High Glucose Induces CCL20 in Proximal Tubular Cells via Activation of the KCa3.1 Channel

Background

Inflammation plays a key role in the development and progression of diabetic nephropathy (DN). KCa3.1, a calcium activated potassium channel protein, is associated with vascular inflammation, atherogenesis, and proliferation of endothelial cells, macrophages, and fibroblasts. We have previously demonstrated that the KCa3.1 channel is activated by TGF-β1 and blockade of KCa3.1 ameliorates renal fibrotic responses in DN through inhibition of the TGF-β1 pathway. The present study aimed to identify the role of KCa3.1 in the inflammatory responses inherent in DN.

Methods

Human proximal tubular cells (HK2 cells) were exposed to high glucose (HG) in the presence or absence of the KCa3.1 inhibitor TRAM34 for 6 days. The proinflammatory cytokine chemokine (C-C motif) ligand 20 (CCL20) expression was examined by real-time PCR and enzyme-linked immunosorbent assay (ELISA). The activity of nuclear factor-κB (NF-κB) was measured by nuclear extraction and electrophoretic mobility shift assay (EMSA). In vivo, the expression of CCL20, the activity of NF-κB and macrophage infiltration (CD68 positive cells) were examined by real-time PCR and/or immunohistochemistry staining in kidneys from diabetic or KCa3.1-/- mice, and in eNOS-/- diabetic mice treated with the KCa3.1 channel inhibitor TRAM34.

Results

In vitro data showed that TRAM34 inhibited CCL20 expression and NF-κB activation induced by HG in HK2 cells. Both mRNA and protein levels of CCL20 significantly decreased in kidneys of diabetic KCa3.1-/- mice compared to diabetic wild type mice. Similarly, TRAM34 reduced CCL20 expression and NF-κB activation in diabetic eNOS-/- mice compared to diabetic controls. Blocking the KCa3.1 channel in both animal models led to a reduction in phosphorylated NF-κB.

Conclusions

Overexpression of CCL20 in human proximal tubular cells is inhibited by blockade of KCa3.1 under diabetic conditions through inhibition of the NF-κB pathway.     

Hypoxia Activates a Ca2+-Permeable Cation Conductance Sensitive to Carbon Monoxide and to GsMTx-4 in Human and Mouse Sickle Erythrocytes

Background

Deoxygenation of sickle erythrocytes activates a cation permeability of unknown molecular identity (Psickle), leading to elevated intracellular [Ca²⁺] ([Ca²⁺]_i) and subsequent activation of K_Ca 3.1. The resulting erythrocyte volume decrease elevates intracellular hemoglobin S (HbSS) concentration, accelerates deoxygenation-induced HbSS polymerization, and increases the likelihood of cell sickling. Deoxygenation-induced currents sharing some properties of Psickle have been recorded from sickle erythrocytes in whole cell configuration.

Methodology/Principal Findings

We now show by cell-attached and nystatin-permeabilized patch clamp recording from sickle erythrocytes of mouse and human that deoxygenation reversibly activates a Ca²⁺- and cation-permeable conductance sensitive to inhibition by Grammastola spatulata mechanotoxin-4 (GsMTx-4; 1 µM), dipyridamole (100 µM), DIDS (100 µM), and carbon monoxide (25 ppm pretreatment). Deoxygenation also elevates sickle erythrocyte [Ca²⁺]_i, in a manner similarly inhibited by GsMTx-4 and by carbon monoxide. Normal human and mouse erythrocytes do not exhibit these responses to deoxygenation. Deoxygenation-induced elevation of [Ca²⁺]_i in mouse sickle erythrocytes did not require KCa3.1 activity.

Conclusions/Significance

The electrophysiological and fluorimetric data provide compelling evidence in sickle erythrocytes of mouse and human for a deoxygenation-induced, reversible, Ca²⁺-permeable cation conductance blocked by inhibition of HbSS polymerization and by an inhibitor of strctch-activated cation channels. This cation permeability pathway is likely an important source of intracellular Ca²⁺ for pathologic activation of KCa3.1 in sickle erythrocytes. Blockade of this pathway represents a novel therapeutic approach for treatment of sickle disease.     

Complementary roles of KCa3.1 channels and β1-integrin during alveolar epithelial repair

Background

Extensive alveolar epithelial injury and remodelling is a common feature of acute lung injury and acute respiratory distress syndrome (ARDS) and it has been established that epithelial regeneration, and secondary lung oedema resorption, is crucial for ARDS resolution. Much evidence indicates that K⁺ channels are regulating epithelial repair processes; however, involvement of the KCa3.1 channels in alveolar repair has never been investigated before.

