The toxin mimic FS48 from the salivary gland of Xenopsylla cheopis functions as a Kv1.3 channel-blocking immunomodulator of T cell activation.

The Kv1.3 channel has been widely demonstrated to play crucial roles in the activation and proliferation of T cells, which suggests that selective blockers could serve as potential therapeutics for autoimmune diseases mediated by T cells. We previously described that the toxin mimic FS48 from salivary gland of Xenopsylla cheopis downregulates the secretion of proinflammatory factors by Raw 264.7 cells by blocking the Kv1.3 channel and the subsequent inactivation of the proinflammatory MAPK/NF-κB pathways. However, the effects of FS48 on human T cells and autoimmune diseases are unclear. Here, we described its immunomodulatory effects on human T cells derived from suppression of Kv1.3 channel. Kv1.3 currents in Jurkat T cells were recorded by whole-cell patch-clamp, and Ca2+ influx, cell proliferation, and TNF-α and IL-2 secretion were measured using Fluo-4, CCK-8, and ELISA assays, respectively. The in vivo immunosuppressive activity of FS48 was evaluated with a rat DTH model. We found that FS48 reduced Kv1.3 currents in Jurkat T cells in a concentration-dependent manner with an IC50 value of about 1.42 µM. FS48 also significantly suppressed Kv1.3 protein expression, Ca2+ influx, MAPK/NF-κB/NFATc1 pathway activation, and TNF-α and IL-2 production in activated Jurkat T cells. Finally, we show that FS48 relieved the DTH response in rats. We therefore conclude that FS48 can block the Kv1.3 channel and inhibit human T cell activation, which most likely contributes to its immunomodulatory actions and highlights the great potential of this evolutionary-guided peptide as a drug template in future studies.

Impact of Diverse Ion Channels on Regulatory T Cell Functions.

The population of regulatory T cells (Tregs) is critical for immunological self-tolerance and homeostasis. Proper ion regulation contributes to Treg lineage identity, regulation, and effector function. Identified ion channels include Ca2+ release-activated Ca2+, transient receptor potential, P2X, volume-regulated anion and K+ channels Kv1.3 and KCa3.1. Ion channel modulation represents a promising therapeutic approach for the treatment of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. This review summarizes studies with gene-targeted mice and pharmacological modulators affecting Treg number and function. Furthermore, participation of ion channels is illustrated and the power of future research possibilities is discussed.

Inhibition of inflammatory cytokine production and proliferation in macrophages by Kunitz-type inhibitors from Echinococcus granulosus.

The genus Echinococcus of cestode parasites includes important pathogens of humans and livestock animals. Transcriptomic and genomic studies on E. granulosus and E. multilocularis uncovered striking expansion of monodomain Kunitz proteins. This expansion is accompanied by the specialization of some family members away from the ancestral protease inhibition function to fulfill cation channel blockade functions. Since cation channels are involved in immune processes, we tested the effects on macrophage physiology of two E. granulosus Kunitz-type inhibitors of voltage-activated cation channels (Kv) that are close paralogs. Both inhibitors, EgKU-1 and EgKU-4, inhibited production of the Th1/Th17 cytokine subunit IL-12/23p40 by macrophages stimulated with the TLR4 agonist LPS. In addition, EgKU-4 but not EgKU-1 inhibited production of the inflammatory cytokine IL-6. These activities were not displayed by EgKU-3, a family member that is a protease inhibitor without known activity on cation channels. EgKU-4 potently inhibited macrophage proliferation in response to M-CSF, whereas EgKU-1 displayed similar activity but with much lower potency, similar to EgKU-3. We discuss structural differences, including a heavily cationic C-terminal extension present in EgKU-4 but not in EgKU-1, that may explain the differential activities of the two close paralogs.

A role for TASK2 channels in the human immunological synapse.

