KCNN4 Channel Research Articles

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.

New KCNN4 Variants Associated With Anemia: Stomatocytosis Without Erythrocyte Dehydration.

The K+ channel activated by the Ca2+, KCNN4, has been shown to contribute to red blood cell dehydration in the rare hereditary hemolytic anemia, the dehydrated hereditary stomatocytosis. We report two de novo mutations on KCNN4, We reported two de novo mutations on KCNN4, V222L and H340N, characterized at the molecular, cellular and clinical levels. Whereas both mutations were shown to increase the calcium sensitivity of the K+ channel, leading to channel opening for lower calcium concentrations compared to WT KCNN4 channel, there was no obvious red blood cell dehydration in patients carrying one or the other mutation. The clinical phenotype was greatly different between carriers of the mutated gene ranging from severe anemia for one patient to a single episode of anemia for the other patient or no documented sign of anemia for the parents who also carried the mutation. These data compared to already published KCNN4 mutations question the role of KCNN4 gain-of-function mutations in hydration status and viability of red blood cells in bloodstream.

KCNN4 Expression Is Elevated in Inflammatory Bowel Disease: This Might Be a Novel Marker and Therapeutic Option Targeting Potassium Channels.

The K + channel KCNN4 is involved in many inflammatory diseases. Previous work has shown that this channel is involved in epithelial ion transport and intestinal restitution. In inflammatory bowel diseases (IBD) a defective epithelial barrier can lead to typical symptoms like secretory diarrhea and the formation of intestinal ulcers. We compared surgical samples from patients with IBD, diverticulitis and controls without inflammation to determine the potential role of KCNN4 as a diagnostic marker and/or therapeutic target. mRNA-levels of KCNN4 and a control K + channel were determined in intestinal epithelial cells (IEC) from patients with IBD, diverticulitis and controls. In addition, we performed a Western blot analysis of KCNN4 and a respective control K + channel in IEC from patients with IBD. Furthermore, we determined epithelial barrier integrity by measuring the flux of fluorescent-labeled dextran beads across a cell monolayer upon incubation with interferon-γ. KCNN4 mRNA and protein levels were elevated in IEC from patients with Crohn`s disease (CD) and ulcerative colitis (UC). Of note, KCNN4 was not elevated in non-IBD intestinal inflammatory conditions e.g. diverticulitis. Of clinical relevance, pharmacological KCNN4 channel openers stabilized epithelial barrier function in vitro. Thus, KCNN4 may have a protective role in IBD and constitute a therapeutic target. Our data demonstrate elevated KCNN4 both at mRNA and protein level in IEC specifically from patients with IBD. Therefore, we conclude that KCNN4 could be used as a novel marker for IBD, especially for the establishment of initial diagnosis. Of therapeutic consequence, we show that pharmacological KCNN4 openers stabilize the epithelial barrier. Thus, KCNN4 might be a novel target to diagnose and treat IBD.

Dexamethasone reduces airway epithelial Cl− secretion by rapid non-genomic inhibition of KCNQ1, KCNN4 and KATP K+ channels

Basolateral membrane K+ channels play a key role in basal and agonist stimulated Cl− transport across airway epithelial cells by generating a favourable electrical driving force for Cl− efflux. The K+ channel sub-types and molecular mechanisms of regulation by hormones and secretagoues are still poorly understood. Here we have identified the type of K+ channels involved in cAMP and Ca2+ stimulated Cl− secretion and uncovered a novel anti-secretory effect of dexamethasone mediated by inhibition of basolateral membrane K+ channels in a human airway cell model of 16HBE14o− cells commonly used for ion transport studies.Dexamethasone produced a rapid inhibition of transepithelial chloride ion secretion under steady state conditions and after stimulation with cAMP agonist (forskolin) or a Ca2+ mobilizing agonist (ATP). Our results show three different types of K+ channels are targeted by dexamethasone to reduce airway secretion, namely Ca2+-activated secretion via KCNN4 (KCa3.1) channels and cAMP-activated secretion via KCNQ1 (Kv7.1) and KATP (Kir6.1,6.2) channels. The down-regulation of KCNN4 and KCNQ1 channel activities by dexamethasone involves rapid non-genomic activation of PKCα and PKA signalling pathways, respectively. Dexamethasone signal transduction for PKC and PKA activation was demonstrated to occur through a rapid non-genomic pathway that did not implicate the classical nuclear receptors for glucocorticoids or mineralocorticoids but occurred via a novel signalling cascade involving sequentially a Gi-protein coupled receptor, PKC, adenylyl cyclase Type IV, cAMP, PKA and ERK1/2 activation.The rapid, non-genomic, effects of dexamethasone on airway epithelial ion transport and cell signalling introduces a new paradigm for glucocorticoid actions in lung epithelia which may serve to augment the anti-inflammatory activity of the steroid and enhance its therapeutic potential in treating airway hypersecretion in asthma and COPD.

