hiPSC-derived cardiomyocytes as a model to study the role of small-conductance Ca2+-activated K+ (SK) ion channel variants associated with atrial fibrillation.

Atrial fibrillation (AF), the most common arrhythmia, has been associated with different electrophysiological, molecular, and structural alterations in atrial cardiomyocytes. Therefore, more studies are required to elucidate the genetic and molecular basis of AF. Various genome-wide association studies (GWAS) have strongly associated different single nucleotide polymorphisms (SNPs) with AF. One of these GWAS identified the rs13376333 risk SNP as the most significant one from the 1q21 chromosomal region. The rs13376333 risk SNP is intronic to the KCNN3 gene that encodes for small conductance calcium-activated potassium channels type 3 (SK3). However, the functional electrophysiological effects of this variant are not known. SK channels represent a unique family of K+ channels, primarily regulated by cytosolic Ca2+ concentration, and different studies support their critical role in the regulation of atrial excitability and consequently in the development of arrhythmias like AF. Since different studies have shown that both upregulation and downregulation of SK3 channels can lead to arrhythmias by different mechanisms, an important goal is to elucidate whether the rs13376333 risk SNP is a gain-of-function (GoF) or a loss-of-function (LoF) variant. A better understanding of the functional consequences associated with these SNPs could influence clinical practice guidelines by improving genotype-based risk stratification and personalized treatment. Although research using native human atrial cardiomyocytes and animal models has provided useful insights, each model has its limitations. Therefore, there is a critical need to develop a human-derived model that represents human physiology more accurately than existing animal models. In this context, research with human induced pluripotent stem cells (hiPSC) and subsequent generation of cardiomyocytes derived from hiPSC (hiPSC-CMs) has revealed the underlying causes of various cardiovascular diseases and identified treatment opportunities that were not possible using in vitro or in vivo studies with animal models. Thus, the ability to generate atrial cardiomyocytes and atrial tissue derived from hiPSCs from human/patients with specific genetic diseases, incorporating novel genetic editing tools to generate isogenic controls and organelle-specific reporters, and 3D bioprinting of atrial tissue could be essential to study AF pathophysiological mechanisms. In this review, we will first give an overview of SK-channel function, its role in atrial fibrillation and outline pathophysiological mechanisms of KCNN3 risk SNPs. We will then highlight the advantages of using the hiPSC-CM model to investigate SNPs associated with AF, while addressing limitations and best practices for rigorous hiPSC studies.

Ca2+ -activated K+ channels regulate cell volume in human glioblastoma cells.

Glioblastoma (GBM), the most lethal form of brain tumors, bases its malignancy on the strong ability of its cells to migrate and invade the narrow spaces of healthy brain parenchyma. Cell migration and invasion are both critically dependent on changes in cell volume and shape driven by the transmembrane transport of osmotically important ions such as K+ and Cl- . However, while the Cl- channels participating in cell volume regulation have been clearly identified, the precise nature of the K+ channels involved is still uncertain. Using a combination of electrophysiological and imaging approaches in GBM U87-MG cells, we found that hypotonic-induced cell swelling triggered the opening of Ca2+ -activated K+ (KCa ) channels of large and intermediate conductance (BKCa and IKCa , respectively), both highly expressed in GBM cells. The influx of Ca2+ mediated by the hypotonic-induced activation of mechanosensitive channels was found to be a key step for opening both the BKCa and the IKCa channels. Finally, the activation of both KCa channels mediated by mechanosensitive channels was found to be essential for the development of the regulatory volume decrease following hypotonic shock. Taken together, these data indicate that KCa channels are the main K+ channels responsible for the volume regulation in U87-MG cells.

Small-conductance calcium-activated potassium channels in the heart: expression, regulation and pathological implications.

