Dendritic cell subsets display different functional role in regulating immune response and lead to various outcomes including Th1 versus Th2 or regulatory versus immunologic response. Administration of Flt3-Ligand prevents and reverses allergic airway inflammation and airway hyperresponsiveness in a mouse model. However, the underlying mechanisms are unclear.
We characterized and examined the role of lung dendritic cell subsets in the therapeutic effect of Flt3-Ligand.
Dendritic cells were isolated from the lungs of OVA-sensitized and challenged mice treated with rhFlt3-Ligand. Two populations of CD11c+ cells labeled with fluorochrome-conjugated antibodies were sorted. The ability of the purified cells to stimulate T cell proliferation and cytokine secretion pattern by different DC subsets was examined. Also, dendritic cells were adoptively transferred in mice to examine their effect on pulmonary function.
Two dendritic cell populations, CD11chighCD11blow and CD11clowCD11bhigh, were identified in the lungs of naïve and OVA-sensitized and challenged mice with and without treatment with Flt3-Ligand. The expression levels of CD8α, B220, CD19, F4/80, MHC II, CCR7, CD40, PDL1, PDL2, CD80, and CD86 were distinctly different between the two DC populations, which supports the notion that, CD11chighCD11blow and CD11clowCD11bhigh dendritic cells, potentially have regulatory and immunogenic properties, respectively. Administration of Flt3-Ligand increased the dendritic cells with regulatory potential in the lungs of antigen-sensitized mice, and CD11chighCD11blow dendritic cells acquired a maximum degree of regulatory capacity after Flt3-Ligand treatment.
These data suggest that Flt3-Ligand reverses airway hyperresponsiveness by regulating the function of lung dendritic cells in a mouse model of allergic airway inflammation.
Flt3-Ligand could be a potential immunomodulator in the treatment of established asthma.
Adoptive transfer; Airway hyperresponsiveness; Asthma; Dendritic cells; Flt3-Ligand; Mouse model; T cell Proliferation; Regulatory dendritic cells
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.
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.
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.
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.
Mast cell recruitment and activation are critical for the initiation and progression of inflammation and fibrosis. Mast cells infiltrate specific structures in many diseased tissues such as the airway smooth muscle (ASM) in asthma. This microlocalisation of mast cells is likely to be key to disease pathogenesis. Human lung mast cells (HLMC) express the Ca2+ activated K+ channel KCa3.1 which modulates mediator release, and is proposed to facilitate the retraction of the cell body during migration of several cell types. A study was undertaken to test the hypothesis that blockade of KCa3.1 would attenuate HLMC proliferation and migration.
HLMC were isolated and purified from lung material resected for bronchial carcinoma. HLMC proliferation was assessed by cell counts at various time points following drug exposure. HLMC chemotaxis was assayed using standard Transwell chambers (8 μm pore size). Ion currents were measured using the single cell patch clamp technique.
KCa3.1 blockade with triarylmethane‐34 (TRAM‐34) did not inhibit HLMC proliferation and clotrimazole had cytotoxic effects. In contrast, HLMC migration towards the chemokine CXCL10, the chemoattractant stem cell factor, and the supernatants from tumour necrosis factor α stimulated asthmatic ASM was markedly inhibited with both the non‐selective KCa3.1 blocker charybdotoxin and the highly specific KCa3.1 blocker TRAM‐34 in a dose dependent manner. Although KCa3.1 blockade inhibits HLMC migration, KCa3.1 is not opened by the chemotactic stimulus, suggesting that it must be involved downstream of the initial receptor‐ligand interactions.
Since modulation of KCa3.1 can inhibit HLMC chemotaxis to diverse chemoattractants, the use of KCa3.1 blockers such as TRAM‐34 could provide new therapeutic strategies for mast cell mediated diseases such as asthma.
mast cell; migration; asthma; CXCL10; KCa3.1
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.
In the present study we evaluated the expression of the intermediate conductance calcium-activated potassium (KCa3.1) channel in human glioblastoma stem-like cells (CSCs) and investigated its role in cell motility. While the KCa3.1 channel is not expressed in neuronal- and glial-derived tissues of healthy individuals, both the KCa3.1 mRNA and protein are present in the glioblastoma tumor population, and are significantly enhanced in CSCs derived from both established cell line U87MG and a primary cell line, FCN9. Consistent with these data, voltage-independent and TRAM-34 sensitive potassium currents imputable to the KCa3.1 channel were recorded in the murine GL261 cell line and several primary human glioblastoma cells lines. Moreover, a significantly higher KCa3.1 current was recorded in U87MG-CD133 positive cells as compared to the U87MG-CD133 negative subpopulation. Further, we found that the tumor cell motility is strongly associated with KCa3.1 channel expression. Blockade of the KCa3.1 channel with the specific inhibitor TRAM-34 has in fact a greater impact on the motility of CSCs (reduction of 75%), which express a high level of KCa3.1 channel, than on the FCN9 parental population (reduction of 32%), where the KCa3.1 channel is expressed at lower level. Similar results were also observed with the CSCs derived from U87MG. Because invasion of surrounding tissues is one of the main causes of treatment failure in glioblastoma, these findings can be relevant for future development of novel cancer therapeutic drugs.
