Pharmacological inhibition of Kv1.3 fails to modulate insulin sensitivity in diabetic mice or human insulin-sensitive tissues

Genetic ablation of the voltage-gated potassium channel Kv1.3 improves insulin sensitivity and increases metabolic rate in mice. Inhibition of Kv1.3 in mouse adipose and skeletal muscle is reported to increase glucose uptake through increased GLUT4 translocation. Since Kv1.3 represents a novel target for the treatment of diabetes, the present study investigated whether Kv1.3 is functionally expressed in human adipose and skeletal muscle and whether specific pharmacological inhibition of the channel is capable of modulating insulin sensitivity in diabetic mouse models. Voltage-gated K(+) channel currents in human skeletal muscle cells (SkMC) were insensitive to block by the specific Kv1.3 blockers 5-(4-phenoxybutoxy)psoralen (PAP-1) and margatoxin (MgTX). Glucose uptake into SkMC and mouse 3T3-L1 adipocytes was also unaffected by treatment with PAP-1 or MgTX. Kv1.3 protein expression was not observed in human adipose or skeletal muscle from normal and type 2 diabetic donors. To investigate the effect of specific Kv1.3 inhibition on insulin sensitivity in vivo, PAP-1 was administered to hyperglycemic mice either acutely or for 5 days prior to an insulin tolerance test. No effect on insulin sensitivity was observed at free plasma PAP-1 concentrations that are specific for inhibition of Kv1.3. Insulin sensitivity was increased only when plasma concentrations of PAP-1 were sufficient to inhibit other Kv1 channels. Surprisingly, acute inhibition of Kv1.3 in the brain was found to decrease insulin sensitivity in ob/ob mice. Overall, these findings are not supportive of a role for Kv1.3 in the modulation of peripheral insulin sensitivity.     

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

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

Characterization of the functional properties of the voltage-gated potassium channel Kv1.3 in human CD4+ T lymphocytes

Previous studies have shown that central memory T (T(CM)) cells predominantly use the calcium-dependent potassium channel KCa3.1 during acute activation, whereas effector memory T (T(EM)) cells use the voltage-gated potassium channel Kv1.3. Because Kv1.3-specific pharmacological blockade selectively inhibited anti-CD3-mediated proliferation, whereas naive T cells and T(CM) cells escaped inhibition due to up-regulation of KCa3.1, this difference indicated a potential for selective targeting of the T(EM) population. We examined the effects of pharmacological Kv1.3 blockers and a dominant-negative Kv1.x construct on T cell subsets to assess the specific effects of Kv1.3 blockade. Our studies indicated both T(CM) and T(EM) CD4+ T cells stimulated with anti-CD3 were inhibited by charybdotoxin, which can block both KCa3.1 and Kv1.3, whereas margatoxin and Stichodactyla helianthus toxin, which are more selective Kv1.3 inhibitors, inhibited proliferation and IFN-gamma production only in the T(EM) subset. The addition of anti-CD28 enhanced proliferation of freshly isolated cells and rendered them refractory to S. helianthus, whereas chronically activated T(EM) cell lines appeared to be costimulation independent because Kv1.3 blockers effectively inhibited proliferation and IFN-gamma regardless of second signal. Transduction of CD4+ T cells with dominant-negative Kv1.x led to a higher expression of CCR7+ T(CM) phenotype and a corresponding depletion of T(EM). These data provide further support for Kv1.3 as a selective target of chronically activated T(EM) without compromising naive or T(CM) immune functions. Specific Kv1.3 blockers may be beneficial in autoimmune diseases such as multiple sclerosis in which T(EM) are found in the target organ.     

Substituted N-{3-[(1,1-dioxido-1,2-benzothiazol-3-yl)(phenyl)amino]propyl}benzamide analogs as potent Kv1.3 ion channel blockers. Part 2

We report the synthesis and in vitro activity of a series of novel substituted N-{3-[(1,1-dioxido-1,2-benzothiazol-3-yl)(phenyl)amino]propyl}benzamide analogs. These analogs showed potent inhibitory activity against Kv1.3. Several demonstrated similar potency to the known Kv1.3 inhibitor PAP-1 when tested under the IonWorks patch clamp assay conditions. Two compounds 13i and 13rr were advanced further as potential tool compounds for in vivo validation studies.     