Integrated expression profiling of potassium channels identifys KCNN4 as a prognostic biomarker of pancreatic cancer

Dysregulated potassium (K+) channels have previously been shown to promote the development and progression of many types of cancers. Meanwhile, K+ channels are particularly important in regulating the endocrine and exocrine functions of pancreas. However, the expression pattern and prognostic significance of K+ channels in pancreatic ductal adenocarcinoma (PDAC) remain unknown. In this study, by screening a GEO dataset containing 36 microdissected PDAC and matching normal pancreatic tissue samples, four differentially expressed K+ channels (KCNJ5, KCNJ16, KCNN4 and KCNK1) were identified in PDAC. by immunohistochemical analysis of pancreatic tissue sections from Pdx1-Cre; LSL-KrasG12D/+ mice (KC), Pdx1-Cre; LSL-KrasG12D/+; LSL-Trp53R172H/+ mice (KPC) and human PDAC tissue microarrays, we found that Ca2+-activated K+ channel KCNN4 was significantly elevated in pancreatic intraepithelial neoplasia (PanIN) and PDAC epithelia compared with untransformed pancreas tissues. Higher epithelial KCNN4 expression was closely correlated with advanced TNM stages and predicted a poor prognosis in patients with PDAC. Elevated KCNN4 expression was significantly associated with shorter survival in univariable and multivariable analyses. Collectively, the identification of expression pattern of K+ channels in PDAC and its precursor PanIN demonstrates the importance of KCNN4 channel during the malignant transformation of PDAC. On the basis of the prognostic signals from two independent cohorts, KCNN4 should be considered as a promising therapeutic target.

Enalapril Normalizes Endothelium-Derived Hyperpolarizing Factor-Mediated Relaxation in Mesenteric Artery of Adult Hypertensive Rats Prenatally Exposed to Testosterone.

Prenatal exposure to elevated testosterone levels induces adult life hypertension associated with selective impairments in endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation in mesenteric arteries. We tested whether the angiotensin-converting enzyme inhibitor enalapril restores EDHF function through regulating the activities of small (Kcnn3) and intermediate (Kcnn4) conductance calcium-activated potassium channels in mesenteric arteries. Pregnant Sprague-Dawley rats were injected subcutaneously with vehicle or testosterone propionate (0.5 mg/kg/day from Gestation Day 15 to 19), and their 6-mo-old adult male offspring were examined. A subset of rats in these two groups was given enalapril (40 mg/kg/day) for 2 wk through drinking water. Blood pressures were assessed through carotid arterial catheter and endothelium-dependent mesenteric arterial EDHF relaxation, using wire myography. Ace and Kcnn3 and Kcnn4 channel expression levels were also examined. Renal and vascular Ace expression and plasma angiotensin II levels were increased in testosterone offspring. Blood pressure levels were significantly higher in testosterone offspring than in controls, and treatment with enalapril significantly attenuated blood pressure in testosterone offspring. EDHF relaxation in testosterone offspring was reduced compared to that in controls, and it was significantly restored by enalapril treatment. Kcnn4 channel expression and function were similar between control and testosterone rats, but it was not affected by enalapril treatment. Relaxation mediated by Kcnn3 was impaired in testosterone offspring, and it was normalized by enalapril treatment. Furthermore, enalapril treatment restored expression levels of Kcnn3 channels. These findings suggest that enalapril has a positive influence on endothelial function with improvement in EDHF relaxation through normalization of Kcnn3 expression and activity.

Differential Expression of Ion Channels and Transporters During Hepatocellular Carcinoma Development.