Natural killer (NK) cells are large granular lymphocytes that participate in both innate and adaptive immune responses against tumors and pathogens. They are also involved in other conditions, including organ rejection, graft-versus-host disease, recurrent spontaneous abortions, and autoimmune diseases such as multiple sclerosis. We demonstrate that human NK cells express the potassium channels Kv1.3 and KCa3.1. Expression of these channels does not vary with expression levels of maturation markers but varies between adherent and non-adherent NK cell subpopulations. Upon activation by mitogens or tumor cells, adherent NK (A-NK) cells preferentially up-regulate KCa3.1 and non-adherent (NA-NK) cells preferentially up-regulate Kv1.3. Consistent with this different phenotype, A-NK and NA-NK do not display the same sensitivity to the selective KCa3.1 blockers TRAM-34 and NS6180 and to the selective Kv1.3 blockers ShK-186 and PAP-1 in functional assays. Kv1.3 block inhibits the proliferation and degranulation of NA-NK cells with minimal effects on A-NK cells. In contrast, blocking KCa3.1 increases the degranulation and cytotoxicity of A-NK cells, but not of NA-NK cells. TRAM-34, however, does not affect their ability to form conjugates with target tumor cells, to migrate, or to express chemokine receptors. TRAM-34 and NS6180 also increase the proliferation of both A-NK and NA-NK cells. This results in a TRAM-34-induced increased ability of A-NK cells to reduce in vivo tumor growth. Taken together, our results suggest that targeting KCa3.1 on NK cells with selective blockers may be beneficial in cancer immunotherapy.
Identifying the factors responsible for relative glucocorticosteroid (GC) resistance present in patients with severe asthma and finding tools to reverse it are of paramount importance. In asthma there is in vivo evidence of GC-resistant pathways in airway smooth muscle (ASM) bundles which can be modelled in vitro by exposing cultured ASM cells to TNFα/IFNγ. This drives GC insensitivity via protein phosphatase-5 (PP5)-dependent impairment of GC receptor (GR) phosphorylation. Here, we investigated whether KCa3.1 ion channels modulate the activity of GC-resistant pathways using our ASM model of GC insensitivity. Immunohistochemical staining of endobronchial biopsies revealed that KCa3.1 channels are localized to the plasma membrane and nucleus of ASM in both healthy controls and asthmatic patients, irrespective of disease severity. Western blot assays and immunofluorescence staining confirmed the nuclear localisation of KCa3.1 channels in ASM cells. The functional importance of KCa3.1 channels in the regulation of GC-resistant chemokines induced by TNFα/IFNγ was assessed using complementary inhibitory strategies including KCa3.1 blockers (TRAM-34 and ICA-17043) or KCa3.1-specific shRNA delivered by adenoviruses. KCa3.1 channel blockade led to a significant reduction of fluticasone-resistant CX3CL1, CCL5 and CCL11 gene and protein expression. KCa3.1 channel blockade also restored fluticasone-induced GRα phosphorylation at ser211 and transactivation properties via the suppression of cytokine-induced PP5 expression. The effect of KCa3.1 blockade was evident in ASM cells from both healthy controls and asthmatic subjects. In summary KCa3.1 channels contribute to the regulation of GC-resistant inflammatory pathways in ASM cells: blocking KCa3.1 channels may enhance corticosteroid activity in severe asthma.
Corticosteroid insensitivity; chemokines; GR phosphorylation; TNFα; transactivation; transrepression; KCa3.1; severe asthma; airway smooth muscle; transcription factors
Atherosclerosis remains a major cause of death in the developed world despite the success of therapies that lower cholesterol and BP. The intermediate-conductance calcium-activated potassium channel KCa3.1 is expressed in multiple cell types implicated in atherogenesis, and pharmacological blockade of this channel inhibits VSMC and lymphocyte activation in rats and mice. We found that coronary vessels from patients with coronary artery disease expressed elevated levels of KCa3.1. In Apoe–/– mice, a genetic model of atherosclerosis, KCa3.1 expression was elevated in the VSMCs, macrophages, and T lymphocytes that infiltrated atherosclerotic lesions. Selective pharmacological blockade and gene silencing of KCa3.1 suppressed proliferation, migration, and oxidative stress of human VSMCs. Furthermore, VSMC proliferation and macrophage activation were reduced in KCa3.1–/– mice. In vivo therapy with 2 KCa3.1 blockers, TRAM-34 and clotrimazole, significantly reduced the development of atherosclerosis in aortas of Apoe–/– mice by suppressing VSMC proliferation and migration into plaques, decreasing infiltration of plaques by macrophages and T lymphocytes, and reducing oxidative stress. Therapeutic concentrations of TRAM-34 in mice caused no discernible toxicity after repeated dosing and did not compromise the immune response to influenza virus. These data suggest that KCa3.1 blockers represent a promising therapeutic strategy for atherosclerosis.
