Blockade Of Kv1 Research Articles

Involvement of KCa3.1 channel activity in immediate perioperative cognitive and neuroinflammatory outcomes

BackgroundPotassium channels (KCa3.1; Kv1.3; Kir2.1) are necessary for microglial activation, a pivotal requirement for the development of Perioperative Neurocognitive Disorders (PNDs). We previously reported on the role of microglial Kv1.3 for PNDs; the present study sought to determine whether inhibiting KCa3.1 channel activity affects neuroinflammation and prevents development of PND.MethodsMice (wild-type [WT] and KCa3.1−/−) underwent aseptic tibial fracture trauma under isoflurane anesthesia or received anesthesia alone. WT mice received either TRAM34 (a specific KCa3.1 channel inhibitor) dissolved in its vehicle (miglyol) or miglyol alone. Spatial memory was assessed in the Y-maze paradigm 6 h post-surgery/anesthesia. Circulating interleukin-6 (IL-6) and high mobility group box-1 protein (HMGB1) were assessed by ELISA, and microglial activitation Iba-1 staining.ResultsIn WT mice surgery induced significant cognitive decline in the Y-maze test, p = 0.019), microgliosis (p = 0.001), and increases in plasma IL-6 (p = 0.002) and HMGB1 (p = 0.001) when compared to anesthesia alone. TRAM34 administration attenuated the surgery-induced changes in cognition, microglial activation, and HMGB1 but not circulating IL-6 levels. In KCa3.1−/− mice surgery neither affected cognition nor microgliosis, although circulating IL-6 levels did increase (p < 0.001).ConclusionSimilar to our earlier report with Kv1.3, perioperative microglial KCa3.1 blockade decreases immediate perioperative cognitive changes, microgliosis as well as the peripheral trauma marker HMGB1 although surgery-induced IL-6 elevation was unchanged. Future research should address whether a synergistic interaction exists between blockade of Kv1.3 and KCa3.1 for preventing PNDs.

Blockade of Kv1.3 Potassium Channel Inhibits Microglia-Mediated Neuroinflammation in Epilepsy

Epilepsy is a chronic neurological disorder whose pathophysiology relates to inflammation. The potassium channel Kv1.3 in microglia has been reported as a promising therapeutic target in neurological diseases in which neuroinflammation is involved, such as multiple sclerosis (MS), Alzheimer's disease (AD), Parkinson's disease (PD), and middle cerebral artery occlusion/reperfusion (MCAO/R). Currently, little is known about the relationship between Kv1.3 and epilepsy. In this study, we found that Kv1.3 was upregulated in microglia in the KA-induced mouse epilepsy model. Importantly, blocking Kv1.3 with its specific small-molecule blocker 5-(4-phenoxybutoxy)psoralen (PAP-1) reduced seizure severity, prolonged seizure latency, and decreased neuronal loss. Mechanistically, we further confirmed that blockade of Kv1.3 suppressed proinflammatory microglial activation and reduced proinflammatory cytokine production by inhibiting the Ca2+/NF-κB signaling pathway. These results shed light on the critical function of microglial Kv1.3 in epilepsy and provided a potential therapeutic target.

Kv1.1 channels inhibition in the rat motor cortex recapitulates seizures associated with anti-LGI1 encephalitis.

Autoimmune encephalitis associated with antibodies directed against the leucine-rich glioma inactivated 1 (LGI1) protein is responsible for specific tonic-dystonic motor seizures. Although dysfunctions in neuronal excitability have been associated with anti-LGI1 autoantibodies, their relation to seizures remain inconclusive. We developed a new in vivo experimental rat model to determine whether inhibition of Kv1.1 channels by dentrotoxin-K (DTX) in the primary motor cortex (M1) could recapitulate the human seizures and to elucidate their subtending cortical mechanisms. Comparing electro-clinical features of DTX-induced seizures in rats with those recorded from a cohort of anti-LGI1 encephalitis patients revealed striking similarities in their electroencephalographic (EEG) signature, frequency of recurrence and semiology. By combining multi-site extracellular and intracellular recordings of M1 pyramidal neurons in DTX rats, we demonstrated that the blockade of Kv1.1 channels induced a sequence of changes in neuronal excitability and synaptic activity, leading to massive suprathreshold membrane depolarizations underlying the paroxysmal EEG activity. Our results suggest the central role of Kv1.1 channels disruption in the emergence of anti-LGI1-associated seizures and suggest that this new rodent model could serve future investigations on ictogenesis in autoimmune encephalitis.

