Investigating the Impact of Electrostatic Interactions on Calmodulin Binding and Ca2+-Dependent Activation of the Calcium-Gated Potassium SK4 Channel.

Ca2+ binding to the ubiquitous Ca2+ sensing protein calmodulin (CaM) activates the intermediate conductance Ca2+-activated SK4 channel. Potential hydrophilic pockets for CaM binding have been identified at the intracellular HA and HB helices in the C-terminal of SK4 from the three published cryo-EM structures of SK4. Single charge reversal substitutions at either site, significantly weakened the pull-down of SK4 by CaM wild-type (CaM), and decreased the TRAM-34 sensitive outward K+ current densities in native HEK293T cells when compared with SK4 WT measured under the same conditions. Only the doubly substituted SK4 R352D/R355D (HB helix) obliterated the CaM-mediated pull-down and thwarted outward K+ currents. However, overexpression of CaM E84K/E87K, which had been predicted to face the arginine doublet, restored the CaM-mediated pull-down of SK4 R352D/R355D and normalized its whole-cell current density. Virtual analysis of the putative salt bridges supports a unique role for the positively charged arginine doublet at the HB helix into anchoring the interaction with the negatively charged CaM glutamate 84 and 87 CaM. Our findings underscore the unique contribution of electrostatic interactions in carrying CaM binding onto SK4 and support the role of the C-terminal HB helix to the Ca2+-dependent gating process.

Kca3.1-Related Cellular Signalling Involved in Cancer Proliferation.

Anomalous expression of potassium channels in cancer tissues is associated with several cancer hallmarks that support deregulated proliferation and tumor progression. Ion channels seem to influence cell proliferation; however, the crucial molecular mechanisms involved remain elusive. Some results show how extracellular mitogenic signals modulate ion channel activity through intracellular secondary messengers. It is relevant because we are beginning to understand how potassium channels can affect the proliferative capacity of cells, either in normal mitogen-dependent proliferation or in mitogen-unresponsive proliferation. Calciumdependent potassium channels have been implicated in cell cycle signaling in many cancerous cell lines. In particular, the so-called intermediate conductance KCa3.1 (IKCa) is reported to play a significant role in uncontrolled cell cycle signaling, among other malignant processes driven by cancer hallmarks. In addition to these features, this channel can be subjected to specific pharmacological regulation, making it a promising cornerstone for understanding the signaling behavior of several types of cancer and as a target for chemotherapeutic approaches. This review is dedicated to the connection of KCa3.1 activity, in canonical and non-canonical ways, to the cell cycle signaling, including the cooperation with calcium channels to generate calcium signals and its role as a mediator of proliferative signals.

The Gárdos Channel and Piezo1 Revisited: Comparison between Reticulocytes and Mature Red Blood Cells.

The Gárdos channel (KCNN4) and Piezo1 are the best-known ion channels in the red blood cell (RBC) membrane. Nevertheless, the quantitative electrophysiological behavior of RBCs and its heterogeneity are still not completely understood. Here, we use state-of-the-art biochemical methods to probe for the abundance of the channels in RBCs. Furthermore, we utilize automated patch clamp, based on planar chips, to compare the activity of the two channels in reticulocytes and mature RBCs. In addition to this characterization, we performed membrane potential measurements to demonstrate the effect of channel activity and interplay on the RBC properties. Both the Gárdos channel and Piezo1, albeit their average copy number of activatable channels per cell is in the single-digit range, can be detected through transcriptome analysis of reticulocytes. Proteomics analysis of reticulocytes and mature RBCs could only detect Piezo1 but not the Gárdos channel. Furthermore, they can be reliably measured in the whole-cell configuration of the patch clamp method. While for the Gárdos channel, the activity in terms of ion currents is higher in reticulocytes compared to mature RBCs, for Piezo1, the tendency is the opposite. While the interplay between Piezo1 and Gárdos channel cannot be followed using the patch clamp measurements, it could be proved based on membrane potential measurements in populations of intact RBCs. We discuss the Gárdos channel and Piezo1 abundance, interdependencies and interactions in the context of their proposed physiological and pathophysiological functions, which are the passing of small constrictions, e.g., in the spleen, and their active participation in blood clot formation and thrombosis.

Overexpression of KCNN4 channels in principal neurons produces an anti-seizure effect without reducing their coding ability.