Ion channels and transporters are potential markers and therapeutic targets for several cancers. However, their expression during hepatocellular carcinoma (HCC) development remains unclear. To investigate the mRNA expression of Na(+), K(+) and Ca(2+) channels and ABC transporters during rat HCC development, as well as Abcc3 protein in human liver biopsies. Wistar rats were treated with diethylnitrosamine (DEN) and developed both cirrhosis (12weeks of treatment) and either pre-neoplastic lesions (16weeks of treatment) or multinodular HCC (16weeks of treatment plus 2weeks DEN-free). The mRNA expression of 12 ion channels and two ABC transporters was studied using real-time RT-PCR. Tumor-containing or tumor-free liver sections were isolated by laser-capture microdissection. Abcc3 protein expression was studied by immunohistochemistry in healthy, cirrhotic and HCC human biopsies. We observed expression changes in seven genes. Kcna3, Kcnn4, Kcnrg and Kcnj11 potassium channel mRNA expression reached peak values at the end of DEN treatment, while Scn2a1 sodium channel, Trpc6 calcium channel and Abcc3 transporter mRNA expression reached their highest levels in the presence of HCC (18weeks). Whereas Kcnn4 and Scn2a1 channel expression was similar in non-tumor and tumor tissue, the Abcc3 transporter and Kcna3 potassium channels were preferentially overexpressed in the tumor sections. We observed differential Abcc3 protein subcellular localization and expression in human samples. The ion channel/transporter expression profile observed suggests that these genes are potential early markers or therapeutic targets of HCC. The differential localization of Abcc3 may be useful in the diagnosis of cirrhosis and HCC.

Upregulation of intermediate-conductance Ca2+-activated K+ channels (KCNN4) in porcine coronary smooth muscle requires NADPH oxidase 5 (NOX5).

AimsNADPH oxidase (NOX) is the primary source of reactive oxygen species (ROS) in vascular smooth muscle cells (SMC) and is proposed to play a key role in redox signaling involved in the pathogenesis of cardiovascular disease. Growth factors and cytokines stimulate coronary SMC (CSMC) phenotypic modulation, proliferation, and migration during atherosclerotic plaque development and restenosis. We previously demonstrated that increased expression and activity of intermediate-conductance Ca2+-activated K+ channels (KCNN4) is necessary for CSMC phenotypic modulation and progression of stenotic lesions. Therefore, the purpose of this study was to determine whether NOX is required for KCNN4 upregulation induced by mitogenic growth factors.Methods and ResultsDihydroethidium micro-fluorography in porcine CSMCs demonstrated that basic fibroblast growth factor (bFGF) increased superoxide production, which was blocked by the NOX inhibitor apocynin (Apo). Apo also blocked bFGF-induced increases in KCNN4 mRNA levels in both right coronary artery sections and CSMCs. Similarly, immunohistochemistry and whole cell voltage clamp showed bFGF-induced increases in CSMC KCNN4 protein expression and channel activity were abolished by Apo. Treatment with Apo also inhibited bFGF-induced increases in activator protein-1 promoter activity, as measured by luciferase activity assay. qRT-PCR demonstrated porcine coronary smooth muscle expression of NOX1, NOX2, NOX4, and NOX5 isoforms. Knockdown of NOX5 alone prevented both bFGF-induced upregulation of KCNN4 mRNA and CSMC migration.ConclusionsOur findings provide novel evidence that NOX5-derived ROS increase functional expression of KCNN4 through activator protein-1, providing another potential link between NOX, CSMC phenotypic modulation, and atherosclerosis.

Multiple mineralocorticoid response elements localized in different introns regulate intermediate conductance K+ (Kcnn4) channel expression in the rat distal colon.

An elevated plasma aldosterone and an increased expression of the intermediate conductance K+ (IK/Kcnn4) channels are linked in colon. This observation suggests that the expression of Kcnn4 gene is controlled through the action of aldosterone on its cognate receptor (i.e., mineralocorticoid receptor; MR). In order to establish this, we performed chromatin immunoprecipitation (ChIP) assay to identify the MR response elements (MREs) in a region that spanned 20 kb upstream and 10 kb downstream of the presumed transcription start site (TSS) using chromatin from the colonic epithelial cells of normal and aldosterone-treated rats. MREs were immunoprecipitated in an approximately 5 kb region that spanned the first and second introns in the aldosterone rats. These regions were individually cloned in luciferase-expression vector lacking enhancer activity. These clones were tested for enhancer activity in vitro by transfecting in HEK293T and CaCo2 cells with MR and aldosterone treatment. At least four regions were found to be responsive to the MR and aldosterone. Two regions were identified to contain MREs using bioinformatics tools. These clones lost their enhancer activity after mutation of the presumptive MREs, and thus, established the functionality of the MREs. The third and fourth clones did not contain any bioinformatically obvious MREs. Further, they lost their activity upon additional sub-cloning, which suggest cooperativity between the regions that were separated upon sub-cloning. These results demonstrate the presence of intronic MREs in Kcnn4 and suggest a highly cooperative interaction between multiple intronic response elements.

