Expression of the Voltage-Gated Potassium Channel Kv1.3 in Lesional Skin from Patients with Cutaneous T-Cell Lymphoma and Benign Dermatitis.

BACKGROUND: The voltage-gated potassium channel Kv1.3 (KCNA3) is expressed by effector memory T cells (TEM) and plays an important role in their activation and proliferation. Mycosis fungoides (MF), the most common subtype of cutaneous T-cell lymphoma (CTCL), was recently proposed to be a malignancy of skin-resident TEM. However, the expression of Kv1.3 in CTCL has not been investigated. OBJECTIVES: This study aims to examine the expression of Kv1.3 in situ and in vitro in CTCL. METHODS: The expression of Kv1.3 was examined by immunohistochemistry in skin lesions from 38 patients with MF, 4 patients with Sézary syndrome (SS), and 27 patients with benign dermatosis. In 4 malignant T-cell lines of CTCL (Myla2059, PB2B, SeAx, and Mac2a) and a non-malignant T-cell line (MyLa1850), the expression of Kv1.3 was determined by flow cytometry. The proliferation of those cell lines treated with various concentrations of Kv1.3 inhibitor ShK was measured by 3H-thymdine incorporation. RESULTS: Half of the MF patients (19/38) displayed partial Kv1.3 expression including 1 patient with moderate Kv1.3 positivity, while the other half (19/38) exhibited Kv1.3 negativity. An almost identical distribution was observed in patients with benign conditions, that is, 44.4% (12/27) were partially positive for Kv1.3 including 1 patient with moderate Kv1.3 positivity, while 55.6% (15/27) were Kv1.3 negative. In contrast, 3 in 4 SS patients displayed partial Kv1.3 positivity including 2 patients with weak staining and 1 with moderate staining, while 1 in 4 SS patients was Kv1.3 negative. In addition, all malignant T-cell lines, and a non-malignant T-cell line, displayed low Kv1.3 surface expression with a similar pattern. Whereas 2 cell lines (PB2B and Mac2a) were sensitive to Kv1.3 blockade, the other 2 (Myla2059 and SeAx) were completely resistant. CONCLUSIONS: We provide the first evidence of a heterogeneous Kv1.3 expression in situ in CTCL lesions.

Myocardin-Dependent Kv1.5 Channel Expression Prevents Phenotypic Modulation of Human Vessels in Organ Culture.

OBJECTIVE: We have previously described that changes in the expression of Kv channels associate to phenotypic modulation (PM), so that Kv1.3/Kv1.5 ratio is a landmark of vascular smooth muscle cells phenotype. Moreover, we demonstrated that the Kv1.3 functional expression is relevant for PM in several types of vascular lesions. Here, we explore the efficacy of Kv1.3 inhibition for the prevention of remodeling in human vessels, and the mechanisms linking the switch in Kv1.3 /Kv1.5 ratio to PM. Approach and Results: Vascular remodeling was explored using organ culture and primary cultures of vascular smooth muscle cells obtained from human vessels. We studied the effects of Kv1.3 inhibition on serum-induced remodeling, as well as the impact of viral vector-mediated overexpression of Kv channels or myocardin knock-down. Kv1.3 blockade prevented remodeling by inhibiting proliferation, migration, and extracellular matrix secretion. PM activated Kv1.3 via downregulation of Kv1.5. Hence, both Kv1.3 blockers and Kv1.5 overexpression inhibited remodeling in a nonadditive fashion. Finally, myocardin knock-down induced vessel remodeling and Kv1.5 downregulation and myocardin overexpression increased Kv1.5, while Kv1.5 overexpression inhibited PM without changing myocardin expression. CONCLUSIONS: We demonstrate that Kv1.5 channel gene is a myocardin-regulated, vascular smooth muscle cells contractile marker. Kv1.5 downregulation upon PM leaves Kv1.3 as the dominant Kv1 channel expressed in dedifferentiated cells. We demonstrated that the inhibition of Kv1.3 channel function with selective blockers or by preventing Kv1.5 downregulation can represent an effective, novel strategy for the prevention of intimal hyperplasia and restenosis of the human vessels used for coronary angioplasty procedures.