Kv1.3 deletion biases T cells toward an immunoregulatory phenotype and renders mice resistant to autoimmune encephalomyelitis

Increasing evidence suggests ion channels have critical functions in the differentiation and plasticity of T cells. Kv1.3, a voltage-gated K(+) channel, is a functional marker and a pharmacological target for activated effector memory T cells. Selective Kv1.3 blockers have been shown to inhibit proliferation and cytokine production by human and rat effector memory T cells. We used Kv1.3 knockout (KO) mice to investigate the mechanism by which Kv1.3 blockade affects CD4(+) T cell differentiation during an inflammatory immune-mediated disease. Kv1.3 KO animals displayed significantly lower incidence and severity of myelin oligodendrocyte glycoprotein (MOG) peptide-induced experimental autoimmune encephalomyelitis. Kv1.3 was the only K(V) channel expressed in MOG 35-55-specific CD4(+) T cell blasts, and no K(V) current was present in MOG-specific CD4(+) T cell-blasts from Kv1.3 KO mice. Fewer CD4(+) T cells migrated to the CNS in Kv1.3 KO mice following disease induction, and Ag-specific proliferation of CD4(+) T cells from these mice was impaired with a corresponding cell-cycle delay. Kv1.3 was required for optimal expression of IFN-γ and IL-17, whereas its absence led to increased IL-10 production. Dendritic cells from Kv1.3 KO mice fully activated wild-type CD4(+) T cells, indicating a T cell-intrinsic defect in Kv1.3 KO mice. The loss of Kv1.3 led to a suppressive phenotype, which may contribute to the mechanism by which deletion of Kv1.3 produces an immunotherapeutic effect. Skewing of CD4(+) T cell differentiation toward Ag-specific regulatory T cells by pharmacological blockade or genetic suppression of Kv1.3 might be beneficial for therapy of immune-mediated diseases such as multiple sclerosis.     

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.     

N-{3-[(1,1-dioxido-1,2-benzothiazol-3-yl)(phenyl)amino]propyl}benzamide analogs as potent Kv1.3 inhibitors. Part 1

We report the synthesis and in vitro activity of a series of novel N-{3-[(1,1-dioxido-1,2-benzothiazol-3-yl)(phenyl)amino]propyl}benzamide analogs. These analogs showed potent inhibitory activity against Kv1.3. Several compounds, including compound 8b, showed similar potency to the known Kv1.3 inhibitor PAP-1 when tested under the IonWorks patch clamp assay conditions.     

Immunosuppressive effects of a Kv1.3 inhibitor

The voltage gated potassium channel (Kv1.3) has been shown to play a role in immune responsiveness. Blockade of the channel led to diminution of T cell activation and delayed type hypersensitivity. Previous in vitro studies of the blockade were focused on T cell activation and proliferation. In this study we examined other T and monocytic cell mediated events to glean the extent of the immunosuppressive effects of a Kv1.3 specific inhibitor, Margatoxin (MgTX). We found that MgTX inhibited the intracellular production of Th-1 as well as Th-2 cytokines. MgTX can also inhibit IL-2 production and proliferation of T cells upon stimulation with anti-CD3 and VCAM-1. Furthermore, a redirected cytolytic activity was also inhibited by MgTX. However, MgTX did not inhibit generation of CTL to EBV transformed lymphoma cells or antibody-dependent cellular cytolysis mediated by monocytes. It appears that a Kv1.3 blockade does not affect all immune responses, particularly those of innate immunity.     

Control of memory CD4 T cell recall by the CD28/B7 costimulatory pathway

The CD28/B7 costimulatory pathway is generally considered dispensable for memory T cell responses, largely based on in vitro studies demonstrating memory T cell activation in the absence of CD28 engagement by B7 ligands. However, the susceptibility of memory CD4 T cells, including central (CD62L(high)) and effector memory (T(EM); CD62L(low)) subsets, to inhibition of CD28-derived costimulation has not been closely examined. In this study, we demonstrate that inhibition of CD28/B7 costimulation with the B7-binding fusion molecule CTLA4Ig has profound and specific effects on secondary responses mediated by memory CD4 T cells generated by priming with Ag or infection with influenza virus. In vitro, CTLA4Ig substantially inhibits IL-2, but not IFN-gamma production from heterogeneous memory CD4 T cells specific for influenza hemagglutinin or OVA in response to peptide challenge. Moreover, IL-2 production from polyclonal influenza-specific memory CD4 T cells in response to virus challenge was completely abrogated by CTLA4Ig with IFN-gamma production partially inhibited. When administered in vivo, CTLA4Ig significantly blocks Ag-driven memory CD4 T cell proliferation and expansion, without affecting early recall and activation. Importantly, CTLA4Ig treatment in vivo induced a striking shift in the phenotype of the responding population from predominantly T(EM) in control-treated mice to predominantly central memory T cells in CTLA4Ig-treated mice, suggesting biased effects of CTLA4Ig on T(EM) responses. Our results identify a novel role for CD28/B7 as a regulator of memory T cell responses, and have important clinical implications for using CTLA4Ig to abrogate the pathologic consequences of T(EM) cells in autoimmunity and chronic disease.     