Ca2+-activated K+ channels are critical to cellular Ca2+ homeostasis and excitability; they couple intracellular Ca2+ and membrane voltage change. Of these, the small, 4-14 pS, conductance SK channels include three, KCNN1-3 encoded, SK1/KCa2.1, SK2/KCa2.2 and SK3/KCa2.3, channel subtypes with characteristic, EC50 ∼ 10 nM, 40 pM, 1 nM, apamin sensitivities. All SK channels, particularly SK2 channels, are expressed in atrial, ventricular and conducting system cardiomyocytes. Pharmacological and genetic modification results have suggested that SK channel block or knockout prolonged action potential durations (APDs) and effective refractory periods (ERPs) particularly in atrial, but also in ventricular, and sinoatrial, atrioventricular node and Purkinje myocytes, correspondingly affect arrhythmic tendency. Additionally, mitochondrial SK channels may decrease mitochondrial Ca2+ overload and reactive oxygen species generation. SK channels show low voltage but marked Ca2+ dependences (EC50 ∼ 300-500 nM) reflecting their α-subunit calmodulin (CaM) binding domains, through which they may be activated by voltage-gated or ryanodine-receptor Ca2+ channel activity. SK function also depends upon complex trafficking and expression processes and associations with other ion channels or subunits from different SK subtypes. Atrial and ventricular clinical arrhythmogenesis may follow both increased or decreased SK expression through decreased or increased APD correspondingly accelerating and stabilizing re-entrant rotors or increasing incidences of triggered activity. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Design, synthesis and biological evaluation of pyrrolopyrimidine derivatives as novel and selective positive modulator of the small conductance Ca2+-activated K+ channels.

The type 2 small conductance Ca2+-activated K+ channels (SK2) have been considered as one of the most promising therapeutic targets for spinocerebellar ataxias type 2 (SCA2) by playing a critical role in the control of normal purkinje cells (PCs) pacemaking. Herein, a novel series of pyrrolopyrimidine derivatives were designed and synthesized from the lead compound NS13001 as subtype-selective modulators of SK channels. Among them, the halogen-substituted compound 12b (EC50 = 0.34 ± 0.044 µM) was identified with a ∼5.4-fold higher potency on potentiating SK2-a channels at submicromolar concentrations as compared to NS13001 (EC50 = 1.83 ± 0.50 µM). Furthermore, compound 12b exhibited selectivity on SK2-a/SK3 subtype by displaying 93.33 ± 3.26% efficacies on SK2-a channels, and 84.54% ± 7.49% on SK3 channels. In addition, compound 12b demonstrated the potential to cross the blood-brain barrier (BBB) with suitable pharmacokinetic properties and low cytotoxicity. Molecular docking study also unveiled the binding interactions of compound 12b with SK2-CaM protein complex. Overall, the novel pyrrolopyrimidines provide an insightful guidance for future structural optimization of SK channel agonists.

Role of organellar Ca2+-activated K+ channels in disease development.

The organellar Ca2+-activated K+ channels share a similar ability to transfer the alteration of Ca2+ concentration to membrane conductance of potassium. Multiple effects of Ca2+-activated K+ channels on cell metabolism and complex signaling pathways during organ development have been explored. The organellar Ca2+-activated K+ channels are able to control the ionic equilibrium and are always associated with oxidative stress in different organelles and the whole cells. Some drugs targeting Ca2+-activated K+ channels have been tested for various diseases in clinical trials. In this review, the known roles of organellar Ca2+-activated K+ channels were described, and their effects on different diseases, particularly on diabetes, cardiovascular diseases, and neurological diseases were discussed. It was attempted to summarize the currently known operational modes with the involvement of organellar Ca2+-activated K+ channels. This review may assist scholars to more comprehensively understand organellar Ca2+-activated K+ channels and related diseases.

Complementary mechanisms of modulation of spontaneous phasic contractions by the gaseous signalling molecules NO, H2S, HNO and the polysulfide Na2S3 in the rat colon.