Temporal profiling of Kv1.3 channel expression in brain mononuclear phagocytes following ischemic stroke.

BACKGROUND: Microglia and CNS-infiltrating monocytes/macrophages (CNS-MPs) perform pro-inflammatory and protective anti-inflammatory functions following ischemic stroke. Selective inhibition of pro-inflammatory responses can be achieved by Kv1.3 channel blockade, resulting in a lower infarct size in the transient middle cerebral artery occlusion (tMCAO) model. Whether beneficial effects of Kv1.3 blockers are mediated by targeting microglia or CNS-infiltrating monocytes/macrophages remains unclear. METHODS: In the 30-min tMCAO mouse model, we profiled functional cell-surface Kv1.3 channels and phagocytic properties of acutely isolated CNS-MPs at various timepoints post-reperfusion. Kv1.3 channels were flow cytometrically detected using fluorescein-conjugated Kv1.3-binding peptide ShK-F6CA as well as by immunohistochemistry. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) was performed to measure Kv1.3 (Kcna3) and Kir2.1 (Kcnj2) gene expression. Phagocytosis of 1-µm microspheres by acutely isolated CNS-MPs was measured by flow cytometry. RESULTS: In flow cytometric assays, Kv1.3 channel expression by CD11b+ CNS-MPs was increased between 24 and 72 h post-tMCAO and decreased by 7 days post-tMCAO. Increased Kv1.3 expression was restricted to CD11b+CD45lowLy6clow (microglia) and CD11b+CD45highLy6Clow CNS-MPs but not CD11b+CD45highLy6chigh inflammatory monocytes/macrophages. In immunohistochemical studies, Kv1.3 protein expression was increased in Iba1+ microglia at 24-48 h post-tMCAO. No change in Kv1.3 mRNA in CNS-MPs was observed following tMCAO. CONCLUSIONS: We conclude that resident microglia and a subset of CD45highLy6clow CNS-MPs are the likely cellular targets of Kv1.3 blockers and the delayed phase of neuroinflammation is the optimal therapeutic window for Kv1.3 blockade in ischemic stroke.

Voltage Gated Potassium Channel Kv1.3 Is Upregulated on Activated Astrocytes in Experimental Autoimmune Encephalomyelitis.

Kv1.3 is a voltage gated potassium channel that has been implicated in pathophysiology of multiple sclerosis (MS). In the present study we investigated temporal and cellular expression pattern of this channel in the lumbar part of spinal cords of animals with experimental autoimmune encephalomyelitis (EAE), animal model of MS. EAE was actively induced in female Dark Agouti rats. Expression of Kv1.3 was analyzed at different time points of disease progression, at the onset, peak and end of EAE. We here show that Kv1.3 increased by several folds at the peak of EAE at both gene and protein level. Double immunofluorescence analyses demonstrated localization of Kv1.3 on activated microglia, macrophages, and reactive astrocytes around inflammatory lesions. In vitro experiments showed that pharmacological block of Kv1.3 in activated astrocytes suppresses the expression of proinflammatory mediators, suggesting a role of this channel in inflammation. Our results support the hypothesis that Kv1.3 may be a therapeutic target of interest for MS and add astrocytes to the list of cells whose activation would be suppressed by inhibiting Kv1.3 in inflammatory conditions.

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.

Determinants of Helix Formation for a Kv1.3 Transmembrane Segment inside the Ribosome Exit Tunnel.

Proteins begin to fold in the ribosome, and misfolding has pathological consequences. Among the earliest folding events in biogenesis is the formation of a helix, an elementary structure that is ubiquitously present and required for correct protein folding in all proteomes. The determinants underlying helix formation in the confined space of the ribosome exit tunnel are relatively unknown. We chose the second transmembrane segment, S2, of a voltage-gated potassium channel, Kv1.3, as a model to probe this issue. Since the N terminus of S2 is initially in an extended conformation in the folding vestibule of the ribosome yet ultimately emerges at the exit port as a helix, S2 is ideally suited for delineating sequential events and folding determinants of helix formation inside the ribosome. We show that S2's extended N terminus inside the tunnel is converted into a helix by a single, distant mutation in the nascent peptide. This transition depends on nascent peptide sequence at specific tunnel locations. Co-translational secondary folding of nascent chains inside the ribosome has profound physiological consequences that bear on correct membrane insertion, tertiary folding, oligomerization, and biochemical modification of the newborn protein during biogenesis.