Idiopathic pulmonary fibrosis is a common and invariably fatal disease with limited therapeutic options. Ca2+-activated KCa3.1 potassium channels play a key role in promoting TGFβ1 and bFGF-dependent profibrotic responses in human lung myofibroblasts (HLMFs). We hypothesised that KCa3.1 channel-dependent cell processes regulate HLMF αSMA expression via Smad2/3 signalling pathways.
In this study we have compared the phenotype of HLMFs derived from non-fibrotic healthy control lungs (NFC) with cells derived from IPF lungs. HLMFs grown in vitro were examined for αSMA expression by immunofluorescence (IF), RT-PCR and flow cytommetry. Basal Smad2/3 signalling was examined by RT-PCR, western blot and immunofluorescence. Two specific and distinct KCa3.1 blockers (TRAM-34 200 nM and ICA-17043 [Senicapoc] 100 nM) were used to determine their effects on HLMF differentiation and the Smad2/3 signalling pathways.
IPF-derived HLMFs demonstrated increased constitutive expression of both α-smooth muscle actin (αSMA) and actin stress fibres, indicative of greater myofibroblast differentiation. This was associated with increased constitutive Smad2/3 mRNA and protein expression, and increased Smad2/3 nuclear localisation. The increased Smad2/3 nuclear localisation was inhibited by removing extracellular Ca2+ or blocking KCa3.1 ion channels with selective KCa3.1 blockers (TRAM-34, ICA-17043). This was accompanied by de-differentiation of IPF-derived HLMFs towards a quiescent fibroblast phenotype as demonstrated by reduced αSMA expression and reduced actin stress fibre formation.
Taken together, these data suggest that Ca2+- and KCa3.1-dependent processes facilitate “constitutive” Smad2/3 signalling in IPF-derived fibroblasts, and thus promote fibroblast to myofibroblast differentiation. Importantly, inhibiting KCa3.1 channels reverses this process. Targeting KCa3.1 may therefore provide a novel and effective approach for the treatment of IPF and there is the potential for the rapid translation of KCa3.1-directed therapy to the clinic.
Idiopathic pulmonary fibrosis (IPF); Fibrosis; Lung; Myofibroblast; KCa3.1; Ion channel; Differentiation; Smad 2; Smad 3
The calcium-activated potassium channel KCa3.1 is critically involved in T cell activation, as well as in the proliferation of smooth muscle cells and fibroblasts. We sought to investigate whether KCa3.1 contributes to the pathogenesis of obliterative airway disease (OAD) and whether knockout or pharmacological blockade would prevent the development of OAD.
Tracheas from CBA donors were heterotopically transplanted into the omentum of C57Bl/6J wild-type or KCa3.1−/− mice. C57Bl/6J recipients were either left untreated or received the KCa3.1 blocker TRAM-34 (120mg/kg/d). Histopathology and immunological assays were performed on postoperative days (POD) 5 or 28.
Subepithelial T cell and macrophage infiltration on POD 5, as seen in untreated allografts, was significantly reduced in the KCa3.1−/− and TRAM-34 groups. Also, systemic Th1 activation was significantly, and Th2 mildly reduced by KCa3.1 knockout or blockade. After 28 days, luminal obliteration of tracheal allografts was reduced from 89±21% in untreated recipients to 53±26% (p=0.010) and 59±33% (p=0.032) in KCa3.1−/− and TRAM-34-treated animals, respectively. The airway epithelium was mostly preserved in syngeneic grafts, mostly destroyed in the KCa3.1−/− and TRAM-34 groups, and absent in untreated allografts. Allografts triggered an antibody response in untreated recipients, which was significantly reduced in KCa3.1−/− animals. KCa3.1 was detected in T cells, airway epithelial cells and myofibroblasts. TRAM-34 dose-dependently suppressed proliferation of wild-type C57B/6J splenocytes, but did not show any effect on KCa3.1−/− splenocytes.
Our findings suggest that KCa3.1 channels are involved in the pathogenesis of OAD and that KCa3.1 blockade holds promise to reduce OAD development.