OBJECTIVES: Reactive oxygen and nitrogen species may be produced during inflammation leading to the formation of NO, H2S or HNO. Enzymes such as iNOS, CSE and CBS might also be responsible for polysulfide production. Since these signalling molecules might have an impact on colonic motility, the aim of this study was to compare their effect on rat colonic slow phasic contractions (SPC). METHODS: Organ bath measurements with strips obtained from rat proximal colon were performed using the polysulfide Na2S3, sodium nitroprusside (NaNP), sodium hydrogen sulfide (NaHS), Angeli's salt as NO, H2S, and HNO donors, respectively. TTX (1 µM) was used to block neuronal activity. RESULTS: All four molecules, concentration-dependently, inhibited the amplitude and frequency of SPC both in the circular and longitudinal muscle layer. The relative potency was NaNP>Angeli's salt>NaHS>Na2S3. The inhibitory response induced by NaNP (1 µM) and Angeli's salt (50 µM) was reversed by ODQ (10 µM) whereas the inhibitory effect of NaHS (1 mM) was reversed by apamin (1 µM) and glibenclamide (10 µM). Na2S3 (1 mM) response was partially reversed by apamin (1 µM) and glibenclamide (10 µM). High concentrations of Na2S3 caused an increase in tone. Low concentrations of NaHS or Na2S3 did not potentiate NaNP responses. CONCLUSIONS: All signalling molecules inhibit SPC in both muscle layers. The effect is independent of neural activity and involves guanylyl cyclase (NO and HNO) and SKCa and KATP channels (NaHS or Na2S3). Other pathways might also be involved in Na2S3 responses. Accordingly, complementary mechanisms of inhibition might be attributable to these signalling molecules.

Comparison of relaxant effects of nifedipine and NS11021 on isolated umbilical arteries of healthy and preeclamptic pregnant women.

OBJECTIVES: Potassium (K+) channel openers and calcium (Ca2+) channel blockers are currently used to treat acute severe hypertension in pregnancy. We aimed to investigate the vasorelaxant effect of NS11021, a potent and specific big-conductance Ca2+-activated K+ (BKCa) channel activator, and to compare it with the vasorelaxant effect of nifedipine on human umbilical arteries (HUAs) isolated from healthy and preeclamptic pregnants. STUDY DESIGN: A total of 29 HUAs were isolated immediately after delivery from 14 healthy and 15 preeclamptic pregnant with severe features. The concentration-dependent relaxation responses were obtained to nifedipine and NS11021 on HUAs precontracted with endothelin-1 (ET-1) (10-8 M) in an isolated tissue bath. RESULTS: Both nifedipine and NS11021 caused concentration-dependent relaxation responses in HUAs from healthy and preeclamptic pregnants. While the maximum responses (Emax) and pD2 values of nifedipine did not change significantly in both groups, the Emax and pD2 values of NS11021 were significantly decreased in the preeclampsia group (Emax ± SEM; %75.57 ± 4.53 and %43.75 ± 14.00 and pD2 ± SEM; 6.92 ± 0.26 and 5.24 ± 0.53 respectively, p < 0.05). In addition, the pD2 value of NS11021 was not significantly different from that of nifedipine in the control group, but decreased significantly in the preeclampsia group (pD2 ± SEM 7.1 ± 0.41 and 5.2 ± 0.53, p < 0.05, respectively). CONCLUSIONS: Efficacy and potency of NS11021 decreased in HUAs from preeclamptic pregnants. Also, NS11021 is less potent than nifedipine in the preeclampsia group. BKCa channels may have a role in the pathogenesis of preeclampsia, however, further experimental studies are needed to elucidate that.

Regulation of Papillary Muscle Contractility by NAD and Ammonia Interplay: Contribution of Ion Channels and Exchangers.