Gene therapy offers a potential alternative to the surgical treatment of epilepsy, which affects millions of people and is pharmacoresistant in ~30% of cases. Aimed at reducing the excitability of principal neurons, the engineered expression of K+ channels has been proposed as a treatment due to the outstanding ability of K+ channels to hyperpolarize neurons. However, the effects of K+ channel overexpression on cell physiology remain to be investigated. Here we report an adeno-associated virus (AAV) vector designed to reduce epileptiform activity specifically in excitatory pyramidal neurons by expressing the human Ca2+-gated K+ channel KCNN4 (KCa3.1). Electrophysiological and pharmacological experiments in acute brain slices showed that KCNN4-transduced cells exhibited a Ca2+-dependent slow afterhyperpolarization that significantly decreased the ability of KCNN4-positive neurons to generate high-frequency spike trains without affecting their lower-frequency coding ability and action potential shapes. Antiepileptic activity tests showed potent suppression of pharmacologically induced seizures in vitro at both single cell and local field potential levels with decreased spiking during ictal discharges. Taken together, our findings strongly suggest that the AAV-based expression of the KCNN4 channel in excitatory neurons is a promising therapeutic intervention as gene therapy for epilepsy.

Endothelial KCa3.1 and KCa2.3 Mediate S1P (Sphingosine-1-Phosphate)-Dependent Vasodilation and Blood Pressure Homeostasis.

BACKGROUND: S1P (sphingosine-1-phosphate) has been reported to possess vasodilatory properties, but the underlying pathways are largely unknown. METHODS: Isolated mouse mesenteric artery and endothelial cell models were used to determine S1P-induced vasodilation, intracellular calcium, membrane potentials, and calcium-activated potassium channels (KCa2.3 and KCa3.1 [endothelial small- and intermediate-conductance calcium-activated potassium channels]). Effect of deletion of endothelial S1PR1 (type 1 S1P receptor) on vasodilation and blood pressure was evaluated. RESULTS: Mesenteric arteries subjected to acute S1P stimulation displayed a dose-dependent vasodilation response, which was attenuated by blocking endothelial KCa2.3 or KCa3.1 channels. In cultured human umbilical vein endothelial cells, S1P stimulated immediate membrane potential hyperpolarization following activation of KCa2.3/KCa3.1 with elevated cytosolic Ca2+. Further, chronic S1P stimulation enhanced expression of KCa2.3 and KCa3.1 in human umbilical vein endothelial cells in dose- and time-dependent manners, which was abolished by disrupting either S1PR1-Ca2+ signaling or downstream Ca2+-activated calcineurin/NFAT (nuclear factor of activated T-cells) signaling. By combination of bioinformatics-based binding site prediction and chromatin immunoprecipitation assay, we revealed in human umbilical vein endothelial cells that chronic activation of S1P/S1PR1 promoted NFATc2 nuclear translocation and binding to promoter regions of KCa2.3 and KCa3.1 genes thus to upregulate transcription of these channels. Deletion of endothelial S1PR1 reduced expression of KCa2.3 and KCa3.1 in mesenteric arteries and exacerbated hypertension in mice with angiotensin II infusion. CONCLUSIONS: This study provides evidence for the mechanistic role of KCa2.3/KCa3.1-activated endothelium-dependent hyperpolarization in vasodilation and blood pressure homeostasis in response to S1P. This mechanistic demonstration would facilitate the development of new therapies for cardiovascular diseases associated with hypertension.

Ca2+-Sensitive Potassium Channels.

The Ca2+ ion is used ubiquitously as an intracellular signaling molecule due to its high external and low internal concentration. Many Ca2+-sensing ion channel proteins have evolved to receive and propagate Ca2+ signals. Among them are the Ca2+-activated potassium channels, a large family of potassium channels activated by rises in cytosolic calcium in response to Ca2+ influx via Ca2+-permeable channels that open during the action potential or Ca2+ release from the endoplasmic reticulum. The Ca2+ sensitivity of these channels allows internal Ca2+ to regulate the electrical activity of the cell membrane. Activating these potassium channels controls many physiological processes, from the firing properties of neurons to the control of transmitter release. This review will discuss what is understood about the Ca2+ sensitivity of the two best-studied groups of Ca2+-sensitive potassium channels: large-conductance Ca2+-activated K+ channels, KCa1.1, and small/intermediate-conductance Ca2+-activated K+ channels, KCa2.x/KCa3.1.