KCa3.1; Obliterative airway disease; TRAM-34; Chronic rejection; Heterotopic tracheal transplantation
The intermediate-conductance Ca2+-activated K+ channel KCa3.1 (also known as KCNN4, IK1, or the Gárdos channel) plays an important role in the activation of T and B cells, mast cells, macrophages, and microglia by regulating membrane potential, cellular volume, and calcium signaling. KCa3.1 is further involved in the proliferation of dedifferentiated vascular smooth muscle cells and fibroblast and endothelium-derived hyperpolarization responses in the vascular endothelium. Accordingly, KCa3.1 inhibitors are therapeutically interesting as immunosuppressants and for the treatment of a wide range of fibroproliferative disorders, whereas KCa3.1 activators constitute a potential new class of endothelial function preserving antihypertensives. Here, we report the development of QPatch assays for both KCa3.1 inhibitors and activators. During assay optimization, the Ca2+ sensitivity of KCa3.1 was studied using varying intracellular Ca2+ concentrations. A free Ca2+ concentration of 1 μM was chosen to optimally test inhibitors. To identify activators, which generally act as positive gating modulators, a lower Ca2+ concentration (∼200 nM) was used. The QPatch results were benchmarked against manual patch-clamp electrophysiology by determining the potency of several commonly used KCa3.1 inhibitors (TRAM-34, NS6180, ChTX) and activators (EBIO, riluzole, SKA-31). Collectively, our results demonstrate that the QPatch provides a comparable but much faster approach to study compound interactions with KCa3.1 channels in a robust and reliable assay.
Key features of asthma include bronchial hyperresponsiveness (BHR), eosinophilic airway inflammation, and bronchial remodeling, characterized by subepithelial collagen deposition, airway fibrosis, and increased bronchial smooth muscle (BSM) mass. The calcium-activated K+ channel KCa3.1 is expressed by many cells implicated in the pathogenesis of asthma, and is involved in both inflammatory and remodeling responses in a number of tissues. The specific KCa3.1 blocker 5-[(2-chlorophenyl)(diphenyl)methyl]-1H-pyrazole (TRAM-34) attenuates BSM cell proliferation, and both mast cell and fibrocyte recruitment in vitro. We aimed to examine the effects of KCa3.1 blockade on BSM remodeling, airway inflammation, and BHR in a murine model of chronic asthma. BALB/c mice were sensitized with intraperitoneal ovalbumin (OVA) on Days 0 and 14, and then challenged with intranasal OVA during Days 14–75. OVA-sensitized/challenged mice received TRAM-34 (120 mg/kg/day, subcutaneous) from Days −7 to 75 (combined treatment), Days −7 to 20 (preventive treatment), or Days 21 to 75 (curative treatment). Untreated mice received daily injections of vehicle (n = 8 per group). Bronchial remodeling was assessed by histological and immunohistochemical analyses. Inflammation was evaluated using bronchoalveolar lavage and flow cytometry. We also determined BHR in both conscious and anesthetized mice via plethysmography. We demonstrated that curative treatment with TRAM-34 abolishes BSM remodeling and subbasement collagen deposition, and attenuates airway eosinophilia. Although curative treatment alone did not significantly reduce BHR, the combined treatment attenuated nonspecific BHR to methacholine. This study indicates that KCa3.1 blockade could provide a new therapeutic strategy in asthma.
asthma; KCa3.1; ion channel; remodeling; smooth muscle
The blood-brain tumor barrier (BTB) impedes the delivery of therapeutic agents to brain tumors. While adequate delivery of drugs occurs in systemic tumors, the BTB limits delivery of anti-tumor agents into brain metastases.
In this study, we examined the function and regulation of calcium-activated potassium (KCa) channels in a rat metastatic brain tumor model. We showed that intravenous infusion of NS1619, a KCa channel agonist, and bradykinin selectively enhanced BTB permeability in brain tumors, but not in normal brain. Iberiotoxin, a KCa channel antagonist, significantly attenuated NS1619-induced BTB permeability increase. We found KCa channels and bradykinin type 2 receptors (B2R) expressed in cultured human metastatic brain tumor cells (CRL-5904, non-small cell lung cancer, metastasized to brain), human brain microvessel endothelial cells (HBMEC) and human lung cancer brain metastasis tissues. Potentiometric assays demonstrated the activity of KCa channels in metastatic brain tumor cells and HBMEC. Furthermore, we detected higher expression of KCa channels in the metastatic brain tumor tissue and tumor capillary endothelia as compared to normal brain tissue. Co-culture of metastatic brain tumor cells and brain microvessel endothelial cells showed an upregulation of KCa channels, which may contribute to the overexpression of KCa channels in tumor microvessels and selectivity of BTB opening.
These findings suggest that KCa channels in metastatic brain tumors may serve as an effective target for biochemical modulation of BTB permeability to enhance selective delivery of chemotherapeutic drugs to metastatic brain tumors.