Aldosterone Stimulates Neutrophils Leading To Increased β-Glucuronidase, Protein Disulfide Isomerase and Myeloperoxidase Secretion

Aldosterone (ALDO) has been shown to play an important role in inflammatory responses in addition to its well described effects on sodium homeostasis via activation of the mineralocorticoid receptor (MR). However, its effects on polymorphonuclear leukocytes (PMNC) are not well described. We isolated untouched circulating human PMNC by immunomagnetic isolation following density gradient sedimentation with PolymorphPrep from otherwise healthy subjects. Flow cytometric analyses showed greater than 97% of PMNC were positive for the myeloid-neutrophil markers, CD45, CD16 and CD66b. We show that PMNC express MR by western blot and RT-PCR analyses. We incubated PMNC with ALDO (10–9–10–7M) for 30 min and observed a dose-dependent rise in β–glucuronidase release with an EC50 of 6.11 nM (P<0.001, n=3), an event that was blocked by pre-incubation of cells with 1μM canrenoic acid (CA), an MR antagonist (P<0.04, n=3). In addition, our results show that incubation of human PMNC with 10-8M ALDO likewise led to increases in myeloperoxidase ([MPO], P<0.05, n=3) and protein disulfide isomerase ([PDI], P<0.01, n=4), a multifunctional enzyme of the thioredoxin superfamily that mediates redox modifications, regulates KCNN4 channel and erythrocyte volume and is up-regulated under hypoxic conditions (Prado, 2013 FASEB J). We then studied the effects of ALDO on HL-60, a human promyelocytic cell line, induced to differentiate into neutrophil-like cells by incubation for 5 days with 1.3% DMSO. Our results likewise show an increase in MPO responses upon 10–8M ALDO stimulation as compared to vehicle (AUC: 1090±147 to 505±48, P<0.02, n=3). We have recently reported that aldosterone stimulates increases of striatin, a scaffolding protein that interacts with caveolin-1, and co-precipitates with striatin and as such may facilitate cross talk of signaling complexes. As there are no pharmacological inhibitors of striatin we used a molecular approach to reduce striatin levels. In differentiated HL-60 cells, siRNA against striatin led to reduced MPO responses (AUC: 590±14 to 528±13, P<0.05, n=3) that were associated with significantly reduced striatin mRNA levels but not when cells were transfected with scrambled siRNA as determined by quantitative RT-PCR with ABI TaqMan detection probes and β-microglobulin used as an endogenous control (P<0.01, n=3). These results suggest that striatin plays an important role in ALDO-stimulated degranulation responses. Of importance we also observed that incubation with ALDO (10–9–10–7M) in differentiated HL60 cells led to increases in the oxidative-respiratory burst [superoxide production] in a dose- and time-dependent manner (P<0.01, n=4). Consistent with these results, we observed that ALDO likewise led to significant increases in the oxidative-respiratory burst in human PMNC (P<0.01, n=3). As there is evidence that activated neutrophils, MPO and PDI are elevated in Sickle Cell Disease, we studied the in vivo effects of MR blockade in BERK sickle transgenic mice, a model of increased oxidative stress. Sickle mice were randomized to receive either normal rodent chow or chow containing eplerenone (156 mg/kg per day), an MR receptor antagonist, and tap water ad libitum for 14 days at which time the mice were sacrificed and blood collected. We observed that mice on eplerenone had significantly lower plasma PDI activity than mice on regular chow (63.7 ± 8.7 control diet to 47.9 ± 2.4 eplerenone, Relative Fluorescence Units [RFU]; P<0.005, n=6 and 9) and lower MPO levels (AUC: 214±11 to 73±20, P<0.03, n=3); events that were associated with increases in both erythrocyte MCV (41.3±2.5 vs 47.4±1.1 fL, P<0.03, n=7) and reticulocyte MCV (53.6.3±2.8 vs 60.1±0.6 fL, P<0.02, n=7). Thus, our results suggest that MR activation by ALDO is a novel mechanism for neutrophil stimulation and as such represents a novel therapeutic target aimed at ameliorating the vascular complications of Sickle Cell Disease. Supported by NIH R01HL090632 (AR) and R01HL096518 (JRR). Disclosures:No relevant conflicts of interest to declare.

