activated receptor 2 on colonic motility in diabetes mice / 上海交通大学学报（医学版）

Objective • To observe the effect of protease activated receptor 2 (PAR2) on the colonic motility in diabetic mice and investigate the mechanism. Methods • The mouse model of type 1 diabetes mellitus was established by intraperitoneal injection of streptozotocin. The smooth muscle strips and segments of colons were isolated. The effects of PAR2 agonist on colonic motility were observed by muscle strip tension contraction and colonic migrating motor complex experiments. The effect of small conductance calcium-activated potassium channel (SK3 channel) antagonist on it was also observed. Results • PAR2 agonist inhibited colonic motility and colonic smooth muscle was more sensitive to PAR2 agonist in diabetic mice. PAR2 agonist-induced inhibition was inhibited by SK3 channel antagonist. Conclusion • PAR2 activity in diabetic mice colons is significantly enhanced, which may inhibit colonic motility through SK3 channel.

The Drosophila Small Conductance Calcium-Activated Potassium Channel Negatively Regulates Nociception.

Inhibition of nociceptor activity is important for the prevention of spontaneous pain and hyperalgesia. To identify the critical K+ channels that regulate nociceptor excitability, we performed a forward genetic screen using a Drosophila larval nociception paradigm. Knockdown of three K+ channel loci, the small conductance calcium-activated potassium channel (SK), seizure, and tiwaz, causes marked hypersensitive nociception behaviors. In more detailed studies of SK, we found that hypersensitive phenotypes can be recapitulated with a genetically null allele. Optical recordings from nociceptive neurons showed a significant increase in mechanically activated Ca2+ signals in SK mutant nociceptors. SK is expressed in peripheral neurons, including nociceptive neurons. Interestingly, SK proteins localize to axons of these neurons but are not detected in dendrites. Our findings suggest a major role for SK channels in the regulation of nociceptor excitation and are inconsistent with the hypothesis that the important site of action is within dendrites.

Metformin restores electrophysiology of small conductance calcium-activated potassium channels in the atrium of GK diabetic rats.

BACKGROUND: Small conductance calcium-activated potassium channels (SK channels) play a critical role in action potential repolarization in cardiomyocytes. Recently, the potential anti-arrhythmic effect of metformin in diabetic patients has been recognized, yet the underlying mechanism remains elusive. METHODS: Diabetic Goto-Kakizaki (GK) rats were untreated or treated with metformin (300 mg/kg/day) for 12 weeks, and age-matched Wistar rats were used as control (n = 6 per group). Electrocardiography, Hematoxylin-eosin staining and Masson's trichome staining were performed to assess cardiac function, histology and fibrosis. The expression levels of the SK channels in the myocardium were determined by real-time PCR and Western blotting. The electrophysiology of the SK channels in the cardiomyocytes isolated from the three groups of rats was examined by patch clamp assay, with specific blockade of the SK channels with apamin. RESULTS: Metformin treatment significantly reduced cardiac fibrosis and alleviated arrhythmia in the diabetic rats. In the atrial myocytes from control, GK and metformin-treated GK rats, the expression of KCa2.2 (SK2 channel) was down-regulated and the expression of KCa2.3 (SK3 channel) was up-regulated in the atrium of GK rats as compared with that of control rats, and metformin reversed diabetes-induced alterations in atrial SK channel expression. Moreover, patch clamp assay revealed that the SK current was markedly reduced and the action potential duration was prolonged in GK atrial myocytes, and the SK channel function was partially restored in the atrial myocytes from metformin-treated GK rats. CONCLUSIONS: Our data suggests an involvement of the SK channels in the development of arrhythmia under diabetic conditions, and supports a potential beneficial effect of metformin on atrial electrophysiology.

Mechanisms of Spontaneous Climbing Fiber Discharge-Evoked Pauses and Output Modulation of Cerebellar Purkinje Cell in Mice.