L-type, voltage-dependent calcium (Ca2+) channels,
ryanodine-sensitive Ca2+ release (RyR) channels, and
large-conductance Ca2+-activated potassium (KCa) channels
comprise a functional unit that regulates smooth muscle contractility. Here, we
investigated whether genetic ablation of caveolin-1 (cav-1), a caveolae protein,
alters Ca2+ spark to KCa channel coupling and
Ca2+ spark regulation by voltage-dependent Ca2+ channels
in murine cerebral artery smooth muscle cells. Caveolae were abundant in the
sarcolemma of control (cav-+/+) cells but were not observed in
cav-1-deficient (cav-1−/−) cells.
Ca2+ spark and transient KCa current frequency were
approximately twofold higher in cav-1−/− than
in cav-1+/+ cells. Although voltage-dependent Ca2+ current
density was similar in cav-1+/+ and
cav-1−/− cells, diltiazem and
Cd2+, voltage-dependent Ca2+ channel blockers, reduced
transient KCa current frequency to ∼55% of control in
cav-1+/+ cells but did not alter transient KCa current
frequency in cav-1−/− cells. Furthermore,
although KCa channel density was elevated in
cav-1−/− cells, transient KCa
current amplitude was similar to that in cav-1+/+ cells. Higher
Ca2+ spark frequency in cav-1−/−
cells was not due to elevated intracellular Ca2+ concentration,
sarcoplasmic reticulum Ca2+ load, or nitric oxide synthase activity.
Similarly, Ca2+ spark amplitude and spread, the percentage of
Ca2+ sparks that activated a transient KCa current,
the amplitude relationship between sparks and transient KCa currents,
and KCa channel conductance and apparent Ca2+ sensitivity
were similar in cav-1+/+ and
cav-1−/− cells. In summary, cav-1 ablation
elevates Ca2+ spark and transient KCa current frequency,
attenuates the coupling relationship between voltage-dependent Ca2+
channels and RyR channels that generate Ca2+ sparks, and elevates
KCa channel density but does not alter transient KCa
current activation by Ca2+ sparks. These findings indicate that cav-1
is required for physiological Ca2+ spark and transient KCa
current regulation in cerebral artery smooth muscle cells.
ryanodine-sensitive Ca2+ release channel; large-conductance Ca2+-activated potassium channel; caveolae; voltage-dependent Ca2+ channel
Idiopathic pulmonary fibrosis (IPF) is a common, progressive and invariably lethal interstitial lung disease with no effective therapy. We hypothesised that KCa3.1 K+ channel-dependent cell processes contribute to IPF pathophysiology.
KCa3.1 expression in primary human lung myofibroblasts was examined using RT-PCR, western blot, immunofluorescence and patch-clamp electrophysiology. The role of KCa3.1 channels in myofibroblast proliferation, wound healing, collagen secretion and contraction was examined using two specific and distinct KCa3.1 blockers (TRAM-34 and ICA-17043 [Senicapoc]).
Both healthy non fibrotic control and IPF-derived human lung myofibroblasts expressed KCa3.1 channel mRNA and protein. KCa3.1 ion currents were elicited more frequently and were larger in IPF-derived myofibroblasts compared to controls. KCa3.1 currents were increased in myofibroblasts by TGFβ1 and basic FGF. KCa3.1 was expressed strongly in IPF tissue. KCa3.1 pharmacological blockade attenuated human myofibroblast proliferation, wound healing, collagen secretion and contractility in
vitro, and this was associated with inhibition of TGFβ1-dependent increases in intracellular free Ca2+.
KCa3.1 activity promotes pro-fibrotic human lung myofibroblast function. Blocking KCa3.1 may offer a novel approach to treating IPF with the potential for rapid translation to the clinic.
In vascular biology, endothelial KCa2.3 and KCa3.1 channels contribute to arterial blood pressure regulation by producing membrane hyperpolarization and smooth muscle relaxation. The role of KCa2.3 and KCa3.1 channels in the pulmonary circulation is not fully established. Using mice with genetically encoded deficit of KCa2.3 and KCa3.1 channels, this study investigated the effect of loss of the channels in hypoxia-induced pulmonary hypertension.
Approach and Result
Male wild type and KCa3.1−/−/KCa2.3T/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 KCa3.1−/−/KCa2.3T/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 KCa2.3 channel by twofold in wild type mice and increased by 2.5-fold the relaxation evoked by the KCa2.3 and KCa3.1 channel activator NS309, whereas the acetylcholine-induced relaxation - sensitive to the combination of KCa2.3 and KCa3.1 channel blockers, apamin and charybdotoxin - was reduced by 2.5-fold in chronic hypoxic mice of either genotype.