Results

Wound-healing assays demonstrated that the repair rates were increased in primary rat alveolar cell monolayers grown on a fibronectin matrix compared to non-coated supports, whereas an anti-β1-integrin antibody reduced it. KCa3.1 inhibition/silencing impaired the fibronectin-stimulated wound-healing rates, as well as cell migration and proliferation, but had no effect in the absence of coating. We then evaluated a putative relationship between KCa3.1 channel and the migratory machinery protein β1-integrin, which is activated by fibronectin. Co-immunoprecipitation and immunofluorescence experiments indicated a link between the two proteins and revealed their cellular co-distribution. In addition, we demonstrated that KCa3.1 channel and β1-integrin membrane expressions were increased on a fibronectin matrix. We also showed increased intracellular calcium concentrations as well as enhanced expression of TRPC4, a voltage-independent calcium channel belonging to the large TRP channel family, on a fibronectin matrix. Finally, wound-healing assays showed additive effects of KCa3.1 and TRPC4 inhibitors on alveolar epithelial repair.

Conclusion

Taken together, our data demonstrate for the first time complementary roles of KCa3.1 and TRPC4 channels with extracellular matrix and β1-integrin in the regulation of alveolar repair processes.     

Modulation of KCa3.1 Channels by Eicosanoids,Omega-3 Fatty Acids,and Molecular Determinants

Background

Cytochrome P450- and ω-hydrolase products (epoxyeicosatrienoic acids (EETs), hydroxyeicosatetraeonic acid (20-HETE)), natural omega-3 fatty acids (ω3), and pentacyclic triterpenes have been proposed to contribute to a wide range of vaso-protective and anti-fibrotic/anti-cancer signaling pathways including the modula-tion of membrane ion channels. Here we studied the modulation of intermediate-conductance Ca²⁺/calmodulin-regulated K⁺ channels (K_Ca3.1) by EETs, 20-HETE, ω3, and pentacyclic triterpenes and the structural requirements of these fatty acids to exert channel blockade.

Methodology/Principal Findings

We studied modulation of cloned human hK_Ca3.1 and the mutant hK_Ca3.1^V275A in HEK-293 cells, of rK_Ca3.1 in aortic endothelial cells, and of mK_Ca3.1 in 3T3-fibroblasts by inside-out and whole-cell patch-clamp experiments, respectively. In inside-out patches, Ca²⁺-activated hK_Ca3.1 were inhibited by the ω3, DHA and α-LA, and the ω6, AA, in the lower µmolar range and with similar potencies. 5,6-EET, 8,9-EET, 5,6-DiHETE, and saturated arachidic acid, had no appreciable effects. In contrast, 14,15-EET, its stable derivative, 14,15-EEZE, and 20-HETE produced channel inhibition. 11,12-EET displayed less inhibitory activity. The K_Ca3.1^V275A mutant channel was insensitive to any of the blocking EETs. Non-blocking 5,6-EET antagonized the inhibition caused by AA and augmented cloned hK_Ca3.1 and rK_Ca3.1 whole-cell currents. Pentacyclic triterpenes did not modulate K_Ca3.1 currents.

Conclusions/Significance

Inhibition of K_Ca3.1 by EETs (14,15-EET), 20-HETE, and ω3 critically depended on the presence of electron double bonds and hydrophobicity within the 10 carbons preceding the carboxyl-head of the molecules. From the physiological perspective, metabolism of AA to non-blocking 5,6,- and 8,9-EET may cause AA-de-blockade and contribute to cellular signal transduction processes influenced by these fatty acids.     

KCa3.1 Channel-Blockade Attenuates Airway Pathophysiology in a Sheep Model of Chronic Asthma

Background

The Ca²⁺-activated K⁺ channel K_Ca3.1 is expressed in several structural and inflammatory airway cell types and is proposed to play an important role in the pathophysiology of asthma. The aim of the current study was to determine whether inhibition of K_Ca3.1 modifies experimental asthma in sheep.

Methodology and Principal Findings

Atopic sheep were administered either 30 mg/kg Senicapoc (ICA-17073), a selective inhibitor of the K_Ca3.1-channel, or vehicle alone (0.5% methylcellulose) twice daily (orally). Both groups received fortnightly aerosol challenges with house dust mite allergen for fourteen weeks. A separate sheep group received no allergen challenges or drug treatment. In the vehicle-control group, twelve weeks of allergen challenges resulted in a 60±19% increase in resting airway resistance, and this was completely attenuated by treatment with Senicapoc (0.25±12%; n = 10, P = 0.0147). The vehicle-control group had a peak-early phase increase in lung resistance of 82±21%, and this was reduced by 58% with Senicapoc treatment (24±14%; n = 10, P = 0.0288). Senicapoc-treated sheep also demonstrated reduced airway hyperresponsiveness, requiring a significantly higher dose of carbachol to increase resistance by 100% compared to allergen-challenged vehicle-control sheep (20±5 vs. 52±18 breath-units of carbachol; n = 10, P = 0.0340). Senicapoc also significantly reduced eosinophil numbers in bronchoalveolar lavage taken 48 hours post-allergen challenge, and reduced vascular remodelling.