The immunological synapse is a transient junction that occurs when the plasma membrane of a T cell comes in close contact with an APC after recognizing a peptide from the antigen-MHC. The interaction starts when CRAC channels embedded in the T cell membrane open, flowing calcium ions into the cell. To counterbalance the ion influx and subsequent depolarization, Kv 1.3 and KCa3.1 channels are recruited to the immunological synapse, increasing the extracellular K+ concentration. These processes are crucial as they initiate gene expression that drives T cell activation and proliferation. The T cell-specific function of the K2P channel family member TASK2 channels and their role in autoimmune processes remains unclear. Using mass spectrometry analysis together with epifluorescence and super-resolution single-molecule localization microscopy, we identified TASK2 channels as novel players recruited to the immunological synapse upon stimulation. TASK2 localizes at the immunological synapse, upon stimulation with CD3 antibodies, likely interacting with these molecules. Our findings suggest that, together with Kv 1.3 and KCa3.1 channels, TASK2 channels contribute to the proper functioning of the immunological synapse, and represent an interesting treatment target for T cell-mediated autoimmune disorders.

Luteolin-mediated Kv1.3 K+ channel inhibition augments BCG vaccine efficacy against tuberculosis by promoting central memory T cell responses in mice.

Despite the availability of multiple antibiotics, tuberculosis (TB) remains a major health problem worldwide, with one third of the population latently infected and ~2 million deaths annually. The only available vaccine for TB, Bacillus Calmette Guérin (BCG), is ineffective against adult pulmonary TB. Therefore, alternate strategies that enhance vaccine efficacy are urgently needed. Vaccine efficacy and long-term immune memory are critically dependent on central memory T (TCM) cells, whereas effector memory T (TEM) cells are important for clearing acute infections. Recently, it has been shown that inhibition of the Kv1.3 K+ ion channel, which is predominantly expressed on TEM but not TCM cells, profoundly enhances TCM cell differentiation. We exploited this phenomenon to improve TCM:TEM cell ratios and protective immunity against Mycobacterium tuberculosis infection in response to BCG vaccination of mice. We demonstrate that luteolin, a plant-derived Kv1.3 K+ channel inhibitor, profoundly promotes TCM cells by selectively inhibiting TEM cells, and significantly enhances BCG vaccine efficacy. Thus, addition of luteolin to BCG vaccination may provide a sustainable means to improve vaccine efficacy by boosting host immunity via modulation of memory T cell differentiation.

ß1-Integrin- and KV1.3 channel-dependent signaling stimulates glutamate release from Th17 cells.

Although the impact of Th17 cells on autoimmunity is undisputable, their pathogenic effector mechanism is still enigmatic. We discovered soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) complex proteins in Th17 cells that enable a vesicular glutamate release pathway that induces local intracytoplasmic calcium release and subsequent damage in neurons. This pathway is glutamine dependent and triggered by binding of ß1-integrin to vascular cell adhesion molecule 1 (VCAM-1) on neurons in the inflammatory context. Glutamate secretion could be blocked by inhibiting either glutaminase or KV1.3 channels, which are known to be linked to integrin expression and highly expressed on stimulated T cells. Although KV1.3 is not expressed in CNS tissue, intrathecal administration of a KV1.3 channel blocker or a glutaminase inhibitor ameliorated disability in experimental neuroinflammation. In humans, T cells from patients with multiple sclerosis secreted higher levels of glutamate, and cerebrospinal fluid glutamine levels were increased. Altogether, our findings demonstrate that ß1-integrin- and KV1.3 channel-dependent signaling stimulates glutamate release from Th17 cells upon direct cell-cell contact between Th17 cells and neurons.

Delayed Rectifier K+-Channel Is a Novel Therapeutic Target for Interstitial Renal Fibrosis in Rats with Unilateral Ureteral Obstruction.

BACKGROUND: Delayed rectifier K+-channel, Kv1.3, is most predominantly expressed in T-lymphocytes and macrophages. In such leukocytes, Kv1.3-channels play pivotal roles in the activation and proliferation of cells, promoting cellular immunity. Since leukocyte-derived cytokines stimulate fibroblasts to produce collagen fibers in inflamed kidneys, Kv1.3-channels expressed in leukocytes would contribute to the progression of tubulointerstitial renal fibrosis. METHODS: Male Sprague-Dawley rats that underwent unilateral ureteral obstruction (UUO) were used at 1, 2, or 3 weeks after the operation. We examined the histological features of the kidneys and the leukocyte expression of Kv1.3-channels. We also examined the therapeutic effects of a selective channel inhibitor, margatoxin, on the progression of renal fibrosis and the proliferation of leukocytes within the cortical interstitium. RESULTS: In rat kidneys with UUO, progression of renal fibrosis and the infiltration of leukocytes became most prominent at 3 weeks after the operation, when Kv1.3-channels were overexpressed in proliferating leukocytes. In the cortical interstitium of margatoxin-treated UUO rat kidneys, immunohistochemistry revealed reduced expression of fibrosis markers. Additionally, margatoxin significantly decreased the numbers of leukocytes and suppressed their proliferation. CONCLUSIONS: This study clearly demonstrated that the numbers of T-lymphocytes and macrophages were markedly increased in UUO rat kidneys with longer postobstructive days. The overexpression of Kv1.3-channels in leukocytes was thought to be responsible for the proliferation of these cells and the progression of renal fibrosis. This study strongly suggested the therapeutic usefulness of targeting lymphocyte Kv1.3-channels in the treatment of renal fibrosis.