Various models, including stem cells derived and isolated cardiomyocytes with overexpressed channels, are utilized to analyze the functional interplay of diverse ion currents involved in cardiac automaticity and excitation-contraction coupling control. Here, we used ß-NAD and ammonia, known hyperpolarizing and depolarizing agents, respectively, and applied inhibitory analysis to reveal the interplay of several ion channels implicated in rat papillary muscle contractility control. We demonstrated that: 4 mM ß-NAD, having no strong impact on resting membrane potential (RMP) and action potential duration (APD90) of ventricular cardiomyocytes, evoked significant suppression of isometric force (F) of paced papillary muscle. Reactive blue 2 restored F to control values, suggesting the involvement of P2Y-receptor-dependent signaling in ß-NAD effects. Meantime, 5 mM NH4Cl did not show any effect on F of papillary muscle but resulted in significant RMP depolarization, APD90 shortening, and a rightward shift of I-V relationship for total steady state currents in cardiomyocytes. Paradoxically, NH4Cl, being added after ß-NAD and having no effect on RMP, APD, and I-V curve, recovered F to the control values, indicating ß-NAD/ammonia antagonism. Blocking of HCN, Kir2.x, and L-type calcium channels, Ca2+-activated K+ channels (SK, IK, and BK), or NCX exchanger reverse mode prevented this effect, indicating consistent cooperation of all currents mediated by these channels and NCX. We suggest that the activation of Kir2.x and HCN channels by extracellular K+, that creates positive and negative feedback, and known ammonia and K+ resemblance, may provide conditions required for the activation of all the chain of channels involved in the interplay. Here, we present a mechanistic model describing an interplay of channels and second messengers, which may explain discovered antagonism of ß-NAD and ammonia on rat papillary muscle contractile activity.

Placental protein 13 dilation of pregnant rat uterine vein is endothelium dependent and involves nitric oxide/calcium activated potassium channels signals.

INTRODUCTION: Accumulating evidence demonstrates the importance of the galectin protein Placental Protein 13 (PP13) in predicting Preeclampsia (PE), a gestational disorder that has no cure and is associated with a compromised uterine vascular adaptation to pregnancy. Uterine vasculature undergoes significant remodeling (growth in length and in circumference) during normal pregnancy to accommodate the increased blood volume to the feto-placental unit. The aim of this study was to demonstrate the role of PP13 on the uterine veins (UVs). METHODS: PP13 was tested on UVs isolated from rat by using a pressurized myograph. The PP13 investigation was carried out in the presence of: a) nitric oxide synthases inhibitors (l-NAME + L-NNA, 2 x 10-4 M); b) small conductance Ca2+-activated K+ channels (SKca) inhibitor (Apamin, 10-7 M); c) intermediate conductance Ca2+-activated K+ channels (IKca) inhibitor (TRAM-34, 10-5 M); d) big conductance Ca2+-activated K+ channels (BKca) inhibitor (Paxilline, 10-5 M) and in the absence of endothelium. RESULTS: Our results showed that in late pregnancy, PP13 induced a significant dilation of UVs that is endothelium dependent. Further, PP13-dilation is mediated by the SKca - NO - BKca pathway. DISCUSSION: For the first time, this study provides evidence that in pregnancy, the UVs are dilated by PP13 and suggests SKCa as a potential target for treatments aimed at restoring pregnancy complication associated with deficiency in uterine adaptation.

Inhibition of platelet aggregation by activation of platelet intermediate conductance Ca2+ -activated potassium channels.