Conclusions

These findings suggest that K_Ca3.1-activity contributes to allergen-induced airway responses, inflammation and vascular remodelling in a sheep model of asthma, and that inhibition of K_Ca3.1 may be an effective strategy for blocking allergen-induced airway inflammation and hyperresponsiveness in humans.     

High Expression of KCa3.1 in Patients with Clear Cell Renal Carcinoma Predicts High Metastatic Risk and Poor Survival

Background

Ca²⁺-activated K⁺ channels have been implicated in cancer cell growth, metastasis, and tumor angiogenesis. Here we hypothesized that high mRNA and protein expression of the intermediate-conductance Ca²⁺-activated K⁺ channel, KCa3.1, is a molecular marker of clear cell Renal Cell Carcinoma (ccRCC) and metastatic potential and survival.

Methodology/Principal Findings

We analyzed channel expression by qRT-PCR, immunohistochemistry, and patch-clamp in ccRCC and benign oncocytoma specimens, in primary ccRCC and oncocytoma cell lines, as well as in two ccRCC cell lines (Caki-1 and Caki-2). CcRCC specimens contained 12-fold higher mRNA levels of KCa3.1 than oncocytoma specimens. The large-conductance channel, KCa1.1, was 3-fold more highly expressed in ccRCC than in oncocytoma. KCa3.1 mRNA expression in ccRCC was 2-fold higher than in the healthy cortex of the same kidney. Disease specific survival trended towards reduction in the subgroup of high-KCa3.1-expressing tumors (p<0.08 vs. low-KCa3.1-expressing tumors). Progression-free survival (time to metastasis/recurrence) was reduced significantly in the subgroup of high-KCa3.1-expressing tumors (p<0.02, vs. low-KCa3.1-expressing tumors). Immunohistochemistry revealed high protein expression of KCa3.1 in tumor vessels of ccRCC and oncocytoma and in a subset of ccRCC cells. Oncocytoma cells were devoid of KCa3.1 protein. In a primary ccRCC cell line and Caki-1/2-ccRCC cells, we found KCa3.1-protein as well as TRAM-34-sensitive KCa3.1-currents in a subset of cells. Furthermore, Caki-1/2-ccRCC cells displayed functional Paxilline-sensitive KCa1.1 currents. Neither KCa3.1 nor KCa1.1 were found in a primary oncocytoma cell line. Yet KCa-blockers, like TRAM-34 (KCa3.1) and Paxilline (KCa1.1), had no appreciable effects on Caki-1 proliferation in-vitro.

Conclusions/Significance

Our study demonstrated expression of KCa3.1 in ccRCC but not in benign oncocytoma. Moreover, high KCa3.1-mRNA expression levels were indicative of low disease specific survival of ccRCC patients, short progression-free survival, and a high metastatic potential. Therefore, KCa3.1 is of prognostic value in ccRCC.     

Pyrrolidinyl caffeamide against ischemia/reperfusion injury in cardiomyocytes through AMPK/AKT pathways

Background

Coronary heart disease is a leading cause of death in the world and therapy to reduce injury is still needed. The uncoupling of glycolysis and glucose oxidation induces lactate accumulation during myocardial ischemia/reperfusion (I/R) injury. Cell death occurs and finally leads to myocardial infarction. Caffeic acid, one of the major phenolic constituents in nature, acts as an antioxidant. Pyrrolidinyl caffeamide (PLCA), a new derivative of caffeic acid, was synthesized by our team. We aimed to investigate the effect of PLCA on hypoxia/reoxygenation (H/R) in neonatal rat ventricular myocytes (NRVM) and on myocardial I/R in rats.

Results

Cardiomyocytes were isolated and subjected to 6 h hypoxia followed by 18 h reperfusion. PLCA (0.1 to 3 μM) and metformin (30 μM) were added before hypoxia was initiated. PLCA at 1 μM and metformin at 30 μM exerted similar effects on the improvement of cell viability and the alleviation of cell apoptosis in NRVM after H/R. PLCA promoted p-AMPK, p-AKT, and GLUT4 upregulation to induce a cardioprotective effect in both cell and animal model. The accumulation of cardiac lactate was attenuated by PLCA during myocardial I/R, and infarct size was smaller in rats treated with PLCA (1 mg/kg) than in those treated with caffeic acid (1 mg/kg).

Conclusions

AMPK and AKT are synergistically activated by PLCA, which lead facilities glucose utilization, thereby attenuating lactate accumulation and cell death. The cardioprotective dose of PLCA was lower than those of metformin and caffeic acid. We provide a new insight into this potential drug for the treatment of myocardial I/R injury.     