A systems pharmacology-based approach to identify novel Kv1.3 channel-dependent mechanisms in microglial activation.

BACKGROUND: Kv1.3 potassium channels regulate microglial functions and are overexpressed in neuroinflammatory diseases. Kv1.3 blockade may selectively inhibit pro-inflammatory microglia in neurological diseases but the molecular and cellular mechanisms regulated by Kv1.3 channels are poorly defined. METHODS: We performed immunoblotting and flow cytometry to confirm Kv1.3 channel upregulation in lipopolysaccharide (LPS)-activated BV2 microglia and in brain mononuclear phagocytes freshly isolated from LPS-treated mice. Quantitative proteomics was performed on BV2 microglia treated with control, LPS, ShK-223 (highly selective Kv1.3 blocker), and LPS+ShK-223. Gene ontology (GO) analyses of Kv1.3-dependent LPS-regulated proteins were performed, and the most representative proteins and GO terms were validated. Effects of Kv1.3-blockade on LPS-activated BV2 microglia were studied in migration, focal adhesion formation, reactive oxygen species production, and phagocytosis assays. In vivo validation of protein changes and predicted molecular pathways were performed in a model of systemic LPS-induced neuroinflammation, employing antigen presentation and T cell proliferation assays. Informed by pathway analyses of proteomic data, additional mechanistic experiments were performed to identify early Kv1.3-dependent signaling and transcriptional events. RESULTS: LPS-upregulated cell surface Kv1.3 channels in BV2 microglia and in microglia and CNS-infiltrating macrophages isolated from LPS-treated mice. Of 144 proteins differentially regulated by LPS (of 3141 proteins), 21 proteins showed rectification by ShK-223. Enriched cellular processes included MHCI-mediated antigen presentation (TAP1, EHD1), cell motility, and focal adhesion formation. In vitro, ShK-223 decreased LPS-induced focal adhesion formation, reversed LPS-induced inhibition of migration, and inhibited LPS-induced upregulation of EHD1, a protein involved in MHCI trafficking. In vivo, intra-peritoneal ShK-223 inhibited LPS-induced MHCI expression by CD11b+CD45low microglia without affecting MHCI expression or trafficking of CD11b+CD45high macrophages. ShK-223 inhibited LPS-induced MHCI-restricted antigen presentation to ovalbumin-specific CD8+ T cells both in vitro and in vivo. Kv1.3 co-localized with the LPS receptor complex and regulated LPS-induced early serine (S727) STAT1 phosphorylation. CONCLUSIONS: We have unraveled novel molecular and functional roles for Kv1.3 channels in pro-inflammatory microglial activation, including a Kv1.3 channel-regulated pathway that facilitates MHCI expression and MHCI-dependent antigen presentation by microglia to CD8+ T cells. We also provide evidence for neuro-immunomodulation by systemically administered ShK peptides. Our results further strengthen the therapeutic candidacy of microglial Kv1.3 channels in neurologic diseases.

Toxins Targeting the Kv1.3 Channel: Potential Immunomodulators for Autoimmune Diseases.

Autoimmune diseases are usually accompanied by tissue injury caused by autoantigen-specific T-cells. KV1.3 channels participate in modulating calcium signaling to induce T-cell proliferation, immune activation and cytokine production. Effector memory T (TEM)-cells, which play major roles in many autoimmune diseases, are controlled by blocking KV1.3 channels on the membrane. Toxins derived from animal venoms have been found to selectively target a variety of ion channels, including KV1.3. By blocking the KV1.3 channel, these toxins are able to suppress the activation and proliferation of TEM cells and may improve TEM cell-mediated autoimmune diseases, such as multiple sclerosis and type I diabetes mellitus.