Aggressive migration in acidic pH of a glioblastoma cancer stem cell line in vitro is independent of ASIC and KCa3.1 ion channels, but involves phosphoinositide 3-kinase.

The microenvironment of proliferative and aggressive tumours, such as the brain tumour glioblastoma multiforme (GBM), is often acidic, hypoxic, and nutrient deficient. Acid-sensing ion channels (ASICs) are proton-sensitive Na+ channels that have been proposed to play a role in pH sensing and in modulation of cancer cell migration. We previously reported that primary glioblastoma stem cells (GSCs), which grow as multicellular tumour spheroids, express functional ASIC1a and ASIC3, whereas ASIC2a is downregulated in GSCs. Using a 2.5D migration assay, here we report that acidic pH dramatically increased migration of GSCs of the pro-neural subtype. Pharmacological blockade as well as CRISPR-Cas9-mediated gene knock-out of ASIC1a or stable overexpression of ASIC2a, however, revealed that neither ASIC1a nor ASIC3, nor downregulation of ASIC2a, mediated the aggressive migration at acidic pH. Therefore, we tested the role of two other proteins previously implicated in cancer cell migration: the Ca2+-activated K+ channel KCa3.1 (KCNN4) and phosphoinositide 3-kinase (PI3K). While pharmacological blockade of KCa3.1 did also not affect migration, blockade of PI3K decreased migration at acidic pH to control levels. In summary, our study reveals a strongly enhanced migration of GSCs at acidic pH in vitro and identifies PI3K as an important mediator of this effect.

Channelopathy of small- and intermediate-conductance Ca2+-activated K+ channels.

Small- and intermediate-conductance Ca2+-activated K+ (KCa2.x/KCa3.1 also called SK/IK) channels are gated exclusively by intracellular Ca2+. The Ca2+ binding protein calmodulin confers sub-micromolar Ca2+ sensitivity to the channel-calmodulin complex. The calmodulin C-lobe is constitutively associated with the proximal C-terminus of the channel. Interactions between calmodulin N-lobe and the channel S4-S5 linker are Ca2+-dependent, which subsequently trigger conformational changes in the channel pore and open the gate. KCNN genes encode four subtypes, including KCNN1 for KCa2.1 (SK1), KCNN2 for KCa2.2 (SK2), KCNN3 for KCa2.3 (SK3), and KCNN4 for KCa3.1 (IK). The three KCa2.x channel subtypes are expressed in the central nervous system and the heart. The KCa3.1 subtype is expressed in the erythrocytes and the lymphocytes, among other peripheral tissues. The impact of dysfunctional KCa2.x/KCa3.1 channels on human health has not been well documented. Human loss-of-function KCa2.2 mutations have been linked with neurodevelopmental disorders. Human gain-of-function mutations that increase the apparent Ca2+ sensitivity of KCa2.3 and KCa3.1 channels have been associated with Zimmermann-Laband syndrome and hereditary xerocytosis, respectively. This review article discusses the physiological significance of KCa2.x/KCa3.1 channels, the pathophysiology of the diseases linked with KCa2.x/KCa3.1 mutations, the structure-function relationship of the mutant KCa2.x/KCa3.1 channels, and potential pharmacological therapeutics for the KCa2.x/KCa3.1 channelopathy.

Pharmacological targeting of the mitochondrial calcium-dependent potassium channel KCa3.1 triggers cell death and reduces tumor growth and metastasis in vivo.