Potentiation of large conductance KCa channels by niflumic, flufenamic, and mefenamic acids.

Large conductance calcium-activated K+ (KCa) channels are rapidly activated by niflumic acid dose-dependently and reversibly. External niflumic acid was about 5 times more potent than internal niflumic acid, and its action was characterized by an increase in the channel affinity for [Ca2+], a parallel left shift of the voltage-activation curve, and a decrease of the channel long-closed states. Niflumic acid applied from the external side did not interfere with channel block by charybdotoxin, suggesting that its site of action is not at or near the charybdotoxin receptor. Accordingly, partial tetraethylammonium blockade did not interfere with channel activation by niflumic acid. Flufenamic acid and mefenamic acid also stimulated KCa channel activity and, as niflumic acid, they were more potent from the external than from the internal side. Fenamates applied from the external side displayed the following potency sequence: flufenamic acid approximately niflumic acid >> mefenamic acid. These results indicate that KCa channels possess at least one fenamatereceptor whose occupancy leads to channel opening.     

Amiodarone Inhibits Apamin-Sensitive Potassium Currents

Background

Apamin sensitive potassium current (I _KAS), carried by the type 2 small conductance Ca²⁺-activated potassium (SK2) channels, plays an important role in post-shock action potential duration (APD) shortening and recurrent spontaneous ventricular fibrillation (VF) in failing ventricles.

Objective

To test the hypothesis that amiodarone inhibits I _KAS in human embryonic kidney 293 (HEK-293) cells.

Methods

We used the patch-clamp technique to study I _KAS in HEK-293 cells transiently expressing human SK2 before and after amiodarone administration.

Results

Amiodarone inhibited I_KAS in a dose-dependent manner (IC₅₀, 2.67±0.25 µM with 1 µM intrapipette Ca²⁺). Maximal inhibition was observed with 50 µM amiodarone which inhibited 85.6±3.1% of I_KAS induced with 1 µM intrapipette Ca²⁺ (n = 3). I_KAS inhibition by amiodarone was not voltage-dependent, but was Ca²⁺-dependent: 30 µM amiodarone inhibited 81.5±1.9% of I _KAS induced with 1 µM Ca²⁺ (n = 4), and 16.4±4.9% with 250 nM Ca²⁺ (n = 5). Desethylamiodarone, a major metabolite of amiodarone, also exerts voltage-independent but Ca²⁺ dependent inhibition of I _KAS.

Conclusion

Both amiodarone and desethylamiodarone inhibit I _KAS at therapeutic concentrations. The inhibition is independent of time and voltage, but is dependent on the intracellular Ca²⁺ concentration. SK2 current inhibition may in part underlie amiodarone''s effects in preventing electrical storm in failing ventricles.     

Receptor Activated Ca2+ Release Is Inhibited by Boric Acid in Prostate Cancer Cells

Background

The global disparity in cancer incidence remains a major public health problem. We focused on prostate cancer since microscopic disease in men is common, but the incidence of clinical disease varies more than 100 fold worldwide. Ca²⁺ signaling is a central regulator of cell proliferation, but has received little attention in cancer prevention. We and others have reported a strong dose-dependent reduction in the incidence of prostate and lung cancer within populations exposed to boron (B) in drinking water and food; and in tumor and cell proliferation in animal and cell culture models.

Methods/Principal Findings

We examined the impact of B on Ca²⁺ stores using cancer and non-cancer human prostate cell lines, Ca²⁺ indicators Rhod-2 AM and Indo-1 AM and confocal microscopy. In DU-145 cells, inhibition of Ca²⁺ release was apparent following treatment with Ringers containing RyR agonists cADPR, 4CmC or caffeine and respective levels of BA (50 µM), (1, 10 µM) or (10, 20, 50,150 µM). Less aggressive LNCaP cancer cells required 20 µM BA and the non-tumor cell line PWR1E required 150 µM BA to significantly inhibit caffeine stimulated Ca²⁺ release. BA (10 µM) and the RyR antagonist dantroline (10 µM) were equivalent in their ability to inhibit ER Ca²⁺ loss. Flow cytometry and confocal microscopy analysis showed exposure of DU-145 cells to 50 µM BA for 1 hr decreased stored [Ca²⁺] by 32%.

Conclusion/Significance

We show B causes a dose dependent decrease of Ca²⁺ release from ryanodine receptor sensitive stores. This occurred at BA concentrations present in blood of geographically disparate populations. Our results suggest higher BA blood levels lower the risk of prostate cancer by reducing intracellular Ca²⁺ signals and storage.     