Despite the deficits of the KCa2.3 and KCa3.1 channels failed to change hypoxia-induced pulmonary hypertension, the up-regulation of KCa2.3-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 KCa2.3/KCa3.1 activators for the treatment of pulmonary hypertension.
The voltage-gated Kv1.3 and the calcium-activated KCa3.1 potassium channel modulate many calcium-dependent cellular processes in immune cells, including T-cell activation and proliferation, and have therefore been proposed as novel therapeutic targets for immunomodulation. Kv1.3 is highly expressed in CCR7− effector memory T cells and is emerging as a target for T-cell mediated diseases like multiple sclerosis, rheumatoid arthritis, type-1 diabetes mellitus, allergic contact dermatitis, and psoriasis. KCa3.1 in contrast is expressed in CCR7+ naïve and central memory T cells, as well as in mast cells, macrophages, dedifferentiated vascular smooth muscle cells, fibroblasts, vascular endothelium, and airway epithelium. Given this expression pattern, KCa3.1 is a potential therapeutic target for conditions ranging from inflammatory bowel disease, multiple sclerosis, arthritis, and asthma to cardiovascular diseases like atherosclerosis and post-angioplasty restenosis. Results from animal studies have been supportive of the therapeutic potential of both Kv1.3 and KCa3.1 blockers and have also not shown any toxicities associated with pharmacological Kv1.3 and KCa3.1 blockade. To date, two compounds targeting Kv1.3 are in preclinical development but, so far, no Kv1.3 blocker has advanced into clinical trials. KCa3.1 blockers, on the other hand, have been evaluated in clinical trials for sickle cell anemia and exercise-induced asthma, but have so far not shown efficacy. However, the trial results support KCa3.1 as a safe therapeutic target, and will hopefully help enable clinical trials for other medical conditions that might benefit from KCa3.1 blockade.
immunosuppression; potassium channel; Kv1.3; KCa3.1; TRAM-34; PAP-1
The calmodulin/calcium-activated K+ channel KCa3.1 is expressed in red and white blood cells, epithelia and endothelia, and possibly central and peripheral neurons. However, our knowledge about its contribution to neurological functions and behavior is incomplete. Here, we investigated whether genetic deficiency or pharmacological activation of KCa3.1 change behavior and cerebral monoamine levels in mice.
In the open field test, KCa3.1-deficiency increased horizontal activity, as KCa3.1−/− mice travelled longer distances (≈145% of KCa3.1+/+) and at higher speed (≈1.5-fold of KCa3.1+/+). Working memory in the Y-maze was reduced by KCa3.1-deficiency. Motor coordination on the rotarod and neuromuscular functions were unchanged. In KCa3.1−/− mice, HPLC analysis revealed that turn-over rates of serotonin were reduced in frontal cortex, striatum and brain stem, while noradrenalin turn-over rates were increased in the frontal cortex. Dopamine turn-over rates were unaltered. Plasma catecholamine and corticosterone levels were unaltered. Intraperitoneal injections of 10 mg/kg of the KCa3.1/KCa2-activator SKA-31 reduced rearing and turning behavior in KCa3.1+/+ but not in KCa3.1−/− mice, while 30 mg/kg SKA-31 caused strong sedation in 50% of the animals of either genotypes. KCa3.1−/− mice were hyperactive (≈+60%) in their home cage and SKA-31-administration reduced nocturnal physical activity in KCa3.1+/+ but not in KCa3.1−/− mice.
KCa3.1-deficiency causes locomotor hyperactivity and altered monoamine levels in selected brain regions, suggesting a so far unknown functional link of KCa3.1 channels to behavior and monoaminergic neurotransmission in mice. The tranquilizing effects of low-dose SKA-31 raise the possibility to use KCa3.1/KCa2 channels as novel pharmacological targets for the treatment of neuropsychiatric hyperactivity disorders.
Human lung mast cells (HLMC) express the Ca2+-activated K+ channel KCa3.1, which opens following IgE-dependent activation. This hyperpolarises the cell membrane and potentiates both Ca2+ influx and degranulation. In addition, blockade of KCa3.1 profoundly inhibits HLMC migration to a variety of diverse chemotactic stimuli. KCa3.1 activation is attenuated by the β2adrenoceptor through a Gαs-coupled mechanism independent of cyclic AMP. Adenosine is an important mediator that both attenuates and enhances HLMC mediator release through the Gαs-coupled A2A and A2B adenosine receptors, respectively. We show that at concentrations that inhibit HLMC degranulation (10–5–10–3 M), adenosine closes KCa3.1 both dose-dependently and reversibly. KCa3.1 suppression by adenosine was reversed partially by the selective adenosine A2A receptor antagonist ZM241385 but not by the A2B receptor antagonist MRS1754, and the effects of adenosine were mimicked by the selective A2A receptor agonist CGS21680. Adenosine also opened a depolarising current carried by non-selective cations. As predicted from the role of KCa3.1 in HLMC migration, adenosine abolished HLMC chemotaxis to asthmatic airway smooth muscle-conditioned medium. In summary, the Gαs-coupled adenosine A2A receptor closes KCa3.1, providing a clearly defined mechanism by which adenosine inhibits HLMC migration and degranulation. A2A receptor agonists with channel-modulating function may be useful for the treatment of mast cell-mediated disease.