Different calcium influx characteristics upon Kv1.3 and IKCa1 potassium channel inhibition in T helper subsets.

Functional imbalance between T helper subsets plays important role in the pathogenesis of autoimmune disorders. Transient increase of cytoplasmic calcium level, and sustention of negative membrane potential by voltage sensitive Kv1.3 and calcium-dependent IKCa1 potassium channels are essential for short-term lymphocyte activation, thus present possible target for selective immunomodulation. We aimed to investigate calcium influx sensitivity to the inhibition of potassium channels in the main T helper subsets. Peripheral blood from 11 healthy individuals was drawn and calcium influx kinetics following activation with phytohemagglutinin in Th1, Th2, Th17, and Treg cells were evaluated. Alteration of calcium influx induced by specific inhibitors of Kv1.3 and IKCa1 potassium channels, and the expression of Kv1.3 channels were also assessed. Highest cytoplasmic calcium concentration was observed in stimulated Th1 cells, while the lowest level was measured in Treg cells. In Th1 and Th17 cells, inhibition of both investigated potassium channels decreased calcium influx. In Th2 cells only the inhibitor of Kv1.3 channels, while in Treg cells none of the inhibitors had significant effect. Upon the inhibition of IKCa1 channels, short-term activation of proinflammatory cells was specifically decreased without affecting anti-inflammatory subsets, indicating that selective immunomodulation is possible in healthy individuals.

Regulation of the voltage gated K channel Kv1.3 by recombinant human klotho protein.

BACKGROUND/AIMS: Klotho, a protein mainly produced in the kidney and released into circulating blood, contributes to the negative regulation of 1,25(OH)2D3 formation and is thus a powerful regulator of mineral metabolism. As ß-glucuronidase, alpha Klotho protein further regulates the stability of several carriers and channels in the plasma membrane and thus regulates channel and transporter activity. Accordingly, alpha Klotho protein participates in the regulation of diverse functions seemingly unrelated to mineral metabolism including lymphocyte function. The present study explored the impact of alpha Klotho protein on the voltage gated K+ channel Kv1.3. METHODS: cRNA encoding Kv1.3 (KCNA3) was injected into Xenopus oocytes and depolarization induced outward current in Kv1.3 expressing Xenopus oocytes determined utilizing dual electrode voltage clamp. Experiments were performed without or with prior treatment with recombinant human Klotho protein (50 ng/ml, 24 hours) in the absence or presence of a ß-glucuronidase inhibitor D-saccharic acid-1,4-lactone (DSAL, 10 µM). Moreover, the voltage gated K+ current was determined in Jcam lymphoma cells by whole cell patch clamp following 24 hours incubation without or with recombinant human Klotho protein (50 ng/ml, 24 hours). Kv1.3 protein abundance in Jcam cells was determined utilising fluorescent antibodies in flow cytometry. RESULTS: In Kv1.3 expressing Xenopus oocytes the Kv1.3 currents and the protein abundance of Kv1.3 were both significantly enhanced after treatment with recombinant human Klotho protein (50 ng/ml, 24 hours), an effect reversed by presence of DSAL. Moreover, treatment with recombinant human Klotho protein increased Kv currents and Kv1.3 protein abundance in Jcam cells. CONCLUSION: Alpha Klotho protein enhances Kv1.3 channel abundance and Kv1.3 currents in the plasma membrane, an effect depending on its ß-glucuronidase activity.

Modulation of voltage-dependent and inward rectifier potassium channels by 15-epi-lipoxin-A4 in activated murine macrophages: implications in innate immunity.