KCa3.1 channels are involved in the infiltrative behavior of glioblastoma in vivo

Glioblastoma multiforme (GBM) is a diffuse brain tumor characterized by high infiltration in the brain parenchyma rendering the tumor difficult to eradicate by neurosurgery. Efforts to identify molecular targets involved in the invasive behavior of GBM suggested ion channel inhibition as a promising therapeutic approach. To determine if the Ca²⁺-dependent K⁺ channel KCa3.1 could represent a key element for GBM brain infiltration, human GL-15 cells were xenografted into the brain of SCID mice that were then treated with the specific KCa3.1 blocker TRAM-34 (1-((2-chlorophenyl) (diphenyl)methyl)-1H-pyrazole). After 5 weeks of treatment, immunofluorescence analyses of cerebral slices revealed reduced tumor infiltration and astrogliosis surrounding the tumor, compared with untreated mice. Significant reduction of tumor infiltration was also observed in the brain of mice transplanted with KCa3.1-silenced GL-15 cells, indicating a direct effect of TRAM-34 on GBM-expressed KCa3.1 channels. As KCa3.1 channels are also expressed on microglia, we investigated the effects of TRAM-34 on microglia activation in GL-15 transplanted mice and found a reduction of CD68 staining in treated mice. Similar results were observed in vitro where TRAM-34 reduced both phagocytosis and chemotactic activity of primary microglia exposed to GBM-conditioned medium. Taken together, these results indicate that KCa3.1 activity has an important role in GBM invasiveness in vivo and that its inhibition directly affects glioma cell migration and reduces astrocytosis and microglia activation in response to tumor-released factors. KCa3.1 channel inhibition therefore constitutes a potential novel therapeutic approach to reduce GBM spreading into the surrounding tissue.     

Low Voltage Activation of KCa1.1 Current by Cav3-KCa1.1 Complexes

Calcium-activated potassium channels of the KCa1.1 class are known to regulate repolarization of action potential discharge through a molecular association with high voltage-activated calcium channels. The current study examined the potential for low voltage-activated Cav3 (T-type) calcium channels to interact with KCa1.1 when expressed in tsA-201 cells and in rat medial vestibular neurons (MVN) in vitro. Expression of the channel α-subunits alone in tsA-201 cells was sufficient to enable Cav3 activation of KCa1.1 current. Cav3 calcium influx induced a 50 mV negative shift in KCa1.1 voltage for activation, an interaction that was blocked by Cav3 or KCa1.1 channel blockers, or high internal EGTA. Cav3 and KCa1.1 channels coimmunoprecipitated from lysates of either tsA-201 cells or rat brain, with Cav3 channels associating with the transmembrane S0 segment of the KCa1.1 N-terminus. KCa1.1 channel activation was closely aligned with Cav3 calcium conductance in that KCa1.1 current shared the same low voltage dependence of Cav3 activation, and was blocked by voltage-dependent inactivation of Cav3 channels or by coexpressing a non calcium-conducting Cav3 channel pore mutant. The Cav3-KCa1.1 interaction was found to function highly effectively in a subset of MVN neurons by activating near –50 mV to contribute to spike repolarization and gain of firing. Modelling data indicate that multiple neighboring Cav3-KCa1.1 complexes must act cooperatively to raise calcium to sufficiently high levels to permit KCa1.1 activation. Together the results identify a novel Cav3-KCa1.1 signaling complex where Cav3-mediated calcium entry enables KCa1.1 activation over a wide range of membrane potentials according to the unique voltage profile of Cav3 calcium channels, greatly extending the roles for KCa1.1 potassium channels in controlling membrane excitability.     

Caffeic acid phenethyl ester and its amide analogue are potent inhibitors of leukotriene biosynthesis in human polymorphonuclear leukocytes

Background

5-lipoxygenase (5-LO) catalyses the transformation of arachidonic acid (AA) into leukotrienes (LTs), which are important lipid mediators of inflammation. LTs have been directly implicated in inflammatory diseases like asthma, atherosclerosis and rheumatoid arthritis; therefore inhibition of LT biosynthesis is a strategy for the treatment of these chronic diseases.

Methodology/Principal Findings

Analogues of caffeic acid, including the naturally-occurring caffeic acid phenethyl ester (CAPE), were synthesized and evaluated for their capacity to inhibit 5-LO and LTs biosynthesis in human polymorphonuclear leukocytes (PMNL) and whole blood. Anti-free radical and anti-oxidant activities of the compounds were also measured. Caffeic acid did not inhibit 5-LO activity or LT biosynthesis at concentrations up to 10 µM. CAPE inhibited 5-LO activity (IC₅₀ 0.13 µM, 95% CI 0.08–0.23 µM) more effectively than the clinically-approved 5-LO inhibitor zileuton (IC₅₀ 3.5 µM, 95% CI 2.3–5.4 µM). CAPE was also more effective than zileuton for the inhibition of LT biosynthesis in PMNL but the compounds were equipotent in whole blood. The activity of the amide analogue of CAPE was similar to that of zileuton. Inhibition of LT biosynthesis by CAPE was the result of the inhibition of 5-LO and of AA release. Caffeic acid, CAPE and its amide analog were free radical scavengers and antioxidants with IC₅₀ values in the low µM range; however, the phenethyl moiety of CAPE was required for effective inhibition of 5-LO and LT biosynthesis.