Endothelial-Specific Knockdown Of Caveolin-1 Lowers Circulating Protein Disulfide Isomerase Levels and Is Associated With Changes In Erythrocyte Volume Homeostasis

We have recently reported that Endothelin-1 (ET1), a potent vasoconstrictor peptide, is implicated in the pathophysiology of Sickle Cell Disease (SCD) via increased circulating Protein Disulfide Isomerase (PDI) activity (Prado, 2013 FASEB J). PDI is a multifunctional enzyme of the thioredoxin superfamily that mediates redox modifications, catalyzes disulfide interchange reactions in the plasma membrane, regulates KCNN4 channel and erythrocyte volume and is up-regulated under hypoxic conditions as commonly observed in SCD. In erythrocytes, ET1 stimulates PDI activity via activation of ET1 receptor B (ETRB). However, the precise mechanisms by which ET1 leads to increases in PDI are not entirely clear. There is evidence that activation of endothelial cells leads to increased PDI secretion and that ETRBs form a complex with caveolin-1 (CAV1) within caveolae to mediate ET1's cellular effects. We tested the hypothesis that reduction of CAV1 would alter PDI secretion. We studied the in vivo effects of endothelial-specific CAV1 knockdown on circulating PDI activity in mice. We optimized conditions to measure circulating PDI using fluorescently labeled GSSG conversion to GSH. We now report that circulating plasma PDI levels were significantly decreased in CAV1 knockdown mice when compared to wild-type littermates (WT) (7.44±0.70 vs 10.93±2.66, n=7, P<0.05). In addition and consistent with our report showing a role for PDI in erythrocyte volume regulation, we also observed lower cell hemoglobin concentration mean (CHCM) and hemoglobin distribution width (HDW) that was associated with increased erythrocyte and reticulocyte mean cell volume (MCV) in blood from CAV1 knockdown mice when compared to WT (n=13 and n=19, respectively, P<0.005). We then isolated early cultures of mouse aortic endothelial cells (MAEC) from these mice and measured PDI activity following 24 hrs of incubation in 0.4% fetal bovine serum. Our results show that MAEC from CAV1 knockdown mice had lower PDI secretion when compared to cells from WT mice (99.4±16 vs 129.9±35, n=5, P<0.03). We then studied the effects of ET1 on PDI secretion from human endothelial cells. We detected PDI and ETRB by western blot analyses in membranes from the human endothelial cell line, EA.hy926 (EA). We observed that incubation of EA cells for 60 mins with 10-7 M ET1 was associated with increased extracellular PDI activity (15.97±7.22 to 34.07±8.89 [RFU/mg protein], n=3, P<0.011) that was sensitive to preincubation with BQ788, a specific ETRB receptor antagonist (15.97±7.22 to 7.97±3.25 (RFU/mg protein), n=3, P<0.02). Similar increases in PDI were observed when cells were treated with the specific ETRB agonist, IRL1620 (1143±137 to 1593 207 RFU/mg protein). In addition, PDI siRNA knockdown was associated with reduced ET1-stimulated PDI activity when compared to scrambled siRNA transfected cells (1731±147 to 757±141 RFU, n=2). We then tested the effects of methyl-β-cyclodextrin to disrupt caveolae in these cells and observed a blunted IRL1620–stimulated PDI response (288±40 to 171±14 RFU/mg protein, n=3, P<0.025). We also characterized the effects of ET1 on PDI expression in EA cells, using quantitative RT-PCR with ABI TaqMan probes and β-actin as an endogenous control and observed that stimulation of EA cells with 10-8 M ET1 for 4 hr was associated with increased PDI mRNA expression levels that were 1.89 fold greater than vehicle treated cells (n=6, P<0.04). Thus our results provide evidence for a heretofore unrecognized role of endothelial specific CAV1 in erythrocyte volume and circulating PDI levels. Supported by NIH R01HL090632 (AR) and R01HL104032 (LHP). Disclosures:No relevant conflicts of interest to declare.

Prenatal Testosterone Induces Sex-Specific Dysfunction in Endothelium-Dependent Relaxation Pathways in Adult Male and Female Rats1

Prenatal testosterone (T) exposure impacts postnatal cardiovascular function, leading to increases in blood pressure with associated decreased endothelium-dependent vascular relaxation in adult females. Endothelial function in males is not known. Furthermore, which of the endothelial pathways contributes to endothelial dysfunction and if there exists sex differences are not known. The objective of this study was to characterize the relative contribution of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) to the impaired endothelium-dependent vasodilation in prenatal T-exposed adult males and females. Offspring of pregnant rats treated with T propionate or its vehicle were examined. Telemetric blood pressure levels and endothelium-dependent vascular reactivity were assessed with wire myography. Levels of nitric oxide synthase (NOS3) and Kcnn3 and Kcnn4 channel expression were examined in mesenteric arteries. Mean arterial pressure was significantly higher in T males and females than in controls. Endothelium-dependent acetylcholine relaxation was significantly lower in both T males and females. EDHF-mediated relaxation was specifically blunted in T males (Emax = 48.64% ± 3.73%) compared to that in control males (Emax = 81.71% ± 3.18%); however, NO-mediated relaxation was specifically impaired in T females (Emax = 36.01% ± 4.29%) compared with that in control females (Emax = 54.56% ± 6.37%). Relaxation to sodium nitroprusside and levcromakalim were unaffected with T-treatment. NOS3 protein was decreased in T females but not in T males. Kcnn3 expression was decreased in both T males and females compared to controls. These findings suggest that prenatal T leads to an increase in blood pressure in the adult offspring, associated with blunting of endothelial cell-associated relaxation and that the effects are sex-specific: EDHF-related in males and NO-related in females.