Ion channels are non-conventional, druggable oncological targets. The intermediate-conductance calcium-dependent potassium channel (KCa3.1) is highly expressed in the plasma membrane and in the inner mitochondrial membrane (mitoKCa3.1) of various cancer cell lines. The role mitoKCa3.1 plays in cancer cells is still undefined. Here we report the synthesis and characterization of two mitochondria-targeted novel derivatives of a high-affinity KCa3.1 antagonist, TRAM-34, which retain the ability to block channel activity. The effects of these drugs were tested in melanoma, pancreatic ductal adenocarcinoma and breast cancer lines, as well as in vivo in two orthotopic models. We show that the mitochondria-targeted TRAM-34 derivatives induce release of mitochondrial reactive oxygen species, rapid depolarization of the mitochondrial membrane, fragmentation of the mitochondrial network. They trigger cancer cell death with an EC50 in the µM range, depending on channel expression. In contrast, inhibition of the plasma membrane KCa3.1 by membrane-impermeant Maurotoxin is without effect, indicating a specific role of mitoKCa3.1 in determining cell fate. At sub-lethal concentrations, pharmacological targeting of mitoKCa3.1 significantly reduced cancer cell migration by enhancing production of mitochondrial reactive oxygen species and nuclear factor-κB (NF-κB) activation, and by downregulating expression of Bcl-2 Nineteen kD-Interacting Protein (BNIP-3) and of Rho GTPase CDC-42. This signaling cascade finally leads to cytoskeletal reorganization and impaired migration. Overexpression of BNIP-3 or pharmacological modulation of NF-κB and CDC-42 prevented the migration-reducing effect of mitoTRAM-34. In orthotopic models of melanoma and pancreatic ductal adenocarcinoma, the tumors at sacrifice were 60% smaller in treated versus untreated animals. Metastasis of melanoma cells to lymph nodes was also drastically reduced. No signs of toxicity were observed. In summary, our results identify mitochondrial KCa3.1 as an unexpected player in cancer cell migration and show that its pharmacological targeting is efficient against both tumor growth and metastatic spread in vivo.

Peptide Lv augments intermediate-conductance calcium-dependent potassium channels (KCa3.1) in endothelial cells to promote angiogenesis.

Peptide Lv is a small endogenous secretory peptide that is expressed in various tissues and conserved across different species. Patients with diabetic retinopathy, an ocular disease with pathological angiogenesis, have upregulated peptide Lv in their retinas. The pro-angiogenic activity of peptide Lv is in part through promoting vascular endothelial cell (EC) proliferation, migration, and sprouting, but its molecular mechanism is not completely understood. This study aimed to decipher how peptide Lv promotes EC-dependent angiogenesis by using patch-clamp electrophysiological recordings, Western immunoblotting, quantitative PCR, and cell proliferation assays in cultured ECs. Endothelial cells treated with peptide Lv became significantly hyperpolarized, an essential step for EC activation. Treatment with peptide Lv augmented the expression and current densities of the intermediate-conductance calcium-dependent potassium (KCa3.1) channels that contribute to EC hyperpolarization but did not augment other potassium channels. Blocking KCa3.1 attenuated peptide Lv-elicited EC proliferation. These results indicate that peptide Lv-stimulated increases of functional KCa3.1 in ECs contributes to EC activation and EC-dependent angiogenesis.

IKCa channels control breast cancer metabolism including AMPK-driven autophagy.

Ca2+-activated K+ channels of intermediate conductance (IK) are frequently overexpressed in breast cancer (BC) cells, while IK channel depletion reduces BC cell proliferation and tumorigenesis. This raises the question, of whether and mechanistically how IK activity interferes with the metabolic activity and energy consumption rates, which are fundamental for rapidly growing cells. Using BC cells obtained from MMTV-PyMT tumor-bearing mice, we show that both, glycolysis and mitochondrial ATP-production are reduced in cells derived from IK-deficient breast tumors. Loss of IK altered the sub-/cellular K+- and Ca2+- homeostasis and mitochondrial membrane potential, ultimately resulting in reduced ATP-production and metabolic activity. Consequently, we find that BC cells lacking IK upregulate AMP-activated protein kinase activity to induce autophagy compensating the glycolytic and mitochondrial energy shortage. Our results emphasize that IK by modulating cellular Ca2+- and K+-dynamics contributes to the remodeling of metabolic pathways in cancer. Thus, targeting IK channel might disturb the metabolic activity of BC cells and reduce malignancy.

Functional expression of mitochondrial KCa3.1 channels in non-small cell lung cancer cells.

Lung cancer is one of the leading causes of cancer-related deaths worldwide. The Ca2+-activated K+ channel KCa3.1 contributes to the progression of non-small cell lung cancer (NSCLC). Recently, KCa3.1 channels were found in the inner membrane of mitochondria in different cancer cells. Mitochondria are the main sources for the generation of reactive oxygen species (ROS) that affect the progression of cancer cells. Here, we combined Western blotting, immunofluorescence, and fluorescent live-cell imaging to investigate the expression and function of KCa3.1 channels in the mitochondria of NSCLC cells. Western blotting revealed KCa3.1 expression in mitochondrial lysates from different NSCLC cells. Using immunofluorescence, we demonstrate a co-localization of KCa3.1 channels with mitochondria of NSCLC cells. Measurements of the mitochondrial membrane potential with TMRM reveal a hyperpolarization following the inhibition of KCa3.1 channels with the cell-permeable blocker senicapoc. This is not the case when cells are treated with the cell-impermeable peptidic toxin maurotoxin. The hyperpolarization of the mitochondrial membrane potential is accompanied by an increased generation of ROS in NSCLC cells. Collectively, our results provide firm evidence for the functional expression of KCa3.1 channels in the inner membrane of mitochondria of NSCLC cells.