Climbing fiber (CF) afferents modulate the frequency and patterns of cerebellar Purkinje cell (PC) simple spike (SS) activity, but its mechanism is unclear. In the present study, we investigated the mechanisms of spontaneous CF discharge-evoked pauses and the output modulation of cerebellar PCs in urethane-anesthetized mice using in vivo whole-cell recording techniques and pharmacological methods. Under voltage-clamp recording conditions, spontaneous CF discharge evoked strong inward currents followed by small conductance calcium-activated potassium (SK) channels that mediated outward currents. The application of a GABAA receptor antagonist did not significantly alter the spontaneous SS firing rate, although an AMPA receptor blocker abolished complex spike (CS) activity and induced significantly increased SS firing rates and a decreased coefficient of variation (CV) SS value. Either removal of extracellular calcium or chelated intracellular calcium induced a decrease in amplitude of CS-evoked after-hyperpolarization (AHP) potential accompanied by an increase in SS firing rate. In addition, blocking SK channels activity with a selective antagonist, dequalinium decreased the amplitude of AHP and increased SS firing rate. Moreover, we found repeated CF stimulation at 1 Hz induced a significant decrease in the spontaneous firing rate of SS, and accompanied with an increase in CV of SS in cerebellar slices, which was also abolished by dequalinium. These results indicated that the spontaneous CF discharge contributed to decreasing SS firing rate via activation of SK channels in the cerebellar PCs in vivo in mice.

The role of extracellular calcium in bone metastasis.

This review summarizes the role of extracellular calcium, as found present in the bone tissue, in the process of bone metastasis.

Hydrogen peroxide modulates neuronal excitability and membrane properties in ventral horn neurons of the rat spinal cord.

Hydrogen peroxide (H2O2), a reactive oxygen species, is an important signaling molecule for synaptic and neuronal activity in the central nervous system; it is produced excessively in brain ischemia and spinal cord injury. Although H2O2-mediated modulations of synaptic transmission have been reported in ventral horn (VH) neurons of the rat spinal cord, the effects of H2O2 on neuronal excitability and membrane properties remain poorly understood. Accordingly, the present study investigated such effects using a whole-cell patch-clamp technique. The bath-application of H2O2 decreased neuronal excitability accompanied by decreased input resistance, firing frequency, and action potential amplitude and by increased rheobase. These H2O2-mediated changes were induced by activation of extrasynaptic, but not synaptic, GABAA receptors. Indeed, GABAergic tonic currents were enhanced by H2O2. On the other hand, the amplitude of medium and slow afterhyperpolarization (mAHP and sAHP), which plays important roles in controlling neuronal excitability and is mediated by small-conductance calcium-activated potassium (SK) channels, was significantly decreased by H2O2. When extrasynaptic GABAA receptors were completely blocked, these decreases of mAHP and sAHP persisted, and H2O2 increased excitability, suggesting that H2O2 per se might have the potential to increase neuronal excitability via decreased SK channel conductance. These findings indicate that activating extrasynaptic GABAA receptors or SK channels may attenuate acute neuronal damage caused by H2O2-induced hyperexcitability and therefore represent a novel therapeutic target for the prevention and treatment of H2O2-induced motor neuron disorders.

The small conductance calcium-activated potassium channel 3 (SK3) is a molecular target for Edelfosine to reduce the invasive potential of urothelial carcinoma cells.

Metastasis is the survival-determining factor in urothelial carcinoma (UC) of the urinary bladder. The small conductance calcium-activated potassium channel 3 (SK3) enhances tumor cell invasion in breast cancer and malignant melanoma. Since Edelfosine, a glycerophospholipid with antitumoral properties, effectively inhibits SK3 channel activity, our goal was to evaluate SK3 as a potential molecular target to inhibit the gain of an invasive phenotype in UC. SK3 protein expression was analyzed in 208 tissue samples and UC cell lines. Effects of Edelfosine on SK3 expression and intracellular calcium levels as well as on cell morphology, cell survival and proliferation were assessed using immunoblotting, potentiometric fluorescence microscopy, and clonogenic/cell survival assay; furthermore, we analyzed the effect of Edelfosine and SK3 RNAi knockdown on tumor cell migration and invasion in vitro and in vivo. We found that SK3 is strongly expressed in muscle-invasive UC and in the RT112 cellular tumor model. Higher concentrations of Edelfosine have a strong antitumoral effect on UC cells, while 1 µM effectively inhibits migration/invasion of UC cells in vitro and in vivo comparable to the SK3 knockdown phenotype. Taken together, our results show strong expression of SK3 in muscle-invasive UC, consistent with the postulated role of the protein in tumor cell invasion. Edelfosine is able to effectively inhibit migration and invasion of UC cells in vitro and in vivo in an SK3-dependent way, pointing towards a possible role for Edelfosine as an antiinvasive drug to effectively inhibit UC cell invasion and metastasis.