BACKGROUND: Within the vasculature platelets and endothelial cells play crucial roles in hemostasis and thrombosis. Platelets, like endothelial cells, possess intermediate conductance Ca2+ -activated K+ (IKCa ) channels and generate nitric oxide (NO). Although NO limits platelet aggregation, the role of IKCa channels in platelet function and NO generation has not yet been explored. OBJECTIVES: We investigated whether IKCa channel activation inhibits platelet aggregation, and per endothelial cells, enhances platelet NO production. METHODS: Platelets were isolated from human volunteers. Aggregometry, confocal microscopy, and a novel flow chamber model, the Quartz Crystal Microbalance (QCM) were used to assess platelet function. Flow cytometry was used to measure platelet NO production, calcium signaling, membrane potential, integrin αIIb /ß3 activation, granule release, and procoagulant platelet formation. RESULTS: Platelet IKCa channel activation with SKA-31 inhibited aggregation in a concentration-dependent manner, an effect reversed by the selective IKCa channel blocker TRAM-34. The QCM model along with confocal microscopy demonstrated that SKA-31 inhibited platelet aggregation under flow conditions. Surprisingly, IKCa activation by SKA-31 inhibited platelet NO generation, but this could be explained by a concomitant reduction in platelet calcium signaling. IKCa activation by SKA-31 also inhibited dense and alpha-granule secretion and integrin αIIb /ß3 activation, but maintained platelet phosphatidylserine surface exposure as a measure of procoagulant response. CONCLUSIONS: Platelet IKCa channel activation inhibits aggregation by reducing calcium-signaling and granule secretion, but not by enhancing platelet NO generation. IKCa channels may be novel targets for the development of antiplatelet drugs that limit atherothrombosis, but not coagulation.

The Inhibition of the Small-Conductance Ca2+-Activated Potassium Channels Decreases the Sinus Node Pacemaking during Beta-Adrenergic Activation.

Sinus pacemaking is based on tight cooperation of intracellular Ca2+ handling and surface membrane ion channels. An important player of this synergistic crosstalk could be the small-conductance Ca2+-activated K+-channel (ISK) that could contribute to the sinoatrial node (SAN) pacemaking driven by the intracellular Ca2+ changes under normal conditions and beta-adrenergic activation, however, the exact role is not fully clarified. SK2 channel expression was verified by immunoblot technique in rabbit SAN cells. Ionic currents and action potentials were measured by patch-clamp technique. The ECG R-R intervals were obtained by Langendorff-perfusion method on a rabbit heart. Apamin, a selective inhibitor of SK channels, was used during the experiments. Patch-clamp experiments revealed an apamin-sensitive current. When 100 nM apamin was applied, we found no change in the action potential nor in the ECG R-R interval. In experiments where isoproterenol was employed, apamin increased the cycle length of the SAN action potentials and enhanced the ECG R-R interval. Apamin did not amplify the cycle length variability or ECG R-R interval variability. Our data indicate that ISK has no role under normal condition, however, it moderately contributes to the SAN automaticity under beta-adrenergic activation.

Acute Cerebellar Inflammation and Related Ataxia: Mechanisms and Pathophysiology.

The cerebellum governs motor coordination and motor learning. Infection with external microorganisms, such as viruses, bacteria, and fungi, induces the release and production of inflammatory mediators, which drive acute cerebellar inflammation. The clinical observation of acute cerebellitis is associated with the emergence of cerebellar ataxia. In our animal model of the acute inflammation of the cerebellar cortex, animals did not show any ataxia but hyperexcitability in the cerebellar cortex and depression-like behaviors. In contrast, animal models with neurodegeneration of the cerebellar Purkinje cells and hypoexcitability of the neurons show cerebellar ataxia. The suppression of the Ca2+-activated K+ channels in vivo is associated with a type of ataxia. Therefore, there is a gap in our interpretation between the very early phase of cerebellar inflammation and the emergence of cerebellar ataxia. In this review, we discuss the hypothesized scenario concerning the emergence of cerebellar ataxia. First, compared with genetically induced cerebellar ataxias, we introduce infection and inflammation in the cerebellum via aberrant immunity and glial responses. Especially, we focus on infections with cytomegalovirus, influenza virus, dengue virus, and SARS-CoV-2, potential relevance to mitochondrial DNA, and autoimmunity in infection. Second, we review neurophysiological modulation (intrinsic excitability, excitatory, and inhibitory synaptic transmission) by inflammatory mediators and aberrant immunity. Next, we discuss the cerebellar circuit dysfunction (presumably, via maintaining the homeostatic property). Lastly, we propose the mechanism of the cerebellar ataxia and possible treatments for the ataxia in the cerebellar inflammation.