Conclusions

CAPE is a potent LT biosynthesis inhibitor that blocks 5-LO activity and AA release. The CAPE structure can be used as a framework for the rational design of stable and potent inhibitors of LT biosynthesis.     

Pulmonary Hypertension in Wild Type Mice and Animals with Genetic Deficit in KCa2.3 and KCa3.1 Channels

Objective

In vascular biology, endothelial K_Ca2.3 and K_Ca3.1 channels contribute to arterial blood pressure regulation by producing membrane hyperpolarization and smooth muscle relaxation. The role of K_Ca2.3 and K_Ca3.1 channels in the pulmonary circulation is not fully established. Using mice with genetically encoded deficit of K_Ca2.3 and K_Ca3.1 channels, this study investigated the effect of loss of the channels in hypoxia-induced pulmonary hypertension.

Approach and Result

Male wild type and K_Ca3.1^−/−/K_Ca2.3^T/T(+DOX) mice were exposed to chronic hypoxia for four weeks to induce pulmonary hypertension. The degree of pulmonary hypertension was evaluated by right ventricular pressure and assessment of right ventricular hypertrophy. Segments of pulmonary arteries were mounted in a wire myograph for functional studies and morphometric studies were performed on lung sections. Chronic hypoxia induced pulmonary hypertension, right ventricular hypertrophy, increased lung weight, and increased hematocrit levels in either genotype. The K_Ca3.1^−/−/K_Ca2.3^T/T(+DOX) mice developed structural alterations in the heart with increased right ventricular wall thickness as well as in pulmonary vessels with increased lumen size in partially- and fully-muscularized vessels and decreased wall area, not seen in wild type mice. Exposure to chronic hypoxia up-regulated the gene expression of the K_Ca2.3 channel by twofold in wild type mice and increased by 2.5-fold the relaxation evoked by the K_Ca2.3 and K_Ca3.1 channel activator NS309, whereas the acetylcholine-induced relaxation - sensitive to the combination of K_Ca2.3 and K_Ca3.1 channel blockers, apamin and charybdotoxin - was reduced by 2.5-fold in chronic hypoxic mice of either genotype.

Conclusion

Despite the deficits of the K_Ca2.3 and K_Ca3.1 channels failed to change hypoxia-induced pulmonary hypertension, the up-regulation of K_Ca2.3-gene expression and increased NS309-induced relaxation in wild-type mice point to a novel mechanism to counteract pulmonary hypertension and to a potential therapeutic utility of K_Ca2.3/K_Ca3.1 activators for the treatment of pulmonary hypertension.     

The Ca(2+)-activated K(+) channel KCa3.1 compartmentalizes in the immunological synapse of human T lymphocytes

T cell receptor engagement results in the reorganization of intracellular and membrane proteins at the T cell-antigen presenting cell interface forming the immunological synapse (IS), an event required for Ca²⁺ influx. KCa3.1 channels modulate Ca²⁺ signaling in activated T cells by regulating the membrane potential. Nothing is known regarding KCa3.1 membrane distribution during T cell activation. Herein, we determined whether KCa3.1 translocates to the IS in human T cells using YFP-tagged KCa3.1 channels. These channels showed electrophysiological and pharmacological properties identical to wild-type channels. IS formation was induced by either anti-CD3/CD28 antibody-coated beads for fixed microscopy experiments or Epstein-Barr virus-infected B cells for fixed and live cell microscopy. In fixed microscopy experiments, T cells were also immunolabeled for F-actin or CD3, which served as IS formation markers. The distribution of KCa3.1 was determined with confocal and fluorescence microscopy. We found that, upon T cell activation, KCa3.1 channels localize with F-actin and CD3 to the IS but remain evenly distributed on the cell membrane when no stimulus is provided. Detailed imaging experiments indicated that KCa3.1 channels are recruited in the IS shortly after antigen presentation and are maintained there for at least 15–30 min. Interestingly, pretreatment of activated T cells with the specific KCa3.1 blocker TRAM-34 blocked Ca²⁺ influx, but channel redistribution to the IS was not prevented. These results indicate that KCa3.1 channels are a part of the signaling complex that forms at the IS upon antigen presentation. T cell activation; ion channels; membrane distribution     

Acute Simvastatin Inhibits KATP Channels of Porcine Coronary Artery Myocytes

Background

Statins (3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors) consumption provides beneficial effects on cardiovascular systems. However, effects of statins on vascular K_ATP channel gatings are unknown.