Potassium channels modulate macrophage physiology. Blockade of voltage-dependent potassium channels (Kv) by specific antagonists decreases macrophage cytokine production and inhibits proliferation. In the presence of aspirin, acetylated cyclooxygenase-2 loses the activity required to synthesize PGs but maintains the oxygenase activity to produce 15R-HETE from arachidonate. This intermediate product is transformed via 5-LOX into epimeric lipoxins, termed 15-epi-lipoxins (15-epi-lipoxin A4 [e-LXA4]). Kv have been proposed as anti-inflammatory targets. Therefore, we studied the effects of e-LXA4 on signaling and on Kv and inward rectifier potassium channels (Kir) in mice bone marrow-derived macrophages (BMDM). Electrophysiological recordings were performed in these cells by the whole-cell patch-clamp technique. Treatment of BMDM with e-LXA4 inhibited LPS-dependent activation of NF-κB and IκB kinase ß activity, protected against LPS activation-dependent apoptosis, and enhanced the accumulation of the Nrf-2 transcription factor. Moreover, treatment of LPS-stimulated BMDM with e-LXA4 resulted in a rapid decrease of Kv currents, compatible with attenuation of the inflammatory response. Long-term treatment of LPS-stimulated BMDM with e-LXA4 significantly reverted LPS effects on Kv and Kir currents. Under these conditions, e-LXA4 decreased the calcium influx versus that observed in LPS-stimulated BMDM. These effects were partially mediated via the lipoxin receptor (ALX), because they were significantly reverted by a selective ALX receptor antagonist. We provide evidence for a new mechanism by which e-LXA4 contributes to inflammation resolution, consisting of the reversion of LPS effects on Kv and Kir currents in macrophages.

Analysis of the K+ current in human CD4+ T lymphocytes in hypercholesterolemic state.

Atherosclerosis involves immune mechanisms: T lymphocytes are found in atherosclerotic plaques, suggesting their activation during atherogenesis. The predominant voltage-gated potassium channel of T cells, Kv1.3 is a key regulator of the Ca(2+)-dependent activation pathway. In the present experiments we studied the proliferation capacity and functional changes of Kv1.3 channels in T cells from healthy and hypercholestaeremic patients. By means of CFSE-assay (carboxyfluorescein succinimidyl ester) we showed that spontaneous activation rate of lymphocytes in hypercholesterolemia was elevated and the antiCD3/antiCD28 co-stimulation was less effective as compared to the healthy group. Using whole-cell patch-clamping we obtained that the activation and deactivation kinetics of Kv1.3 channels were faster in hypercholesterolemic state but no change in other parameters of Kv1.3 were found (inactivation kinetics, steady-state activation, expression level). We suppose that incorporation of oxLDL species via its raft-rupturing effect can modify proliferative rate of T cells as well as the gating of Kv1.3 channels.

Kv1.3 channels can modulate cell proliferation during phenotypic switch by an ion-flux independent mechanism.

OBJECTIVE: Phenotypic modulation of vascular smooth muscle cells has been associated with a decreased expression of all voltage-dependent potassium channel (Kv)1 channel encoding genes but Kcna3 (which encodes Kv1.3 channels). In fact, upregulation of Kv1.3 currents seems to be important to modulate proliferation of mice femoral vascular smooth muscle cells in culture. This study was designed to explore if these changes in Kv1 expression pattern constituted a landmark of phenotypic modulation across vascular beds and to investigate the mechanisms involved in the proproliferative function of Kv1.3 channels. METHODS AND RESULTS: Changes in Kv1.3 and Kv1.5 channel expression were reproduced in mesenteric and aortic vascular smooth muscle cells, and their correlate with protein expression was electrophysiologicaly confirmed using selective blockers. Heterologous expression of Kv1.3 and Kv1.5 channels in HEK cells has opposite effects on the proliferation rate. The proproliferative effect of Kv1.3 channels was reproduced by "poreless" mutants but disappeared when voltage-dependence of gating was suppressed. CONCLUSIONS: These findings suggest that the signaling cascade linking Kv1.3 functional expression to cell proliferation is activated by the voltage-dependent conformational change of the channels without needing ion conduction. Additionally, the conserved upregulation of Kv1.3 on phenotypic modulation in several vascular beds makes this channel a good target to control unwanted vascular remodeling.