Effects of tetraethylammonium-sensitive K+ channel blockade on cholinergic and thermal sweating in endurance-trained and untrained men.

NEW FINDINGS: What is the central question of this study? Does inhibition of K+ channels modulate the exercise-training-induced augmentation in cholinergic and thermal sweating? What is the main finding and its importance? Iontophoretic administration of tetraethylammonium, a K+ channel blocker, blunted sweating induced by a low dose (0.001%) of the cholinergic agent pilocarpine, but not heat-induced sweating. However, no differences in the cholinergic sweating were observed between young endurance-trained and untrained men. Thus, while K+ channels play a role in the regulation of eccrine sweating, they do not contribute to the increase in sweating commonly observed in endurance-trained adults. Our findings provide important new insights into the mechanisms underlying the regulation of sweating by endurance conditioning. ABSTRACT: We evaluated the hypothesis that the activation of K+ channels mediates the exercise-training-induced augmentation of cholinergic and thermal sweating. On separate days, 11 endurance-trained and 10 untrained men participated in two experimental protocols. Prior to each protocol, we administered 2% tetraethylammonium (TEA, K+ channels blocker) and saline (Control) at forearm skin sites on both arms via transdermal iontophoresis. In protocol 1, low (0.001%) and high (1%) doses of pilocarpine were administered at the TEA-treated and Control sites over a 60-min period. In protocol 2, participants were passively heated by immersing their lower limbs in hot water (43°C) until core (rectal) temperature (Tc ) increased by 0.8°C above resting levels. Administration of TEA attenuated cholinergic sweating (P = 0.001) during the initial 20 min after the treatment of low dose of pilocarpine only whilst the response was similar between the groups (P = 0.163). Cholinergic and thermal sweating were higher in the trained relative to the untrained men (all P ≤ 0.033). Thermal sweating reached ∼90% of the response at a Tc elevation of 0.8°C during the initial 20 min of passive heating, which corresponds to the period wherein TEA attenuated cholinergic sweating in protocol 1. However, sweating did not differ between the Control and TEA sites in either group (P = 0.704). We showed that activation of K+ channels does not appear to mediate the elevated sweating response induced by a low dose of pilocarpine in trained men. We also demonstrated that K+ channels do not contribute to sweating during heat stress in either group.

Defining a role of NADPH oxidase in myogenic tone development.

OBJECTIVE: The myogenic response sets the foundation for blood flow control. Recent findings suggest a role for G protein-coupled receptors (GPCR) and signaling pathways tied to the generation of reactive oxygen species (ROS). In this regard, this study ascertained the impact of NADPH oxidase (Nox) on myogenic tone in rat cerebral resistance arteries. METHODS: The study employed real-time qPCR (RT-qPCR), pressure myography, and immunohistochemistry. RESULTS: Gq blockade abolished myogenic tone in rat cerebral arteries, linking GPCR to mechanosensation. Subsequent work revealed that general (TEMPOL) and mitochondrial specific (MitoTEMPO) ROS scavengers had little impact on myogenic tone, whereas apocynin, a broad spectrum Nox inhibitor, initiated transient dilation. RT-qPCR revealed Nox1 and Nox2 mRNA expression in smooth muscle cells. Pressure myography defined Nox1 rather than Nox2 is facilitating myogenic tone. We rationalized that Nox1-generated ROS was initiating this response by impairing the ability of the CaV 3.2 channel to elicit negative feedback via BKCa . This hypothesis was confirmed in functional experiments. The proximity ligation assay further revealed that Nox1 and CaV 3.2 colocalize within 40 nm of one another. CONCLUSIONS: Our data highlight that vascular pressurization augments Nox1 activity and ensuing ROS production facilitates myogenic tone by limiting Ca2+ influx via CaV 3.2.

cGMP and mitochondrial K+ channels-Compartmentalized but closely connected in cardioprotection.