Kv1.3 in psoriatic disease: PAP-1, a small molecule inhibitor of Kv1.3 is effective in the SCID mouse psoriasis--xenograft model.

Kv1.3 channels regulate the activation/proliferation of effector memory T cells and thus play a critical role in the pathogenesis of autoimmune diseases. Using a combination of immunohistochemistry, confocal microscopy, flow cytometry and electrophysiology methods we observed a significant enrichment of activated Kv1.3(+) memory T cells in psoriasis plaques and synovial fluid from patients with psoriasis/psoriatic arthritis (PsA) compared to non-lesional psoriatic skin, normal skin or peripheral blood lympho-mononuclear cells. In in vitro studies performed with lesional mononuclear cells or T cells derived from skin and joints of psoriatic disease, the small molecule Kv1.3 blocker PAP-1 dose-dependently inhibited proliferation and suppressed IL-2 and IFN-γ production. To further substantiate the pathologic role of Kv1.3 high TEM cells in psoriatic disease we tested whether PAP-1 is able to improve psoriatic disease pathology in the SCID mouse-psoriasis skin xenograft model. Following four weeks of daily treatment with 2% PAP-1 ointment we noticed about 50% reduction in the epidermal thickness (rete peg length) and the number of CD3(+) lymphocytes/mm(2) of dermis decreased by 85%. Vehicle treated and untreated plaques in contrast remained unchanged and showed no reduction in epidermis thickness and infiltrating CD3(+) T cells and HLA-DR(+) T cells. Based on these results we propose the development of Kv1.3 targeted topical immunotherapy for psoriasis and possibly for other inflammatory skin conditions, where effector memory T cells are involved in the pathogenesis.

Blocking KV1.3 channels inhibits Th2 lymphocyte function and treats a rat model of asthma.

Allergic asthma is a chronic inflammatory disease of the airways. Of the different lower airway-infiltrating immune cells that participate in asthma, T lymphocytes that produce Th2 cytokines play important roles in pathogenesis. These T cells are mainly fully differentiated CCR7(-) effector memory T (TEM) cells. Targeting TEM cells without affecting CCR7(+) naïve and central memory (TCM) cells has the potential of treating TEM-mediated diseases, such as asthma, without inducing generalized immunosuppression. The voltage-gated KV1.3 potassium channel is a target for preferential inhibition of TEM cells. Here, we investigated the effects of ShK-186, a selective KV1.3 channel blocker, for the treatment of asthma. A significant proportion of T lymphocytes in the lower airways of subjects with asthma expressed high levels of KV1.3 channels. ShK-186 inhibited the allergen-induced activation of peripheral blood T cells from those subjects. Immunization of F344 rats against ovalbumin followed by intranasal challenges with ovalbumin induced airway hyper-reactivity, which was reduced by the administration of ShK-186. ShK-186 also reduced total immune infiltrates in the bronchoalveolar lavage and number of infiltrating lymphocytes, eosinophils, and neutrophils assessed by differential counts. Rats with the ovalbumin-induced model of asthma had elevated levels of the Th2 cytokines IL-4, IL-5, and IL-13 measured by ELISA in their bronchoalveolar lavage fluids. ShK-186 administration reduced levels of IL-4 and IL-5 and induced an increase in the production of IL-10. Finally, ShK-186 inhibited the proliferation of lung-infiltrating ovalbumin-specific T cells. Our results suggest that KV1.3 channels represent effective targets for the treatment of allergic asthma.

Targeting the Kv1.3 potassium channel for immunosuppression in vascularized composite allotransplantation - a pilot study.