Disruption of kv1.3 channel forward vesicular trafficking by hypoxia in human T lymphocytes.

Hypoxia in solid tumors contributes to decreased immunosurveillance via down-regulation of Kv1.3 channels in T lymphocytes and associated T cell function inhibition. However, the mechanisms responsible for Kv1.3 down-regulation are not understood. We hypothesized that chronic hypoxia reduces Kv1.3 surface expression via alterations in membrane trafficking. Chronic hypoxia decreased Kv1.3 surface expression and current density in Jurkat T cells. Inhibition of either protein synthesis or degradation and endocytosis did not prevent this effect. Instead, blockade of clathrin-coated vesicle formation and forward trafficking prevented the Kv1.3 surface expression decrease in hypoxia. Confocal microscopy revealed an increased retention of Kv1.3 in the trans-Golgi during hypoxia. Expression of adaptor protein-1 (AP1), responsible for clathrin-coated vesicle formation at the trans-Golgi, was selectively down-regulated by hypoxia. Furthermore, AP1 down-regulation increased Kv1.3 retention in the trans-Golgi and reduced Kv1.3 currents. Our results indicate that hypoxia disrupts AP1/clathrin-mediated forward trafficking of Kv1.3 from the trans-Golgi to the plasma membrane thus contributing to decreased Kv1.3 surface expression in T lymphocytes.

Voltage-gated potassium channel Kv1.3 blocker as a potential treatment for rat anti-glomerular basement membrane glomerulonephritis.

The voltage-gated potassium channel Kv1.3 has been recently identified as a molecular target that allows the selective pharmacological suppression of effector memory T cells (T(EM)) without affecting the function of naïve T cells (T(N)) and central memory T cells (T(CM)). We found that Kv1.3 was expressed on glomeruli and some tubules in rats with anti-glomerular basement membrane glomerulonephritis (anti-GBM GN). A flow cytometry analysis using kidney cells revealed that most of the CD4(+) T cells and some of the CD8(+) T cells had the T(EM) phenotype (CD45RC(-)CD62L(-)). Double immunofluorescence staining using mononuclear cell suspensions isolated from anti-GBM GN kidney showed that Kv1.3 was expressed on T cells and some macrophages. We therefore investigated whether the Kv1.3 blocker Psora-4 can be used to treat anti-GBM GN. Rats that had been given an injection of rabbit anti-rat GBM antibody were also injected with Psora-4 or the vehicle intraperitoneally. Rats given Psora-4 showed less proteinuria and fewer crescentic glomeruli than rats given the vehicle. These results suggest that T(EM) and some macrophages expressing Kv1.3 channels play a critical role in the pathogenesis of crescentic GN and that Psora-4 will be useful for the treatment of rapidly progressive glomerulonephritis.

Activated T-cells inhibit neurogenesis by releasing granzyme B: rescue by Kv1.3 blockers.

There is a great need for pharmacological approaches to enhance neural progenitor cell (NPC) function particularly in neuroinflammatory diseases with failed neuroregeneration. In diseases such as multiple sclerosis and stroke, T-cell infiltration occurs in periventricular zones where NPCs are located and is associated with irreversible neuronal loss. We studied the effect of T-cell activation on NPC functions. NPC proliferation and neuronal differentiation were impaired by granzyme B (GrB) released by the T-cells. GrB mediated its effects by the activation of a Gi-protein-coupled receptor leading to decreased intracellular levels of cAMP and subsequent expression of the voltage-dependent potassium channel, Kv1.3. Importantly, blocking channel activity with margatoxin or blocking its expression reversed the inhibitory effects of GrB on NPCs. We have thus identified a novel pathway in neurogenesis. The increased expression of Kv1.3 in pathological conditions makes it a novel target for promoting neurorestoration.

A folding zone in the ribosomal exit tunnel for Kv1.3 helix formation.