Hypoxia regulates expression and activity of Kv1.3 channels in T lymphocytes: a possible role in T cell proliferation

T lymphocytes are exposed to hypoxia during their development and also when they migrate to hypoxic pathological sites such as tumors and wounds. Although hypoxia can affect T cell development and function, the mechanisms by which immune cells sense and respond to changes in O(2)-availability are poorly understood. K(+) channels encoded by the Kv1.3 subtype of the voltage-dependent Kv1 gene family are highly expressed in lymphocytes and are involved in the control of membrane potential and cell function. In this study, we investigate the sensitivity of Kv1.3 channels to hypoxia in freshly isolated human T lymphocytes and leukemic Jurkat T cells. Acute exposure to hypoxia (20 mmHg, 2 min) inhibits Kv1.3 currents in both cell types by 20%. Prolonged exposure to hypoxia (1% O(2) for 24 h) selectively decreases Kv1.3 protein levels in Jurkat T cells by 47%, but not Kvbeta2 and SK2 Ca-activated K(+) channel subunit levels. The decrease in Kv1.3 protein levels occurs with no change in Kv1.3 mRNA expression and is associated with a significant decrease in K(+) current density. A decrease in Kv1.3 polypeptide levels similar to that obtained during hypoxia is produced by Kv1.3 channel blockage. Our results indicate that hypoxia produces acute and long-term inhibition of Kv1.3 channels in T lymphocytes. This effect could account for the inhibition of lymphocyte proliferation during hypoxia. Indeed, we herein present evidence showing that hypoxia selectively inhibits TCR-mediated proliferation and that this inhibition is associated with a decrease in Kv1.3 proteins.     

Blocking KCa3.1 Channels Increases Tumor Cell Killing by a Subpopulation of Human Natural Killer Lymphocytes

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.     

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.     

Correolide and derivatives are novel immunosuppressants blocking the lymphocyte Kv1.3 potassium channels

The voltage-gated potassium channel, Kv1.3, is specifically expressed on human lymphocytes, where it controls membrane potential and calcium influx. Blockade of Kv1.3 channels by margatoxin was previously shown to prevent T cell activation and attenuate immune responses in vivo. In the present study, a triterpene natural product, correolide, was found to block Kv1.3 channels in human and miniswine T cells by electrophysiological characterization. T cell activation events, such as anti-CD3-induced calcium elevation, IL-2 production, and proliferation were inhibited by correolide in a dose-dependent manner. More potent analogs were evaluated for pharmacokinetic profiles and subsequently tested in a delayed-type hypersensitivity (DTH) response to tuberculin in the miniswine. Two compounds were dosed orally, iv, or im, and both compounds suppressed DTH responses, demonstrating that small molecule blockers of Kv1.3 channels can act as immunosuppressive agents in vivo. These studies establish correolide and its derivatives as novel immunosuppressants.     

PAP-1, a novel target protein of phosphorylation by pim-1 kinase.

Protooncogene, pim-1, has been reported to be a predisposition for lymphomagenesis along with myc, and its protein product, Pim-1, has been shown to be a serine/threonine protein kinase, whose activity is involved in proliferation and differentiation of blood cells. The signal transduction pathways neither to nor from Pim-1, however, have been clarified. We have cloned a cDNA encoding a novel Pim-1 binding protein, PAP-1, comprising 213 amino acids with a basic amino-acid cluster near the C-terminus. PAP-1 was colocalized with Pim-1 in human HeLa cell nuclei. The in vitro binding assays using GST fusion proteins of the wild-type and various deletion mutants revealed that the whole molecule of Pim-1 is required for the binding activity to PAP-1 and that Pim-1 binds to the region from amino-acid numbers 1-147 of PAP-1, or to two segments in the region. The association of PAP-1 with Pim-1 was also shown in vivo in transfected cells. Furthermore, PAP-1 was phosphorylated in vitro by Pim-1, but not a kinase-negative Pim-1 mutant. The two serine residues of PAP-1 at amino acids 204 and 206 near the C-terminus were phosphorylated by Pim-1. PAP-1 is thus thought to be a target protein for Pim-1 kinase.     

The D-diastereomer of ShK toxin selectively blocks voltage-gated K+ channels and inhibits T lymphocyte proliferation