Kv1.3-channels are critically involved in activation and function of effector memory T cells. Blocking Kv1.3-channels was investigated for its effect on skin rejection in a rat limb-transplantation-model. Animals received the Kv1.3-blocker correolide C systemically or locally as intra-graft-treatment in combination with tacrolimus. Systemic (intraperitoneal) administration of correolide C resulted in slight, but significant prolongation of allograft survival compared with untreated and placebo treated controls. In 4/6 correolide C treated animals, histology showed an intact epidermis and a mild infiltrate by day 10. High correolide C plasma trough levels correlated with prolonged allograft survival. A decrease in CD4+ and CD8+ effector memory T cells was observed in allograft skin, peripheral blood and the spleen on day 5. When applied subcutaneously in combination with systemic tacrolimus (30 days+/-anti-lymphocyte serum) detectable, but insignificant prolongation of graft survival was achieved. 2/5 animals showed an intact epidermis and a mild infiltrate until day 45. Tapering systemic tacrolimus and weaning on day 50 resulted in rejection by day 55, regardless of local correolide C treatment. Subcutaneous injection did not lead to systemic plasma levels. The Kv1.3-channel is a potential drug target worth exploring in more detail for immunosuppression in vascularized composite allotransplantation.

Specific Kv1.3 blockade modulates key cholesterol-metabolism-associated molecules in human macrophages exposed to ox-LDL.

Cholesterol-metabolism-associated molecules, including scavenger receptor class A (SR-A), lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), CD36, ACAT1, ABCA1, ABCG1, and scavenger receptor class B type I, can modulate cholesterol metabolism in the transformation from macrophages to foam cells. Voltage-gated potassium channel Kv1.3 has increasingly been demonstrated to play an important role in the modulation of macrophage function. Here, we investigate the role of Kv1.3 in modulating cholesterol-metabolism-associated molecules in human acute monocytic leukemia cell-derived macrophages (THP-1 macrophages) and human monocyte-derived macrophages exposed to oxidized LDL (ox-LDL). Human Kv1.3 and Kv1.5 channels (hKv1.3 and hKv1.5) are expressed in macrophages and form a heteromultimeric channel. The hKv1.3-E314 antibody that we had generated as a specific hKv1.3 blocker inhibited outward delayed rectifier potassium currents, whereas the hKv1.5-E313 antibody that we had generated as a specific hKv1.5 blocker failed. Accordingly, the hKv1.3-E314 antibody reduced percentage of cholesterol ester and enhanced apoA-I-mediated cholesterol efflux in THP-1 macrophages and human monocyte-derived macrophages exposed to ox-LDL. The hKv1.3-E314 antibody downregulated SR-A, LOX-1, and ACAT1 expression and upregulated ABCA1 expression in THP-1 macrophages and human monocyte-derived macrophages. Our results reveal that specific Kv1.3 blockade represents a novel strategy modulating cholesterol metabolism in macrophages, which benefits the treatment of atherosclerotic lesions.

Selective inhibition of CCR7(-) effector memory T cell activation by a novel peptide targeting Kv1.3 channel in a rat experimental autoimmune encephalomyelitis model.

The voltage-gated Kv1.3 K(+) channel in effector memory T cells serves as a new therapeutic target for multiple sclerosis. In our previous studies, the novel peptide ADWX-1 was designed and synthesized as a specific Kv1.3 blocker. However, it is unclear if and how ADWX-1 alleviates experimental autoimmune encephalomyelitis, a model for multiple sclerosis. In this study, the administration of ADWX-1 significantly ameliorated the rat experimental autoimmune encephalomyelitis model by selectively inhibiting CD4(+)CCR7(-) phenotype effector memory T cell activation. In contrast, the Kv1.3-specific peptide had little effect on CD4(+)CCR7(+) cells, thereby limiting side effects. Furthermore, we determined that ADWX-1 is involved in the regulation of NF-κB signaling through upstream protein kinase C-θ (PKCθ) in the IL-2 pathway of CD4(+)CCR7(-) cells. The elevated expression of Kv1.3 mRNA and protein in activated CD4(+)CCR7(-) cells was reduced by ADWX-1 engagement; however, an apparent alteration in CD4(+)CCR7(+) cells was not observed. Moreover, the selective regulation of the Kv1.3 channel gene expression pattern by ADWX-1 provided a further and sustained inhibition of the CD4(+)CCR7(-) phenotype, which depends on the activity of Kv1.3 to modulate its activation signal. In addition, ADWX-1 mediated the activation of differentiated Th17 cells through the CCR7(-) phenotype. The efficacy of ADWX-1 is supported by multiple functions, which are based on a Kv1.3(high) CD4(+)CCR7(-) T cell selectivity through two different pathways, including the classic channel activity-associated IL-2 pathway and the new Kv1.3 channel gene expression pathway.