Methods

Pig left anterior descending coronary artery and human left internal mammary artery were isolated and endothelium-denuded for tension measurements and Western immunoblots. Enzymatically-dissociated/cultured arterial myocytes were used for patch-clamp electrophysiological studies and for [Ca²⁺]_i, [ATP]_i and [glucose]_o uptake measurements.

Results

The cromakalim (10 nM to 10 µM)- and pinacidil (10 nM to 10 µM)-induced concentration-dependent relaxation of porcine coronary artery was inhibited by simvastatin (3 and 10 µM). Simvastatin (1, 3 and 10 µM) suppressed (in okadaic acid (10 nM)-sensitive manner) cromakalim (10 µM)- and pinacidil (10 µM)-mediated opening of whole-cell K_ATP channels of arterial myocytes. Simvastatin (10 µM) and AICAR (1 mM) elicited a time-dependent, compound C (1 µM)-sensitive [³H]-2-deoxy-glucose uptake and an increase in [ATP]_i levels. A time (2–30 min)- and concentration (0.1–10 µM)-dependent increase by simvastatin of p-AMPKα-Thr¹⁷² and p-PP2A-Tyr³⁰⁷ expression was observed. The enhanced p-AMPKα-Thr¹⁷² expression was inhibited by compound C, ryanodine (100 µM) and KN93 (10 µM). Simvastatin-induced p-PP2A-Tyr³⁰⁷ expression was suppressed by okadaic acid, compound C, ryanodine, KN93, phloridzin (1 mM), ouabain (10 µM), and in [glucose]_o-free or [Na⁺]_o-free conditions.

Conclusions

Simvastatin causes ryanodine-sensitive Ca²⁺ release which is important for AMPKα-Thr¹⁷² phosphorylation via Ca²⁺/CaMK II. AMPKα-Thr¹⁷² phosphorylation causes [glucose]_o uptake (and an [ATP]_i increase), closure of K_ATP channels, and phosphorylation of AMPKα-Thr¹⁷² and PP2A-Tyr³⁰⁷ resulted. Phosphorylation of PP2A-Tyr³⁰⁷ occurs at a site downstream of AMPKα-Thr¹⁷² phosphorylation.     

Intravitreal Docosahexaenoic Acid in a Rabbit Model: Preclinical Safety Assessment

Purpose

The purpose of the present study was to evaluate the retinal toxicity of a single dose of intravitreal docosahexaenoic acid (DHA) in rabbit eyes over a short-term period.

Methods

Sixteen New Zealand albino rabbits were selected for this pre-clinical study. Six concentrations of DHA (Brudy Laboratories, Barcelona, Spain) were prepared: 10 mg/50 µl, 5 mg/50 µl, 2’5 mg/50 µl, 50 µg/50 µl, 25 µg/50 µl, and 5 µg/50 µl. Each concentration was injected intravitreally in the right eye of two rabbits. As a control, the vehicle solution was injected in one eye of four animals. Retinal safety was studied by slit-lamp examination, and electroretinography. All the rabbits were euthanized one week after the intravitreal injection of DHA and the eyeballs were processed to morphologic and morphometric histological examination by light microscopy. At the same time aqueous and vitreous humor samples were taken to quantify the concentration of omega-3 acids by gas chromatography. Statistical analysis was performed by SPSS 21.0.

Results

Slit-lamp examination revealed an important inflammatory reaction on the anterior chamber of the rabbits injected with the higher concentrations of DHA (10 mg/50 µl, 5 mg/50 µl, 2''5 mg/50 µ) Lower concentrations showed no inflammation. Electroretinography and histological studies showed no significant difference between control and DHA-injected groups except for the group injected with 50 µg/50 µl.

Conclusions

Our results indicate that administration of intravitreal DHA is safe in the albino rabbit model up to the maximum tolerated dose of 25 µg/50 µl. Further studies should be performed in order to evaluate the effect of intravitreal injection of DHA as a treatment, alone or in combination, of different retinal diseases.     