Downregulation of IL-8 and IL-10 by the Activation of Ca2+-Activated K+ Channel KCa3.1 in THP-1-Derived M2 Macrophages.

THP-1-differentiated macrophages are useful for investigating the physiological significance of tumor-associated macrophages (TAMs). In the tumor microenvironment (TME), TAMs with the M2-like phenotype play a critical role in promoting cancer progression and metastasis by inhibiting the immune surveillance system. We examined the involvement of Ca2+-activated K+ channel KCa3.1 in TAMs in expressing pro-tumorigenic cytokines and angiogenic growth factors. In THP-1-derived M2 macrophages, the expression levels of IL-8 and IL-10 were significantly decreased by treatment with the selective KCa3.1 activator, SKA-121, without changes in those of VEGF and TGF-ß1. Furthermore, under in vitro experimental conditions that mimic extracellular K+ levels in the TME, IL-8 and IL-10 levels were both significantly elevated, and these increases were reversed by combined treatment with SKA-121. Among several signaling pathways potentially involved in the transcriptional regulation of IL-8 and IL-10, respective treatments with ERK and JNK inhibitors significantly repressed their transcriptions, and treatment with SKA-121 significantly reduced the phosphorylated ERK, JNK, c-Jun, and CREB levels. These results strongly suggest that the KCa3.1 activator may suppress IL-10-induced tumor immune surveillance escape and IL-8-induced tumorigenicity and metastasis by inhibiting their production from TAMs through ERK-CREB and JNK-c-Jun cascades.

Subtype-Selective Positive Modulation of KCa2.3 Channels Increases Cilia Length.

Small-conductance Ca2+-activated potassium (KCa2.x) channels are gated exclusively by intracellular Ca2+. The activation of KCa2.3 channels induces hyperpolarization, which augments Ca2+ signaling in endothelial cells. Cilia are specialized Ca2+ signaling compartments. Here, we identified compound 4 that potentiates human KCa2.3 channels selectively. The subtype selectivity of compound 4 for human KCa2.3 over rat KCa2.2a channels relies on an isoleucine residue in the HA/HB helices. Positive modulation of KCa2.3 channels by compound 4 increased flow-induced Ca2+ signaling and cilia length, while negative modulation by AP14145 reduced flow-induced Ca2+ signaling and cilia length. These findings were corroborated by the increased cilia length due to the expression of Ca2+-hypersensitive KCa2.3_G351D mutant channels and the reduced cilia length resulting from the expression of Ca2+-hyposensitive KCa2.3_I438N channels. Collectively, we were able to associate functions of KCa2.3 channels and cilia, two crucial components in the flow-induced Ca2+ signaling of endothelial cells, with potential implications in vasodilation and ciliopathic hypertension.

Allosteric inhibitors targeting the calmodulin-PIP2 interface of SK4 K+ channels for atrial fibrillation treatment.

The Ca2+-activated SK4 K+ channel is gated by Ca2+-calmodulin (CaM) and is expressed in immune cells, brain, and heart. A cryoelectron microscopy (cryo-EM) structure of the human SK4 K+ channel recently revealed four CaM molecules per channel tetramer, where the apo CaM C-lobe and the holo CaM N-lobe interact with the proximal carboxyl terminus and the linker S4-S5, respectively, to gate the channel. Here, we show that phosphatidylinositol 4-5 bisphosphate (PIP2) potently activates SK4 channels by docking to the boundary of the CaM-binding domain. An allosteric blocker, BA6b9, was designed to act to the CaM-PIP2-binding domain, a previously untargeted region of SK4 channels, at the interface of the proximal carboxyl terminus and the linker S4-S5. Site-directed mutagenesis, molecular docking, and patch-clamp electrophysiology indicate that BA6b9 inhibits SK4 channels by interacting with two specific residues, Arg191 and His192 in the linker S4-S5, not conserved in SK1-SK3 subunits, thereby conferring selectivity and preventing the Ca2+-CaM N-lobe from properly interacting with the channel linker region. Immunohistochemistry of the SK4 channel protein in rat hearts showed a widespread expression in the sarcolemma of atrial myocytes, with a sarcomeric striated Z-band pattern, and a weaker occurrence in the ventricle but a marked incidence at the intercalated discs. BA6b9 significantly prolonged atrial and atrioventricular effective refractory periods in rat isolated hearts and reduced atrial fibrillation induction ex vivo. Our work suggests that inhibition of SK4 K+ channels by targeting drugs to the CaM-PIP2-binding domain provides a promising anti-arrhythmic therapy.