Durable pharmacological responses from the peptide ShK-186, a specific Kv1.3 channel inhibitor that suppresses T cell mediators of autoimmune disease.

The Kv1.3 channel is a recognized target for pharmaceutical development to treat autoimmune diseases and organ rejection. ShK-186, a specific peptide inhibitor of Kv1.3, has shown promise in animal models of multiple sclerosis and rheumatoid arthritis. Here, we describe the pharmacokinetic-pharmacodynamic relationship for ShK-186 in rats and monkeys. The pharmacokinetic profile of ShK-186 was evaluated with a validated high-performance liquid chromatography-tandem mass spectrometry method to measure the peptide's concentration in plasma. These results were compared with single-photon emission computed tomography/computed tomography data collected with an ¹¹¹In-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-conjugate of ShK-186 to assess whole-blood pharmacokinetic parameters as well as the peptide's absorption, distribution, and excretion. Analysis of these data support a model wherein ShK-186 is absorbed slowly from the injection site, resulting in blood concentrations above the Kv1.3 channel-blocking IC50 value for up to 7 days in monkeys. Pharmacodynamic studies on human peripheral blood mononuclear cells showed that brief exposure to ShK-186 resulted in sustained suppression of cytokine responses and may contribute to prolonged drug effects. In delayed-type hypersensitivity, chronic relapsing-remitting experimental autoimmune encephalomyelitis, and pristane-induced arthritis rat models, a single dose of ShK-186 every 2 to 5 days was as effective as daily administration. ShK-186's slow distribution from the injection site and its long residence time on the Kv1.3 channel contribute to the prolonged therapeutic effect of ShK-186 in animal models of autoimmune disease.

The antibody targeting the E314 peptide of human Kv1.3 pore region serves as a novel, potent and specific channel blocker.

Selective blockade of Kv1.3 channels in effector memory T (T(EM)) cells was validated to ameliorate autoimmune or autoimmune-associated diseases. We generated the antibody directed against one peptide of human Kv1.3 (hKv1.3) extracellular loop as a novel and possible Kv1.3 blocker. One peptide of hKv1.3 extracellular loop E3 containing 14 amino acids (E314) was chosen as an antigenic determinant to generate the E314 antibody. The E314 antibody specifically recognized 63.8KD protein stably expressed in hKv1.3-HEK 293 cell lines, whereas it did not recognize or cross-react to human Kv1.1(hKv1.1), Kv1.2(hKv1.2), Kv1.4(hKv1.4), Kv1.5(hKv1.5), KCa3.1(hKCa3.1), HERG, hKCNQ1/hKCNE1, Nav1.5 and Cav1.2 proteins stably expressed in HEK 293 cell lines or in human atrial or ventricular myocytes by Western blotting analysis and immunostaining detection. By the technique of whole-cell patch clamp, the E314 antibody was shown to have a directly inhibitory effect on hKv1.3 currents expressed in HEK 293 or Jurkat T cells and the inhibition showed a concentration-dependence. However, it exerted no significant difference on hKv1.1, hKv1.2, hKv1.4, hKv1.5, hKCa3.1, HERG, hKCNQ1/hKCNE1, L-type Ca(2+) or voltage-gated Na(+) currents. The present study demonstrates that the antibody targeting the E314 peptide of hKv1.3 pore region could be a novel, potent and specific hKv1.3 blocker without affecting a variety of closely related K(v)1 channels, KCa3.1 channels and functional cardiac ion channels underlying central nervous system (CNS) disorders or drug-acquired arrhythmias, which is required as a safe clinic-promising channel blocker.

Functional blockade of the voltage-gated potassium channel Kv1.3 mediates reversion of T effector to central memory lymphocytes through SMAD3/p21cip1 signaling.