Although it is now clear that protein secondary structure can be acquired early, while the nascent peptide resides within the ribosomal exit tunnel, the principles governing folding of native polytopic proteins have not yet been elucidated. We now report an extensive investigation of native Kv1.3, a voltage-gated K(+) channel, including transmembrane and linker segments synthesized in sequence. These native segments form helices vectorially (N- to C-terminus) only in a permissive vestibule located in the last 20 A of the tunnel. Native linker sequences similarly fold in this vestibule. Finally, secondary structure acquired in the ribosome is retained in the translocon. These findings emerge from accessibility studies of a diversity of native transmembrane and linker sequences and may therefore be applicable to protein biogenesis in general.

Developmental switch of the expression of ion channels in human dendritic cells.

Modulation of the expression and activity of plasma membrane ion channels is one of the mechanisms by which immune cells can regulate their intracellular Ca(2+) signaling pathways required for proliferation and/or differentiation. Voltage-gated K+ channels, inwardly rectifying K+ channels, and Ca(2+)-activated K+ channels have been described to play a major role in controlling the membrane potential in lymphocytes and professional APCs, such as monocytes, macrophages, and dendritic cells (DCs). Our study aimed at the characterization and identification of ion channels expressed in the course of human DC differentiation from monocytes. We report in this study for the first time that immature monocyte-derived DCs express voltage-gated Na+ channels in their plasma membrane. The analysis of the biophysical and pharmacological properties of the current and PCR-based cloning revealed the presence of Nav1.7 channels in immature DCs. Transition from the immature to a mature differentiation state, however, was accompanied by the down-regulation of Nav1.7 expression concomitant with the up-regulation of voltage-gated Kv1.3 K+ channel expression. The presence of Kv1.3 channels seems to be common for immune cells; hence, selective Kv1.3 blockers may emerge as candidates for inhibiting various functions of mature DCs that involve their migratory, cytokine-secreting, and T cell-activating potential.

Potassium channel expression in human CD4+ regulatory and naïve T cells from healthy subjects and multiple sclerosis patients.

The membrane potential of human T cells is regulated by two potassium channels: the voltage-gated K(V)1.3 and the Ca2+-activated K(Ca)3.1. These two channels are essential for efficient antigenic activation and proliferation of T cells and are expressed at different levels in naïve, central memory and effector memory T cells. This provides the opportunity to inhibit the proliferation of the targeted subtype by channel-specific blocking compounds. Regulatory T cells (Tregs) also represent a unique subtype of T cells that perform highly specialized tasks in controlling immune responses, which raises the possibility that they too have a distinctive channel expression pattern. Using whole-cell patch-clamp we tested this hypothesis and determined the ion channel expression of CD4+CD25(hi)CD127(lo) regulatory and CD4+CD25(lo)CD127(hi) naïve T cells from the peripheral blood of healthy volunteers and multiple sclerosis (MS) patients sorted by flow cytometry. We have found that naïve and Treg cells from healthy controls expressed equal numbers of K(V)1.3 channels, while Tregs had a greater membrane surface as assessed by capacitance measurements, and consequentially lower channel density than naïve cells, indicating an "incomplete activation state" of Tregs. In contrast, Tregs from MS patients had fewer K(V)1.3 channels than naïve cells and there was no difference in the membrane capacitance or channel density between the two subtypes of cells. The expression level of K(Ca)3.1 channels was similar in all cell subsets. The observed differences in K(V)1.3 channel expression density may contribute to the varying responses upon antigenic stimulation by these cell types in health and disease.

Altered dynamics of Kv1.3 channel compartmentalization in the immunological synapse in systemic lupus erythematosus.