Role of S3 and S4 transmembrane domain charged amino acids in channel biogenesis and gating of KCa2.3 and KCa3.1

The role of positively charged arginines in the fourth transmembrane domain (S4) and a single negatively charged amino acid in the third transmembrane domain (S3) on channel biogenesis and gating of voltage-gated K(+) channels (Kv) has been well established. Both intermediate (KCa3.1) and small (KCa2.x) conductance, Ca(2+)-activated K(+) channels have two conserved arginines in S4 and a single conserved glutamic acid in S3, although these channels are voltage-independent. We demonstrate that mutation of any of these charged amino acids in KCa3.1 or KCa2.3 to alanine, glutamine, or charge reversal mutations results in a rapid degradation (<30 min) of total protein, confirming the critical role of these amino acids in channel biogenesis. Mutation of the S4 arginine closest to the cytosolic side of KCa3.1 to histidine resulted in expression at the cell surface. Excised patch clamp experiments revealed that this Arg/His mutation had a dramatically reduced open probability (P(o)), relative to wild type channels. Additionally, we demonstrate, using a combination of short hairpin RNA, dominant negative, and co-immunoprecipitation studies, that both KCa3.1 and KCa2.3 are translocated out of the endoplasmic reticulum associated with Derlin-1. These misfolded channels are poly-ubiquitylated, recognized by p97, and targeted for proteasomal degradation. Our results suggest that S3 and S4 charged amino acids play an evolutionarily conserved role in the biogenesis and gating of KCa channels. Furthermore, these improperly folded K(+) channels are translocated out of the endoplasmic reticulum in a Derlin-1- and p97-dependent fashion, poly-ubiquitylated, and targeted for proteasomal degradation.     

The Ca2+-Activated K+ Channel KCa3.1 as a Potential New Target for the Prevention of Allograft Vasculopathy

Allograft vasculopathy (AV) remains one of the major challenges to the long-term functioning of solid organ transplants. Although its exact pathogenesis remains unclear, AV is characterized by both fibromuscular proliferation and infiltration of CD4⁺ memory T cells. We here tested whether two experimental immunosuppressants targeting K⁺ channels might be useful for preventing AV. PAP-1 inhibits the voltage-gated Kv1.3 channel, which is overexpressed on CCR7⁻ memory T cells and we therefore hypothesize that it should suppress the memory T cell component of AV. Based on its previous efficacy in restenosis and kidney fibrosis we expected that the KCa3.1 blocker TRAM-34 would primarily affect smooth muscle and fibroblast proliferation and thus reduce intimal hyperplasia. Using immunohistochemistry we demonstrated the presence of Kv1.3 on infiltrating T cells and of KCa3.1 on lymphocytes as well as on proliferating neointimal smooth muscle cells in human vasculopathy samples and in a rat aorta transplant model developing chronic AV. Treatment of PVG rats receiving orthotopically transplanted aortas from ACI rats with TRAM-34 dose-dependently reduced aortic luminal occlusion, intimal hyperplasia, mononuclear cell infiltration and collagen deposition 120 days after transplantation. The Kv1.3 blocker PAP-1 in contrast did not reduce intima hyperplasia despite drastically reducing plasma IFN-γ levels and inhibiting lymphocyte infiltration. Our findings suggest that KCa3.1 channels play an important role in the pathogenesis of chronic AV and constitute an attractive target for the prevention of arteriopathy.     

Investigating CFTR and KCa3.1 Protein/Protein Interactions

In epithelia, Cl^- channels play a prominent role in fluid and electrolyte transport. Of particular importance is the cAMP-dependent cystic fibrosis transmembrane conductance regulator Cl^- channel (CFTR) with mutations of the CFTR encoding gene causing cystic fibrosis. The bulk transepithelial transport of Cl^- ions and electrolytes needs however to be coupled to an increase in K⁺ conductance in order to recycle K⁺ and maintain an electrical driving force for anion exit across the apical membrane. In several epithelia, this K⁺ efflux is ensured by K⁺ channels, including KCa3.1, which is expressed at both the apical and basolateral membranes. We show here for the first time that CFTR and KCa3.1 can physically interact. We first performed a two-hybrid screen to identify which KCa3.1 cytosolic domains might mediate an interaction with CFTR. Our results showed that both the N-terminal fragment M1-M40 of KCa3.1 and part of the KCa3.1 calmodulin binding domain (residues L345-A400) interact with the NBD2 segment (G1237-Y1420) and C- region of CFTR (residues T1387-L1480), respectively. An association of CFTR and F508del-CFTR with KCa3.1 was further confirmed in co-immunoprecipitation experiments demonstrating the formation of immunoprecipitable CFTR/KCa3.1 complexes in CFBE cells. Co-expression of KCa3.1 and CFTR in HEK cells did not impact CFTR expression at the cell surface, and KCa3.1 trafficking appeared independent of CFTR stimulation. Finally, evidence is presented through cross-correlation spectroscopy measurements that KCa3.1 and CFTR colocalize at the plasma membrane and that KCa3.1 channels tend to aggregate consequent to an enhanced interaction with CFTR channels at the plasma membrane following an increase in intracellular Ca²⁺ concentration. Altogether, these results suggest 1) that the physical interaction KCa3.1/CFTR can occur early during the biogenesis of both proteins and 2) that KCa3.1 and CFTR form a dynamic complex, the formation of which depends on internal Ca²⁺.