IgE-dependent mast cell hyperplasya in the intestine of cystic fibrosis mouse model needs KCNN4 channel activity.

Event Abstract Back to Event IgE-dependent mast cell hyperplasya in the intestine of cystic fibrosis mouse model needs KCNN4 channel activity. Texia T. Riquelme1, L.Pablo Cid1, Francisco V. Sepulveda1 and Carlos A. Flores1* 1 Centro de Estudios Científicos, Chile Cystic fibrosis (CF) primary dysfunction in epithelial transport concurs with deregulation of the immune system in human patients and animal models. We observed that when the KCNN4 channel is genetically silenced in the CF mouse lethality is drastically reduced with no improvement of intestinal function. The KCNN4 channel is broadly expressed including the immune system. We aim to test if KCNN4 channel inhibition is involved in immune system dysfunctions associated with CF. CF animals presented a 4-fold increase of intestinal mast cells, the value obtained in the KCNN4-null/CF mice was lower and comparable to controls. IgE-induced migration was 2-fold increased in control and CF mast cells compared with non-stimulated cells. Migration was blocked when the KCNN4 specific inhibitor TRAM-34 was used. Migration was completely impaired in the KCNN4-null and KCNN4-null/CF cells. Serum IgE level was increased in the CF as in the KCNN4-null/CF mice. Inhibition of KCNN4 blocks migration in control and CF mast cells. Hyperplasia of mast cells in the CF mice is localized in the intestinal tissue and can be due to increased migration induced by the higher levels of IgE. Mast cell reduction on intestine of KCNN4-null/CF mice is due to the impairment in migration of mast cells and not to reduced IgE levels. The origin of IgE in the CF mice remains a matter of investigation. KCNN4 inhibtion can be a useful pharmacological target on inflammatory CF disease where mast cells can act as enhancers of inflammation. Acknowledgements FONDECYT 111408 and CONICYT-BFP Keywords: IgE, Mast Cells, Kcnn4, Cystic Fibrosis, mice models Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Innate immunity Citation: Riquelme TT, Cid L, Sepulveda FV and Flores CA (2013). IgE-dependent mast cell hyperplasya in the intestine of cystic fibrosis mouse model needs KCNN4 channel activity.. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01071 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 09 Jul 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Carlos A Flores, Centro de Estudios Científicos, Valdivia, Los Rios, Chile, cflores@cecs.cl Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Texia T Riquelme L.Pablo Cid Francisco V Sepulveda Carlos A Flores Google Texia T Riquelme L.Pablo Cid Francisco V Sepulveda Carlos A Flores Google Scholar Texia T Riquelme L.Pablo Cid Francisco V Sepulveda Carlos A Flores PubMed Texia T Riquelme L.Pablo Cid Francisco V Sepulveda Carlos A Flores Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Cyclic AMP-induced K+ secretion occurs independently of Cl− secretion in rat distal colon

cAMP induces both active Cl(-) and active K(+) secretion in mammalian colon. It is generally assumed that a mechanism for K(+) exit is essential to maintain cells in the hyperpolarized state, thus favoring a sustained Cl(-) secretion. Both Kcnn4c and Kcnma1 channels are located in colon, and this study addressed the questions of whether Kcnn4c and/or Kcnma1 channels mediate cAMP-induced K(+) secretion and whether cAMP-induced K(+) secretion provides the driving force for Cl(-) secretion. Forskolin (FSK)-enhanced short-circuit current (indicator of net electrogenic ion transport) and K(+) fluxes were measured simultaneously in colonic mucosa under voltage-clamp conditions. Mucosal Na(+) orthovanadate (P-type ATPase inhibitor) inhibited active K(+) absorption normally present in rat distal colon. In the presence of mucosal Na(+) orthovanadate, serosal FSK induced both K(+) and Cl(-) secretion. FSK-induced K(+) secretion was 1) not inhibited by either mucosal or serosal 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34; a Kcnn4 channel blocker), 2) inhibited (92%) by mucosal iberiotoxin (Kcnma1 channel blocker), and 3) not affected by mucosal cystic fibrosis transmembrane conductance regulator inhibitor (CFTR(inh)-172). By contrast, FSK-induced Cl(-) secretion was 1) completely inhibited by serosal TRAM-34, 2) not inhibited by either mucosal or serosal iberiotoxin, and 3) completely inhibited by mucosal CFTR(inh)-172. These results indicate that cAMP-induced colonic K(+) secretion is mediated via Kcnma1 channels located in the apical membrane and most likely contributes to stool K(+) losses in secretory diarrhea. On the other hand, cAMP-induced colonic Cl(-) secretion requires the activity of Kcnn4b channels located in the basolateral membrane and is not dependent on the concurrent activation of apical Kcnma1 channels.