Retroviral glycoprotein-mediated immune suppression via the potassium channel KCa3.1 - A new strategy for amelioration of inflammatory bowel diseases.

Peptides derived from retroviral envelope proteins have been shown to possess a wide range of immunosuppressive and anti-inflammatory activities. We have previously reported identification of such a peptide derived from the envelope protein coded by a human endogenous retrovirus (HERV). In this study, we identify that in vitro the peptide inhibits the KCa3.1 potassium channel, a potential target for therapy of immune diseases. We describe in vitro ENV59-GP3 effects with respect to potency of inhibition on KCa3.1 channels and calcium influx. Furthermore, we asses in vivo the effect of blocking KCa3.1 with ENV59-GP3 peptide or KCa3.1-blocker NS6180 on protection against DSS-induced acute colitis. ENV59-GP3 peptide treatment showed reduction of the disease score in the DSS-induced acute colitis mice model, which was comparable to effects of the KCa3.1 channel blocker NS6180. Analysis of cytokine production from DSS-mice model treated animals revealed equipotent inhibitory effects of the ENV59-GP3 and NS6180 compounds on the production of IL-6, TNF-α, IL-1ß. These findings altogether suggest that ENV59-GP3 functions as a KCa3.1 channel inhibitor and underline the implications of using virus derived channel blockers for treatment of autoimmune diseases. Additionally, they open the possibilities whether KCa3.1 inhibition is efficacious in patients with inflammatory bowel diseases.

KCNN4 Promotes the Stemness Potentials of Liver Cancer Stem Cells by Enhancing Glucose Metabolism.

The presence of liver cancer stem cells (LCSCs) is one of the reasons for the treatment failure of hepatocellular carcinoma (HCC). For LCSCs, one of their prominent features is metabolism plasticity, which depends on transporters and ion channels to exchange metabolites and ions. The K+ channel protein KCNN4 (Potassium Calcium-Activated Channel Subfamily N Member 4) has been reported to promote cell metabolism and malignant progression of HCCs, but its influence on LCSC stemness has remained unclear. Here, we demonstrated that KCNN4 was highly expressed in L-CSCs by RT-PCR and Western blot. Then, we illustrated that KCNN4 promoted the stemness of HC-C cells by CD133+CD44+ LCSC subpopulation ratio analysis, in vitro stemness transcription factor detection, and sphere formation assay, as well as in vivo orthotopic liver tumor formation and limiting dilution tumorigenesis assays. We also showed that KCNN4 enhanced the glucose metabolism in LCSCs by metabolic enzyme detections and seahorse analysis, and the KCNN4-promoted increase in LCSC ratios was abolished by glycolysis inhibitor 2-DG or OXPHOS inhibitor oligomycin. Collectively, our results suggested that KCNN4 promoted LCSC stemness via enhancing glucose metabolism, and that KCNN4 would be a potential molecular target for eliminating LCSCs in HCC.

Dextran sodium sulfate potentiates NLRP3 inflammasome activation by modulating the KCa3.1 potassium channel in a mouse model of colitis.

Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, has increased in incidence and prevalence in recent decades. Both clinical and animal studies are critical for understanding the pathogenesis of this disease. Dextran sodium sulfate (DSS)-induced colitis is a frequently used animal model of IBD, but the underlying mechanism of the model remains incompletely understood. In this study, we found that NOD-like receptor family pyrin containing 3 (NLRP3) depletion markedly mitigated DSS-induced colitis and was accompanied by decreased activation of the inflammasome in the colons of mice. However, in vitro assays showed that DSS did not directly trigger but instead potentiated NLRP3 inflammasome assembly in macrophages in response to suboptimal ATP or nigericin stimulation. Mechanistically, DSS potentiated NLRP3 inflammasome activation in macrophages by augmenting KCa3.1-mediated potassium ion (K+) efflux. Furthermore, we found that pharmacologic blockade of the K+ channel KCa3.1 with TRAM-34 or genetic depletion of the Kcnn4 gene (encoding KCa3.1) not only ameliorated the severity of DSS-induced colitis but also attenuated in vivo inflammasome assembly in the colonic tissues of mice, suggesting a causal link between KCa3.1-mediated augmentation of the NLRP3 inflammasome and DSS-induced inflammatory injuries. Collectively, these results indicate that KCa3.1 plays a critical role in mediating DSS-induced colitis in mice by potentiating NLRP3 inflammasome activation. Our data provide a previously unknown mechanism by which DSS induces colitis in mice and suggests that KCa3.1 is an alternative therapeutic target for treating IBD.