The maintenance of T cell memory is critical for the development of rapid recall responses to pathogens, but may also have the undesired side effect of clonal expansion of T effector memory (T(EM)) cells in chronic autoimmune diseases. The mechanisms by which lineage differentiation of T cells is controlled have been investigated, but are not completely understood. Our previous work demonstrated a role of the voltage-gated potassium channel Kv1.3 in effector T cell function in autoimmune disease. In the present study, we have identified a mechanism by which Kv1.3 regulates the conversion of T central memory cells (T(CM)) into T(EM). Using a lentiviral-dominant negative approach, we show that loss of function of Kv1.3 mediates reversion of T(EM) into T(CM), via a delay in cell cycle progression at the G2/M stage. The inhibition of Kv1.3 signaling caused an up-regulation of SMAD3 phosphorylation and induction of nuclear p21(cip1) with resulting suppression of Cdk1 and cyclin B1. These data highlight a novel role for Kv1.3 in T cell differentiation and memory responses, and provide further support for the therapeutic potential of Kv1.3 specific channel blockers in T(EM)-mediated autoimmune diseases.

Peripherally induced human regulatory T cells uncouple Kv1.3 activation from TCR-associated signaling.

Peripherally induced Tregs (iTregs) are being recognized as a functional and physiologically relevant T-cell subset. Understanding the molecular basis of their development is a necessary step before the therapeutic potential of iTreg manipulation can be exploited. In this study, we report that the differentiation of primary human T cells to suppressor iTregs involves the relocation of key proximal TCR signaling elements to the highly active IL-2-Receptor (IL-2-R) pathway. In addition to the recruitment of lymphocyte-specific protein tyrosine kinase (Lck) to the IL-2-R complex, we identified the dissociation of the voltage-gated K(+) channel Kv1.3 from the TCR pathway and its functional coupling to the IL-2-R. The regulatory switch of Kv1.3 activity in iTregs may constitute an important contributing factor in the signaling rewiring associated with the development of peripheral human iTregs and sheds new light upon the reciprocal crosstalk between the TCR and the IL-2-R pathways.

The beneficial effect of blocking Kv1.3 in the psoriasiform SCID mouse model.

The Kv1.3 channel is important in the activation and function of effector memory T cells. Recently, specific blockers of the Kv1.3 channel have been developed as a potential therapeutic option for diverse autoimmune diseases. In psoriatic lesions, most lymphocytes are memory effector T cells. The aim of the present study was to detect the expression of Kv1.3 channels in these cells in psoriatic lesions as well as in human psoriasiform skin grafts using the severe combined immunodeficient (SCID) mouse model. Histological and immunohistochemical staining for Kv1.3 expression and various inflammatory markers was performed in sections obtained from six psoriatic patients and 18 beige-SCID mice with psoriasiform human skin grafts. Six grafted mice were treated with Stichodactyla helianthus neurotoxin (ShK), a known Kv1.3 blocker. The results showed an increased number of Kv1.3+ cells in the psoriatic skin as well as in the psoriasiform skin grafts as compared with normal skin and normal skin grafts. Injections of ShK showed a marked therapeutic effect in three of six psoriasiform skin grafts. A significantly decreased number of Kv1.3+ cells was observed in the responders compared with the control grafts. This pilot study, although performed in a small number of mice, reveals the possible beneficial effect of Kv1.3 blockers in psoriasis patients.

Lymphocyte calcium influx characteristics and their modulation by Kv1.3 and IKCa1 channel inhibitors in healthy pregnancy and preeclampsia.

PROBLEM: Calcium handling of T lymphocytes is altered in healthy pregnancy (HP) and preeclampsia (PE) compared to non-pregnant (non-P) women. We compared the activation-elicited calcium influx in T lymphocytes in HP, PE and non-P women and tested its alteration upon inhibition of Kv1.3 and IKCa1 potassium channels. METHOD OF STUDY: The alteration of calcium influx was measured in major T-lymphocyte subsets of 9 non-P, HP and PE women with flow cytometry with or without treatment of cells with potassium channel inhibitors. RESULTS: The elicited calcium response was lower in HP compared to non-P. In HP, calcium influx was sensitive to potassium channel inhibition in CD8 and Th1, but not in Th2 cells. In PE, calcium influx and its sensitivity to inhibition were comparable to non-P. CONCLUSION: There is a characteristic pattern of calcium influx in T lymphocytes and its sensitivity to potassium channel inhibition in HP that is missing in PE, raising the notion that T-lymphocyte calcium handling may have a role in the characteristic immune status of HP.