Aldosterone induces active K+ secretion by enhancing mucosal expression of Kcnn4c and Kcnma1 channels in rat distal colon

Although both Kcnn4c and Kcnma1 channels are present on colonic mucosal membranes, only Kcnma1 has been suggested to mediate K(+) secretion in the colon. Therefore, studies were initiated to investigate the relative roles of Kcnn4c and Kcnma1 in mediating aldosterone (Na-free diet)-induced K(+) secretion. Mucosal to serosal (m-s), serosal to mucosal (s-m), and net (86)Rb(+) (K(+) surrogate) fluxes as well as short circuit currents (I(sc); measure of net ion movement) were measured under voltage clamp condition in rat distal colon. Active K(+) absorption, but not K(+) secretion, is present in normal, while aldosterone induces active K(+) secretion (1.04 ± 0.26 vs. -1.21 ± 0.15 μeq·h(-1)·cm(-2); P < 0.001) in rat distal colon. Mucosal VO(4) (a P-type ATPase inhibitor) inhibited the net K(+) absorption in normal, while it significantly enhanced net K(+) secretion in aldosterone animals. The aldosterone-induced K(+) secretion was inhibited by the mucosal addition of 1) either Ba(2+) (a nonspecific K(+) channel blocker) or charybdotoxin (CTX; a common Kcnn4 and Kcnma1 channel blocker) by 89%; 2) tetraethyl ammonium (TEA) or iberiotoxin (IbTX; a Kcnma1 channel blocker) by 64%; and 3) TRAM-34 (a Kcnn4 channel blocker) by 29%. TRAM-34, but not TEA, in the presence of IbTX further significantly inhibited the aldosterone-induced K(+) secretion. Thus the aldosterone-induced Ba(2+)/CTX-sensitive K(+) secretion consists of IbTX/TEA-sensitive (Kcnma1) and IbTX/TEA-insensitive fractions. TRAM-34 inhibition of the IbTX-insensitive fraction is consistent with the aldosterone-induced K(+) secretion being mediated partially via Kcnn4c. Western and quantitative PCR analyses indicated that aldosterone enhanced both Kcnn4c and Kcnma1α protein expression and mRNA abundance. In vitro exposure of isolated normal colonic mucosa to aldosterone also enhanced Kcnn4c and Kcnma1α mRNA levels, and this was prevented by exposure to actinomycin D (an RNA synthesis inhibitor). These observations indicate that aldosterone induces active K(+) secretion by enhancing mucosal Kcnn4c and Kcnma1 expression at the transcriptional level.

Cloning and identification of tissue-specific expression of KCNN4 splice variants in rat colon

KCNN4 channels that provide the driving force for cAMP- and Ca(2+)-induced anion secretion are present in both apical and basolateral membranes of the mammalian colon. However, only a single KCNN4 has been cloned. This study was initiated to identify whether both apical and basolateral KCNN4 channels are encoded by the same or different isoforms. Reverse transcriptase-PCR (RT-PCR), real-time quantitative-PCR (RT-QPCR), and immunofluorescence studies were used to clone and identify tissue-specific expression of KCNN4 isoforms. Three distinct KCNN4 cDNAs that are designated as KCNN4a, KCNN4b, and KCNN4c encoding 425, 424, and 395 amino acid proteins, respectively, were isolated from the rat colon. KCNN4a differs from KCNN4b at both the nucleotide and the amino acid level with distinct 628 bp at the 3'-untranslated region and an additional glutamine at position 415, respectively. KCNN4c differs from KCNN4b by lacking the second exon that encodes a 29 amino acid motif. KCNN4a and KCNN4b/c are identified as smooth muscle- and epithelial cell-specific transcripts, respectively. KCNN4b and KCNN4c transcripts likely encode basolateral (40 kDa) and apical (37 kDa) membrane proteins in the distal colon, respectively. KCNN4c, which lacks the S2 transmembrane segment, requires coexpression of a large conductance K(+) channel beta-subunit for plasma membrane expression. The KCNN4 channel blocker TRAM-34 inhibits KCNN4b- and KCNN4c-mediated (86)Rb (K(+) surrogate) efflux with an apparent inhibitory constant of 0.6 +/- 0.1 and 7.8 +/- 0.4 muM, respectively. We conclude that apical and basolateral KCNN4 K(+) channels that regulate K(+) and anion secretion are encoded by distinct isoforms in colonic epithelial cells.