A de novo mutation in KCNN3 associated with autosomal dominant idiopathic non-cirrhotic portal hypertension.

Non-cirrhotic portal hypertension is characterized by histopathological abnormalities in the liver, mostly affecting small intrahepatic portal veins that cause portal hypertension in the absence of cirrhosis. It can be secondary to coagulation disorders or toxic agents. However, most cases are idiopathic non-cirrhotic portal hypertension (INCPH) and familial cases are rare. We report a family in which a father and three of his four children conceived with three different mothers are affected by INCPH. Whole exome and Sanger sequencing showed the father to have a de novo single nucleotide substitution c.1348G>C in the KCNN3 gene that was transmitted to all three of his affected offspring. The KCNN3 gene encodes small conductance calcium-activated potassium (SK) channel 3. SK channels are involved in the regulation of arterial and venous vascular tone by causing smooth muscle relaxation on activation. No data exist on the expression and function of SK channels in portal veins. The autosomal dominant inheritance in this unique pedigree and the single de novo mutation identified, strongly suggests that KCNN3 mutations have a pathogenetic role in INCPH.

Reduced activity of SKC a and Na-K ATPase underlies the accelerated impairment of EDH-type relaxations in mesenteric arteries of aging spontaneously hypertensive rats.

Aging is accompanied by endothelial dysfunction due to reduced bioavailability of nitric oxide (NO) and/or reduced endothelium-dependent hyperpolarizations (EDH). This study examines the hypothesis that hypertension aggravates the impairment of EDH-type relaxation due to aging. EDH-type relaxations were studied in superior mesenteric arteries isolated from Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats of 12, 36, 60, and 72 weeks of age. EDH-type relaxations in WKY were reduced with aging, and this was associated with an impairment of the function of small-conductance calcium-activated potassium channels (SKC a) and sodium-potassium ATPase (Na-K ATPase). EDH-type relaxation in SHR was smaller than that in WKY arteries, and further reduction occurred with aging. Pharmacological experiments suggested a reduced involvement of SKC a and Na-K ATPase and activation of adenosine monophosphate-activated protein kinase and silent information regulator T1 (sirtuin-1; SIRT1) in mesenteric arteries of 12-week-old SHR. These pharmacological findings suggest that in superior mesenteric arteries of the rat, the reduction in EDH-type relaxation occurs with aging and that such a reduction is exacerbated in hypertension. The latter exacerbation appears to involve proteins associated with the process of cellular senescence and is related to impaired function of SKC a and Na-K ATPase, a phenomenon that is also observed in mesenteric arteries of older normotensive rats.

Small-conductance Ca2+ -activated K+ channels and cardiac arrhythmias.

Small-conductance Ca2+ -activated K+ (SK, KCa2) channels are unique in that they are gated solely by changes in intracellular Ca2+ and, hence, function to integrate intracellular Ca2+ and membrane potentials on a beat-to-beat basis. Recent studies have provided evidence for the existence and functional significance of SK channels in the heart. Indeed, our knowledge of cardiac SK channels has been greatly expanded over the past decade. Interests in cardiac SK channels are further driven by recent studies suggesting the critical roles of SK channels in human atrial fibrillation, the SK channel as a possible novel therapeutic target in atrial arrhythmias, and upregulation of SK channels in heart failure in animal models and in human heart failure. However, there remain critical gaps in our knowledge. Specifically, blockade of SK channels in cardiac arrhythmias has been shown to be both antiarrhythmic and proarrhythmic. This contemporary review provides an overview of the literature on the role of cardiac SK channels in cardiac arrhythmias and serves as a discussion platform for the current clinical perspectives. At the translational level, development of SK channel blockers as a new therapeutic strategy in the treatment of atrial fibrillation and the possible proarrhythmic effects merit further considerations and investigations.

Small-conductance calcium-activated potassium (SK) channels contribute to action potential repolarization in human atria.