Adenosine; Chemotaxis; Ion channel; KCa3.1; Mast cell
Calcium-activated potassium (KCa) channels are present throughout the central nervous system as well as many peripheral tissues. Activation of KCa channels contribute to maintenance of the neuronal membrane potential and was shown to underlie the afterhyperpolarization (AHP) that regulates action potential firing and limits the firing frequency of repetitive action potentials. Different subtypes of KCa channels were anticipated on the basis of their physiological and pharmacological profiles, and cloning revealed two well defined but phylogenetic distantly related groups of channels. The group subject of this review includes both the small conductance KCa2 channels (KCa2.1, KCa2.2, and KCa2.3) and the intermediate-conductance (KCa3.1) channel. These channels are activated by submicromolar intracellular Ca2+ concentrations and are voltage independent. Of all KCa channels only the KCa2 channels can be potently but differentially blocked by the bee-venom apamin. In the past few years modulation of KCa channel activation revealed new roles for KCa2 channels in controlling dendritic excitability, synaptic functioning, and synaptic plasticity. Furthermore, KCa2 channels appeared to be involved in neurodegeneration, and learning and memory processes. In this review, we focus on the role of KCa2 and KCa3 channels in these latter mechanisms with emphasis on learning and memory, Alzheimer’s disease and on the interplay between neuroinflammation and different neurotransmitters/neuromodulators, their signaling components and KCa channel activation.
small conductance calcium-activated potassium channels; SK channels; learning and memory; neurodegeneration
Leukocyte Immunoglobulin-like Receptor B4 (LILRB4) null mice have an exacerbated T helper cell type 2 (Th2) immune response and pulmonary inflammation compared with Lilrb4+/+ animals when sensitized intranasally with ovalbumin (OVA) and low-dose lipopolysaccharide (LPS) followed by challenge with OVA. Moreover, OVA-challenged Lilrb4−/− mice exhibit greater migration of antigen (Ag)-bearing dendritic cells (DCs) to lymph nodes and accumulation of interleukin 4- and interleukin 5-producing lymph node lymphocytes. The main objective of this study was to determine how the absence of LILRB4 leads to a greater number of DCs in the lymph nodes of Ag-challenged mice and increased lung Th2 inflammation. Mice were sensitized intranasally with PBS alone or containing OVA and LPS; additional cohorts were subsequently challenged with OVA. Expression of chemokine (C-C motif) ligand 21 (CCL21) in the lung was assessed immunohistologically. OVA ingestion and expression of LILRB4 and chemokine (C-C motif) receptor 7 (CCR7) were quantified by flow cytometry. Inhalation of OVA and LPS induced upregulation of LILRB4 selectively on lung Ag-bearing DCs. After sensitization and challenge, the lung lymphatic vessels of Lilrb4−/− mice expressed more CCL21, a chemokine that directs the migration of DCs from peripheral tissue to draining lymph nodes, compared with Lilrb4+/+ mice. In addition, lung DCs of challenged Lilrb4−/− mice expressed more CCR7, the CCL21 receptor. The lungs of challenged Lilrb4−/− mice also contained significantly greater numbers of CD4+ cells expressing interleukin-4 or interleukin-5, consistent with the greater number of Ag-bearing DCs and Th2 cells in lymph nodes and the attendant exacerbated Th2 lung pathology. Our data establish a new mechanism by which LILRB4 can downregulate the development of pathologic allergic inflammation: reduced upregulation of key molecules needed for DC migration leading to decreases in Th2 cells in lymph nodes and their target tissue.
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.