The 3',5'-cGMP pathway triggers cytoprotective responses and improves cardiomyocyte survival during myocardial ischaemia and reperfusion (I/R) injury. These beneficial effects were attributed to NO-sensitive GC induced cGMP production leading to activation of cGMP-dependent protein kinase I (cGKI). cGKI in turn phosphorylates many substrates, which eventually facilitate opening of mitochondrial ATP-sensitive potassium channels (mitoKATP ) and Ca2+ -activated potassium channels of the BK type (mitoBK). Accordingly, agents activating mitoKATP or mitoBK provide protection against I/R-induced damages. Here, we provide an up-to-date summary of the infarct-limiting actions exhibited by the GC/cGMP axis and discuss how mitoKATP and mitoBK, which are present at the inner mitochondrial membrane, confer mito- and cytoprotective effects on cardiomyocytes exposed to I/R injury. In view of this, we believe that the functional connection between the cGMP cascade and mitoK+ channels should be exploited further as adjunct to reperfusion therapy in myocardial infarction. LINKED ARTICLES: This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.

Subunits of BK channels promote breast cancer development and modulate responses to endocrine treatment in preclinical models.

BACKGROUND AND PURPOSE: Pore-forming α subunits of the voltage- and Ca2+ -activated K+ channel with large conductance (BKα) promote malignant phenotypes of breast tumour cells. Auxiliary subunits such as the leucine-rich repeat containing 26 (LRRC26) protein, also termed BKγ1, may be required to permit activation of BK currents at a depolarized resting membrane potential that frequently occur in non-excitable tumour cells. EXPERIMENTAL APPROACH: Anti-tumour effects of BKα loss were investigated in breast tumour-bearing MMTV-PyMT transgenic BKα knockout (KO) mice, primary MMTV-PyMT cell cultures, and in a syngeneic transplantation model of breast cancer derived from these cells. The therapeutic relevance of BK channels in the context of endocrine treatment was assessed in human breast cancer cell lines expressing either low (MCF-7) or high (MDA-MB-453) levels of BKα and BKγ1, as well as in BKα-negative MDA-MB-157. KEY RESULTS: BKα promoted breast cancer onset and overall survival in preclinical models. Conversely, lack of BKα and/or knockdown of BKγ1 attenuated proliferation of murine and human breast cancer cells in vitro. At low concentrations, tamoxifen and its major active metabolites stimulated proliferation of BKα/γ1-positive breast cancer cells, independent of the genomic signalling controlled by the oestrogen receptor. Finally, tamoxifen increased the relative survival time of BKα KO but not of wild-type tumour cell recipient mice. CONCLUSION AND IMPLICATIONS: Breast cancer initiation, progression, and tamoxifen sensitivity depend on functional BK channels thereby providing a rationale for the future exploration of the oncogenic actions of BK channels in clinical outcomes with anti-oestrogen therapy. LINKED ARTICLES: This article is part of a themed issue on New avenues in cancer prevention and treatment (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.12/issuetoc.

Downregulation of large-conductance Ca2+-activated K+ channels in human umbilical arterial smooth muscle cells in gestational diabetes mellitus.