AIMS: Small-conductance calcium-activated potassium (SK) channels are expressed in the heart of various species, including humans. The aim of the present study was to address whether SK channels play a functional role in human atria. METHODS AND RESULTS: Quantitative real-time PCR analyses showed higher transcript levels of SK2 and SK3 than that of the SK1 subtype in human atrial tissue. SK2 and SK3 were reduced in chronic atrial fibrillation (AF) compared with sinus rhythm (SR) patients. Immunohistochemistry using confocal microscopy revealed widespread expression of SK2 in atrial myocytes. Two SK channel inhibitors (NS8593 and ICAGEN) were tested in heterologous expression systems revealing ICAGEN as being highly selective for SK channels, while NS8593 showed less selectivity for these channels. In isolated atrial myocytes from SR patients, both inhibitors decreased inwardly rectifying K(+) currents by ∼15% and prolonged action potential duration (APD), but no effect was observed in myocytes from AF patients. In trabeculae muscle strips from right atrial appendages of SR patients, both compounds increased APD and effective refractory period, and depolarized the resting membrane potential, while only NS8593 induced these effects in tissue from AF patients. SK channel inhibition did not alter any electrophysiological parameter in human interventricular septum tissue. CONCLUSIONS: SK channels are present in human atria where they participate in repolarization. SK2 and SK3 were down-regulated and had reduced functional importance in chronic AF. As SK current was not found to contribute substantially to the ventricular AP, pharmacological inhibition of SK channels may be a putative atrial-selective target for future antiarrhythmic drug therapy.

Sarcoplasmic reticulum Ca²âº release is both necessary and sufficient for SK channel activation in ventricular myocytes.

SK channels are upregulated in human patients and animal models of heart failure (HF). However, their activation mechanism and function in ventricular myocytes remain poorly understood. We aim to test the hypotheses that activation of SK channels in ventricular myocytes requires Ca(2+) release from sarcoplasmic reticulum (SR) and that SK currents contribute to reducing triggered activity. SK2 channels were overexpressed in adult rat ventricular myocytes using adenovirus gene transfer. Simultaneous patch clamp and confocal Ca(2+) imaging experiments in SK2-overexpressing cells demonstrated that depolarizations resulted in Ca(2+)-dependent outward currents sensitive to SK inhibitor apamin. SR Ca(2+) release induced by rapid application of 10 mM caffeine evoked repolarizing SK currents, whereas complete depletion of SR Ca(2+) content eliminated SK currents in response to depolarizations, despite intact Ca(2+) influx through L-type Ca(2+) channels. Furthermore, voltage-clamp experiments showed that SK channels can be activated by global spontaneous SR Ca(2+) release events Ca(2+) waves (SCWs). Current-clamp experiments revealed that SK overexpression reduces the amplitude of delayed afterdepolarizations (DADs) resulting from SCWs and shortens action potential duration. Immunolocalization studies showed that overexpressed SK channels are distributed both at external sarcolemmal membranes and along the Z-lines, resembling the distribution of endogenous SK channels. In summary, SR Ca(2+) release is both necessary and sufficient for the activation of SK channels in rat ventricular myocytes. SK currents contribute to repolarization during action potentials and attenuate DADs driven by SCWs. Thus SK upregulation in HF may have an anti-arrhythmic effect by reducing triggered activity.

Critical roles of a small conductance Ca²âº-activated K⁺ channel (SK3) in the repolarization process of atrial myocytes.

AIMS: Small conductance Ca(2+)-activated K(+) channels (K(Ca)2 or SK channels) have been reported in excitable cells, where they aid in integrating changes in intracellular Ca(2+) (Ca(i)²âº) 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. METHODS AND RESULTS: 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. CONCLUSION: SK3 channel contributes importantly towards atrial action potential repolarization. Our data suggest the important role of the SK3 isoform in atrial myocytes.

The characterization of the increase of membrane conductance after depolarization in single rat adrenal chromaffin cells

The conductance change evoked by step depolarization was studied in primarily cultured rat adrenal chromaffin cells using patch-clamp and capacitance measurement techniques. When we applied a depolarizing pulse to a chromaffin cell, the inward calcium current was followed by an outward current and depolarization-induced exocytosis was accompanied by an increase in conductance trace. The slow inward tail current which has the same time course as the conductance change was observed in current recording. The activation of slow tail current was calcium-dependent. Reversal potentials agreed with Nernst equation assuming relative permeability of Cs+ to K+ is 0.095. The outward current and tail current were blocked by apamin (200 nM) and d-tubocurarine (2 mM). The conductance change was blocked by apamin and did not affect membrane capacitance recording. We confirmed that conductance change after depolarization comes from the activation of the SK channel and can be blocked by application of the SK channel blockers. Consequently, it is necessary to consider blocking of the SK channel during membrane capacitance recording.