Aberrant T cell responses during T cell activation and immunological synapse (IS) formation have been described in systemic lupus erythematosus (SLE). Kv1.3 potassium channels are expressed in T cells where they compartmentalize at the IS and play a key role in T cell activation by modulating Ca(2+) influx. Although Kv1.3 channels have such an important role in T cell function, their potential involvement in the etiology and progression of SLE remains unknown. This study compares the K channel phenotype and the dynamics of Kv1.3 compartmentalization in the IS of normal and SLE human T cells. IS formation was induced by 1-30 min exposure to either anti-CD3/CD28 Ab-coated beads or EBV-infected B cells. We found that although the level of Kv1.3 channel expression and their activity in SLE T cells is similar to normal resting T cells, the kinetics of Kv1.3 compartmentalization in the IS are markedly different. In healthy resting T cells, Kv1.3 channels are progressively recruited and maintained in the IS for at least 30 min from synapse formation. In contrast, SLE, but not rheumatoid arthritis, T cells show faster kinetics with maximum Kv1.3 recruitment at 1 min and movement out of the IS by 15 min after activation. These kinetics resemble preactivated healthy T cells, but the K channel phenotype of SLE T cells is identical to resting T cells, where Kv1.3 constitutes the dominant K conductance. The defective temporal and spatial Kv1.3 distribution that we observed may contribute to the abnormal functions of SLE T cells.

Kv1 potassium channel C-terminus constant HRETE region: arginine substitution affects surface protein level and conductance level of subfamily members differentially.

We have shown previously that truncating all of the variable cytoplasmic C-terminus of Kv1.1 potassium channels to G421stop had only a small inhibitory effect on their cell surface conductance density levels and cell surface protein levels. Here we investigated the role of a highly conserved cytoplasmic C-terminal charged region of five amino acids (HRETE) of the S6 transmembrane domain in the protein and conductance expression of Kv1.1, Kv1.2, and Kv1.4 channels. For Kv1.1 we found that E420stop, T419stop, and E418stop showed cell surface conductance densities and cell surface protein levels similar to full length control, whereas R417stop and H416stop exhibited essentially no conductance but their surface protein levels were similar to full length control. A bulky non-negatively charged hydrophilic amino acid at position 417 appeared to be critical for wild type gating of Kv1.1 because R417K and R417Q rescued conductance levels whereas R417A or R417E did not. The R417A mutation in the full length Kv1.1 also exhibited surface protein levels similar to control but it did not exhibit significant conductance. In contrast, mutation of the equivalent arginine to alanine in full length Kv1.2 and Kv1.4 appeared to have little or no effect on channel conductance but rather decreased cell surface protein levels by inducing partial high ER retention. These findings are consistent with the notion that the arginine amino acid in the HRETE region plays a different role in affecting conductance levels or cell surface protein levels of very closely related Kv1 potassium channels.

Inhibitory effects of blocking voltage-dependent potassium channel 1.3 on human monocyte-derived macrophage differentiation into foam cells.

OBJECTIVE: To investigate the expression of voltage-dependent potassium channel 1.3 (Kv1.3) mRNA and protein during human monocyte-derived macrophage differentiation into foam cells and its function in foam cell formation. METHODS: Human peripheral blood monocytes were isolated from healthy male volunteers by density gradient centrifugation and then by adherent method. The obtained monocytes were cultured for 5 days to differentiate into macrophages. Based on establishment of the human macrophage-derived foam cell model, the expression of Kv1.3 channel was investigated by immunocytochemical staining, reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Furthermore, the effects of rMargatoxin, a Kv 1.3 channel-specific inhibitor, on cholesterol metabolism in macrophages incepting oxidized low density lipoprotein (OxLDL) were studied. RESULTS: After the macrophages co-incubated with 30 mg/L OxLDL at 37 degrees C for 60 hours, the cellular volume obviously enlarged and many red lipid granules were deposited in cytoplasm. The total amount of cholesterol (TC), free cholesterol (FC) and cholesterol ester (CE) in cells markedly increased and the ratio of CE/TC rose from (14.4+/-6.8)% to (57.9+/-3.5)% (n=7, P<0.05). However, the expression of Kv1.3 channel had no significant change. rMargatoxin (0.1 nmol/L and 10 nmol/L) markedly reduced the contents of TC, FC and CE in macrophages and the ratios of CE/TC decreased to (42.8+/-11.6)% and (22.6+/-8.0)%, respectively (n=7, P<0.05). Meanwhile, the red lipid granules deposited in the cytoplasm of macrophages also decreased. CONCLUSION: These data clearly show that the expression of Kv1.3 channel does not change obviously during human monocyte-derived macrophage differentiation into foam cells and the blocking of it would prevent foam cell formation.