AIMS: We investigated the changes in large-conductance Ca2+-activated K+ (BKCa) channels from human umbilical arterial smooth muscle cells experiencing gestational diabetes mellitus (GDM). MAIN METHODS: Whole-cell patch-clamp technique, arterial tone measurement, RT-PCR, Quantitative real-time PCR, western blot were performed in human umbilical arterial smooth muscle cells. KEY FINDINGS: Whole-cell BKCa current density was decreased in the GDM group compared with the normal group. The vasorelaxant effects of the synthetic BKCa channel activator NS-1619 (10 µM) were impaired in the GDM group compared with the normal group. Reverse-transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, and western blot analyses suggested that the mRNA, total RNA, and protein expression levels of the BKCa channel were decreased in the GDM group relative to the normal group. In addition, the expression levels of protein kinase A and protein kinase G, which regulate BKCa channel activity, remained unchanged between the groups. Applying the BKCa channel inhibitor paxilline (10 µM) induced vasoconstriction and membrane depolarization of isolated umbilical arteries in the normal group but showed less of an effect on umbilical arteries in the GDM group. SIGNIFICANCE: Our results demonstrate for the first time impaired BKCa current and BKCa channel-induced vasorelaxation activities that were not caused by impaired BKCa channel-regulated protein kinases, but by decreased expression of the BKCa channels, in the umbilical arteries of GDM patients.

In vivo morphological alterations of TAMs during KCa3.1 inhibition-by using in vivo two-photon time-lapse technology.

Tumor associated macrophages (TAMs) are the mostprevalent cells recruited in the tumor microenvironment (TME). Once recruited, TAMs acquire a pro-tumor phenotype characterized by a typical morphology: ameboid in the tumor core and with larger soma and thick branches in the tumor periphery. Targeting TAMs by reverting them to an anti-tumor phenotype is a promising strategy for cancer immunotherapy. Taking advantage of Cx3cr1GFP/WT heterozygous mice implanted with murine glioma GL261-RFP cells we investigated the role of Ca2+-activated K+ channel (KCa3.1) on the phenotypic shift of TAMs at the late stage of glioma growth through in vivo two-photon imaging. We demonstrated that TAMs respond promptly to KCa3.1 inhibition using a selective inhibitor of the channel (TRAM-34) in a time-dependent manner by boosting ramified projections attributable to a less hypertrophic phenotype in the tumor core. We also revealed a selective effect of drug treatment by reducing both glioma cells and TAMs in the tumor core with no interference with surrounding cells. Taken together, our data indicate a TRAM-34-dependent progressive morphological transformation of TAMs toward a ramified and anti-tumor phenotype, suggesting that the timing of KCa3.1 inhibition is a key point to allow beneficial effects on TAMs.

Polarity of action in salivary gland acinar cells: Local and preferential Ca2+ signalling.

Salivary secretion is important for digestion and paramount for oral health. Both exocytotic secretion of proteins (including salivary amylase and mucins) and fluid secretion contribute to the formation of saliva. A recent study by T. Takano and colleagues [1] has revealed interesting patterns of Ca2+ responses with implications for important modifications to the established model of fluid secretion.

Activation of SK3 channel plays a pivotal role in modulation of trigeminal neuralgia.

Objective: To investigate whether small conductance Ca2+ activatedK+ channels; Trigeminal ganglion; Trigeminal neuralgia. (SK3) exists in normal rats' trigeminal ganglions (TG) and its effect on their pain thresholds.Methods: In total, 110 healthy adult male Sprague-Dawley (SD) rats were involved in this study. Ten rats were dissected to collect their liver tissues, TG and DRG. The rest of the rats were randomly assigned to either the experimental group or the control group. The animal model of trigeminal neuralgia (TN) was established by infraorbital nerve ligation. The expression of SK3 channels in their livers, TG and dorsal root ganglions (DRG) were detected. And different doses of SK3 channel activator and inhibitor were administered to the rats in both groups 15 days after the operation; meanwhile, their pain thresholds were also measured.Results: The expression of SK3 channel was found in TG. In the experimental group, the pain threshold was significantly decreased and there was a decreased level of SK3 than that in the control group at 15 days after operation. The administration of SK3 channel agonist (CyPPA) could significantly improve the pain threshold, while, the pain threshold decreased after administration of SK3 channel antagonist (Apamin).Conclusion: The SK3 channel may play a pivotal role in the pathogenesis of trigeminal neuralgia, and it may be one of the potential targets for the treatment of trigeminal neuralgia.