The polypeptide toxin ShK is a potent blocker of Kv1.3 potassium channels, which are crucial in the activation of human effector memory T cells (T(EM)); selective blockers constitute valuable therapeutic leads for the treatment of autoimmune diseases mediated by T(EM) cells, such as multiple sclerosis, rheumatoid arthritis, and type-1 diabetes. The critical motif on the toxin for potassium channel blockade consists of neighboring lysine and tyrosine residues. Because this motif is sufficient for activity, an ShK analogue was designed based on D-amino acids. D-allo-ShK has a structure essentially identical with that of ShK and is resistant to proteolysis. It blocked Kv1.3 with K(d) 36 nm (2,800-fold lower affinity than ShK), was 2-fold selective for Kv1.3 over Kv1.1, and was inactive against other K(+) channels tested. D-allo-ShK inhibited human T(EM) cell proliferation at 100-fold higher concentration than ShK. Its circulating half-life was only slightly longer than that of ShK, implying that renal clearance is the major determinant of its plasma levels. D-allo-ShK did not bind to the closed state of the channel, unlike ShK. Models of D-allo-ShK bound to Kv1.3 show that it can block the pore as effectively as ShK but makes different interactions with the vestibule, some of which are less favorable than for native ShK. The finding that an all-D analogue of a polypeptide toxin retains biological activity and selectivity is highly unusual. Being resistant to proteolysis and nonantigenic, this analogue should be useful in K(+) channel studies; all-d analogues with improved Kv1.3 potency and specificity may have therapeutic advantages.     

Benzamide derivatives as blockers of Kv1.3 ion channel

The voltage-gated potassium channel, Kv1.3, is present in human T-lymphocytes. Blockade of Kv1.3 results in T-cell depolarization, inhibition of T-cell activation, and attenuation of immune responses in vivo. A class of benzamide Kv1.3 channel inhibitors has been identified. The structure-activity relationship within this class of compounds in two functional assays, Rb_Kv and T-cell proliferation, is presented. In in vitro assays, trans isomers display moderate selectivity for binding to Kv1.3 over other Kv1.x channels present in human brain.     

Dysregulation of the polo-like kinase pathway in CD4+ T cells is characteristic of pathogenic simian immunodeficiency virus infection

CD4(+) T-cell dysfunction highlighted by defects within the intracellular signaling cascade and cell cycle has long been characterized as a direct and/or indirect consequence of human immunodeficiency virus (HIV) infection in humans and simian immunodeficiency virus (SIV) infection in rhesus macaques (RM). Dysregulation of the M phase of the cell cycle is a well-documented effect of HIV or SIV infection both in vivo and in vitro. In this study the effect of SIV infection on the modulation of two important regulators of the M phase-polo-like kinases Plk3 and Plk1-was investigated. We have previously shown that Plk3 is markedly downregulated in CD4(+) T cells from SIV-infected disease-susceptible RM but not SIV-infected disease-resistant sooty mangabeys (SM), denoting an association of downregulation with disease progression. Here we show that, in addition to the downregulation, Plk3 exhibits aberrant activation patterns in the CD4(+) T cells from SIV-infected RM following T-cell receptor stimulation. Interestingly, in vitro SIV infection of CD4(+) T cells leads to the upregulation, rather than downregulation, of Plk3, suggesting that different mechanisms operate in vitro and in vivo. In addition, CD4(+) T cells from RM with high viral loads exhibited consistent and significant upregulation of Plk1, concurrent with an aberrant activation-induced Plk1 response, suggesting complex mechanisms of SIV-induced M-phase abnormalities in vivo. Altogether this study presents a novel mechanism underlying M-phase defects observed in CD4(+) T cells from HIV or SIV-infected disease-susceptible humans and RM which may contribute to aberrant T-cell responses and disease pathogenesis.     

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.     

Potent Kv1.3 inhibitors from correolide-modification of the C18 position

Kv1.3, the voltage-gated potassium channel in human T cells, represents a new target for treating immunosuppression and autoimmune diseases. Correolide (1), a pentacyclic natural product, is a potent and selective Kv1.3 channel blocker. Simplification of correolide via removal of its E-ring generates enone 4, whose modification produced a new series of tetracyclic Kv1.3 blockers. The structure-activity relationship for this class of compounds in two functional assays, Rb_Kv and human T cell proliferation, is presented herein. The most potent analog 43 is 15-fold more potent than correolide as inhibitor of human T cell proliferation.     

Compensatory anion currents in Kv1.3 channel-deficient thymocytes

Kv1.3 is a voltage-gated potassium channel with roles in human T cell activation/proliferation, cell-mediated cytotoxicity, and volume regulation and is thus a target for therapeutic control of T cell responses. Kv1.3 is also present in some mouse thymocyte subsets and splenocytes, but its role in the mouse is less well understood. We report the generation and characterization of Kv1.3-deficient (Kv1.3-/-) mice. In contrast to wild-type cells, the majority of Kv1.3-/- thymocytes had no detectable voltage-dependent potassium current, although RNA and protein for several potassium channel subunits were found in the thymocyte population. Surprisingly, the level of chloride current in the Kv1.3-/- thymocytes was increased approximately 50-fold over that in wild-type cells. There were no abnormalities in lymphocyte types or absolute numbers in thymus, spleen, and lymph nodes and no obvious defect in thymocyte apoptosis or T cell proliferation in the Kv1.3-/- animals. The compensatory effects of the enhanced chloride current may account for the apparent lack of immune system defects in Kv1.3-/-mice.