Corticotropin Releasing Factor Mediates KCa3.1 Inhibition, Hyperexcitability, and Seizures in Acquired Epilepsy.

Temporal lobe epilepsy (TLE), the most common focal seizure disorder in adults, can be instigated in experimental animals by convulsant-induced status epilepticus (SE). Principal hippocampal neurons from SE-experienced epileptic male rats (post-SE neurons) display markedly augmented spike output compared with neurons from nonepileptic animals (non-SE neurons). This enhanced firing results from a cAMP-dependent protein kinase A-mediated inhibition of KCa3.1, a subclass of Ca2+-gated K+ channels generating the slow afterhyperpolarizing Ca2+-gated K+ current (IsAHP). The inhibition of KCa3.1 in post-SE neurons leads to a marked reduction in amplitude of the IsAHP that evolves during repetitive firing, as well as in amplitude of the associated Ca2+-dependent component of the slow afterhyperpolarization potential (KCa-sAHP). Here we show that KCa3.1 inhibition in post-SE neurons is induced by corticotropin releasing factor (CRF) through its Type 1 receptor (CRF1R). Acute application of CRF1R antagonists restores KCa3.1 activity in post-SE neurons, normalizing KCa-sAHP/IsAHP amplitudes and neuronal spike output, without affecting these variables in non-SE neurons. Moreover, pharmacological antagonism of CRF1Rs in vivo reduces the frequency of spontaneous recurrent seizures in post-SE chronically epileptic rats. These findings may provide a new vista for treating TLE.SIGNIFICANCE STATEMENT Epilepsy, a common neurologic disorder, often develops following a brain insult. Identifying key cellular mechanisms underlying acquired epilepsy is critical for developing effective antiepileptic therapies. In an experimental model of acquired epilepsy, principal hippocampal neurons manifest hyperexcitability because of downregulation of KCa3.1, a subtype of Ca2+-gated K+ ion channels. We show that KCa3.1 downregulation is mediated by corticotropin releasing factor (CRF) acting through its Type 1 receptor (CRF1R). Congruently, acute application of selective CRF1R antagonists restores KCa3.1 channel activity, leading to normalization of neuronal excitability. In the same model, injection of a CRF1R antagonist to epileptic animals markedly decreases the frequency of electrographic seizures. Therefore, targeting CRF1Rs may provide a new strategy in the treatment of acquired epilepsy.

KCNN4-mediated Ca2+/MET/AKT axis is promising for targeted therapy of pancreatic ductal adenocarcinoma.

As a member of the potassium calcium-activated channel subfamily, increasing evidence suggests that KCNN4 was associated with malignancies. However, the roles and regulatory mechanisms of KCNN4 in PDAC have been little explored. In this work, we demonstrated that the level of KCNN4 in PDAC was abnormally elevated, and the overexpression of KCNN4 was induced by transcription factor AP-1. KCNN4 was closely correlated with unfavorable clinicopathologic characteristics and poor survival. Functionally, we found that overexpression of KCNN4 promoted PDAC cell proliferation, migration and invasion. Conversely, the knockdown of KCNN4 attenuated the growth and motility of PDAC cells. In addition to these, knockdown of KCNN4 promoted PDAC cell apoptosis and led to cell cycle arrest in the S phase. In mechanistic investigations, RNA-sequence revealed that the MET-mediated AKT axis was essential for KCNN4, encouraging PDAC cell proliferation and migration. Collectively, these findings reveal a function of KCNN4 in PDAC and suggest it's an attractive therapeutic target and tumor marker. Our studies underscore a better understanding of the biological mechanism of KCNN4 in PDAC and suggest novel strategies for cancer therapy.