T1845 Enhanced Mucosal Expression of KCNN4 and Bk Channels Contribute to Aldosterone Induced K+ Secretion in Distal Colon

Background: Mammalian colon plays important roles in body K+ homeostasis, as it both actively absorbs and secretes K+. Aldosterone (aldo) stimulates both active electroneutral K+ absorption and active electrogenic K+ secretion in rat distal colon. Increased H,K-ATPase expression has been shown as a mechanism for enhanced K+ absorption. However, although both intermediate conductance (KCNN4) and large conductance (BK) K+ channels are present on the mucosal membranes, a mechanism responsible for aldo increased K+ secretion is not known. Aim: Studies were initiated to identify whether KCNN4 and/or BK channels mediate the aldo enhanced K+ secretion in rat distal colon. Methods: Aldo animals were produced by feeding Na-free diet for 6-7 days. Mucosal to serosal (m-s) and serosal to mucosal (s-m) 86Rb (K+ surrogate) fluxes were measured in stripped colonic mucosa mounted under voltage clamp condition in presence of mucosal benzamil (50 μM; ENaC blocker). Western blot analyses were performed using anti-KCNN4-abc (developed in our laboratory) and BKα (Santa Cruz) antibodies. RTQ-PCR analyses were performed using standard technique. Results: Under basal condition, mucosal addition of VO4 (H,K-ATPase inhibitor) inhibited the net K+ absorption (0.42 ± 0.06 vs -0.04 ± 0.03 μEq/h.cm2; p < 0.001) in normal rat distal colon. Serosal addition of carbachol (CCH) induced net K secretion (-0.04 ± 0.03 vs 0.48 ± 0.06 μEq/h.cm2; p < 0.001). The CCH-induced net K+ secretion was inhibited by 60% and 40% by mucosal TRAM-34 (KCNN4 channel blocker) and iberiotoxin (IbTX; BK channel blocker), respectively. Either simultaneous presence of TRAM-34 and IbTX or Ba2+ (nonspecific K+ channel blocker) completely inhibited the CCH-induced K secretion. Aldo reversed the net K absorption in normal colon to net K+ secretion (0.42 ± 0.06 vs -1.34 ± 0.22 μEq/h.cm2; p < 0.001). Mucosal addition of TRAM34 and IbTX inhibited the aldo-induced net K+ secretion by 33% and 55%, respectively. Simultaneous presence of TRMA-34 and IbTX, and Ba2+ inhibited the aldo induced net K secretion by 84% and 80%, respectively. Western blot analyses indicated that aldo enhanced mucosal KCNN4 and BKα specific protein expression by 2.2 and 3.1 fold, respectively. RTQ-PCR analyses indicated that mucosal KCNN4 and BKα channel specific mRNA abundances were increased by 3.0 and 3.5 fold in aldo animals, respectively. Conclusions: 1) Both KCNN4 and BK channels mediate CCHand aldo-induced K+ secretion; 2) Enhanced expression of both mucosal KCNN4 and BK channels mediate the aldo induced K+ secretion; and 3) Aldo regulates both KCNN4 and BK channel expression at transcriptional level.

Blocking ion channel KCNN4 alleviates the symptoms of experimental autoimmune encephalomyelitis in mice

The KCNN4 potassium-ion channel has been reported to play an important role in regulating antigen-induced T cell effector functions in vitro. This study presents the first evidence that a selective KCNN4 blocker, TRAM-34, confers protection against experimental autoimmune encephalomyelitis (EAE) in the mouse model. Treatment with the KCNN4 blocker did not prevent infiltration of T cells in the spinal cord, but resulted in the reduction of both the protein and the message levels of TNF-alpha and IFN-gamma as well as the message levels of several other pro-inflammatory molecules in the spinal cord. Plasma concentrations of TRAM-34 within a 24-h period were between the in vitro IC(50) and IC(90) values for the KCNN4 channel. The effect of TRAM-34 was reversible, as indicated by the development of clinical EAE symptoms within 48 h after withdrawal of treatment. In summary, our data support the idea that KCNN4 channels play a critical role in the immune response during the development of MOG-induced EAE in C57BL/6 mice.