Ca2+-dependent K+channels; glioblastoma; invasion; astrocytes; microglia
Small conductance calcium-activated potassium channels (KCa2.1,2.2,2.3) are blocked with high affinity by both peptide toxins (e.g. apamin) and small molecule blockers (e.g. UCL 1848). In electrophysiological experiments, apamin shows subtype selectivity with IC50s of ∼100 pM and ∼1 nM for block KCa2.2 and KCa2.3 respectively. In binding studies, however, apamin appears not to discriminate between KCa2.2 and 2.3 and is reported to have a significantly higher (∼20–200-fold) affinity (∼5 pM). This discrepancy between binding and block has been suggested to reflect an unusual mode of action of apamin. However, these binding and electrophysiological block experiments have not been conducted in the same ionic conditions, so it is also possible that the discrepancy arises simply because of differences in experimental conditions. We have now examined this latter possibility. Thus, we measured 125I-apamin binding to intact HEK 293 cells expressing KCa2 channels under the same ionic conditions (i.e. normal physiological conditions) that we also used for current block measurements. We find that binding and block experiments agree well if the same ionic conditions are used. Further, the binding of apamin and other blockers showed subtype selectivity when measured in normal physiological solutions (e.g.125I-apamin bound to KCa2.2 with KL 91±40 pM and to KCa2.3 with KL 711±126 pM, while inhibiting KCa2.2 current at IC50 103±2 pM). We also examined KCa2 channel block in Ca2+ and Mg2+ free solutions that mimic conditions reported in the literature for binding experiments. Under these (non-physiological) conditions the IC50 for apamin block of KCa2.2 was reduced to 20±3 pM. Our results therefore suggest that the apparent discrepancy between blocking and binding reported in the literature can be largely accounted for by the use of non-physiological ionic conditions in binding experiments.
Small conductance Ca2+-activated K+ channels (SK, KCa2.1, KCa2.2, KCa2.3) are expressed at high levels in brain regions critical for learning and memory. The activation of dendritic SK channels limits the induction of synaptic plasticity that may underlie hippocampal and amygdala dependent memory. EBIO facilitates SK channel activation by increasing their sensitivity to calcium. The compound CyPPA selectively activates SK2 and SK3 channels in a similar manner. To date there has been no report of the effects of SK channel activators on memory. Therefore, the present study examined the effects of systemic EBIO on mice in a behavioral task battery. Significant effects of EBIO on memory and motor activity were validated and extended by examining the effects of systemic CyPPA. Systemic EBIO and CyPPA both produced a transient decline in locomotor behavior. Neither SK channel activator affected anxiety. EBIO (17.5 mg/kg) impaired the encoding, but not retrieval, of object memory in a spontaneous object recognition task. A similar impairment of object memory encoding was observed in CyPPA (15 mg/kg)-treated mice. These memory-impairing effects were not due to changes in motivation, attention or movement. Systemic EBIO did not affect contextual or cued fear memory after conditioning with a 3 tone (CS)-footshock (US) pairing protocol or a 1 CS-US pairing protocol. Interestingly, apamin (0.4 mg/kg) enhanced contextual fear memory in mice conditioned with a 1 CS-US pairing protocol. These results suggest that SK channel activation impairs the encoding of non-aversive memory but not memory for aversive events. These data support converging evidence that SK channels regulate cellular mechanisms of memory encoding.
memory encoding; object recognition; apamin; mice; fear conditioning; SK channel; EBIO; CyPPA
The Ca2+-activated K+ channel, KCa3.1 (KCNN4/IK1/SK4), contributes to “classical,” pro-inflammatory activation of microglia, and KCa3.1 blockers have improved the outcome in several rodent models of CNS damage. For instance, blocking KCa3.1 with TRAM-34 rescued retinal ganglion neurons after optic nerve damage in vivo and, reduced p38 MAP kinase activation, production of reactive oxygen and nitrogen species, and neurotoxicity by microglia in vitro. In pursuing the therapeutic potential of KCa3.1 blockers, it is crucial to assess KCa3.1 contributions to other microglial functions and activation states, especially the IL-4-induced “alternative” activation state that can counteract pro-inflammatory states. We recently found that IL-4 increases microglia migration – a crucial function in the healthy and damaged CNS – and that KCa3.1 contributes to P2Y2 receptor-stimulated migration. Here, we discovered that KCa3.1 is greatly increased in alternative-activated rat microglia and then contributes to an enhanced migratory capacity. IL-4 up-regulated KCNN4 mRNA (by 6 h) and greatly increased the KCa3.1 current by 1 day, and this required de novo protein synthesis. The increase in current was sustained for at least 6 days. IL-4 increased microglial migration and this was reversed by blocking KCa3.1 with TRAM-34. A panel of inhibitors of signal-transduction mediators was used to analyze contributions of IL-4-related signaling pathways. Induction of KCNN4 mRNA and KCa3.1 current was mediated specifically through IL-4 binding to the type I receptor and, surprisingly, it required JAK3, Ras/MEK/ERK signaling and the transcription factor, activator protein-1, rather than JAK2, STAT6, or phosphatidylinositol 3-kinase.The same receptor subtype and pathway were required for the enhanced KCa3.1-dependent migration. In providing the first direct signaling link between an IL-4 receptor, expression and roles of an ion channel, this study also highlights the potential importance of KCa3.1 in alternative-activated microglia.
alternative microglial activation; AP-1 transcription factor; KCa3.1/SK4 channel; IL-4 signaling; M2 macrophage activation; microglial migration; Ras/MEK/ERK signaling; type I IL-4 receptor