Inhibitory effect of the selective serotonin reuptake inhibitor paroxetine on human Kv1.3 channels

Paroxetine is one of the most effective selective serotonin reuptake inhibitors used to treat depressive and panic disorders that reduce the viability of human T lymphocytes, in which Kv1.3 channels are highly expressed. We examined whether paroxetine could modulate human Kv1.3 channels acutely and directly with the aim of understanding the biophysical effects and the underlying mechanisms of the drug. Kv1.3 channel proteins were expressed in Xenopus oocytes. Paroxetine rapidly inhibited the steady-state current and peak current of these channels within 6 min in a concentration-dependent manner; IC50s were 26.3 μM and 53.9 μM, respectively, and these effects were partially reversed by washout, which excluded the possibility of genomic regulation. At the same test voltage, paroxetine blockade of the steady-state currents was higher than that of the peak currents, and the inhibition of the steady-state current increased relative to the degree of depolarization. Paroxetine decreased the inactivation time constant in a concentration-dependent manner, but it did not affect the activation time constant, which resulted in the acceleration of intrinsic inactivation without changing ultrarapid activation. Blockade of Kv1.3 channels by paroxetine exhibited more rapid inhibition at higher activation frequencies showing the use-dependency of the blockade. Overall, these results show that paroxetine directly suppresses human Kv1.3 channels in an open state and accelerates the process of steady-state inactivation; thus, we have revealed a biophysical mechanism for possible acute immunosuppressive effects of paroxetine.

IL-17 Inhibits Oligodendrocyte Progenitor Cell Proliferation and Differentiation by Increasing K+ Channel Kv1.3

Interleukin 17 (IL-17) is a signature cytokine of Th17 cells. IL-17 level is significantly increased in inflammatory conditions of the CNS, including but not limited to post-stroke and multiple sclerosis. IL-17 has been detected direct toxicity on oligodendrocyte (Ol) lineage cells and inhibition on oligodendrocyte progenitor cell (OPC) differentiation, and thus promotes myelin damage. The cellular mechanism of IL-17 in CNS inflammatory diseases remains obscure. Voltage-gated K+ (Kv) channel 1.3 is the predominant Kv channel in Ol and potentially involved in Ol function and cell cycle regulation. Kv1.3 of T cells involves in immunomodulation of inflammatory progression, but the role of Ol Kv1.3 in inflammation-related pathogenesis has not been fully investigated. We hypothesized that IL-17 induces myelin injury through Kv1.3 activation. To test the hypothesis, we studied the involvement of OPC/Ol Kv1.3 in IL-17-induced Ol/myelin injury in vitro and in vivo. Kv1.3 currents and channel expression gradually decreased during the OPC development. Application of IL-17 to OPC culture increased Kv1.3 expression, leading to a decrease of AKT activation, inhibition of proliferation and myelin basic protein reduction, which were prevented by a specific Kv1.3 blocker 5-(4-phenoxybutoxy) psoralen. IL-17-caused myelin injury was validated in LPC-induced demyelination mouse model, particularly in corpus callosum, which was also mitigated by aforementioned Kv1.3 antagonist. IL-17 altered Kv1.3 expression and resultant inhibitory effects on OPC proliferation and differentiation may by interrupting AKT phosphorylating activation. Taken together, our results suggested that IL-17 impairs remyelination and promotes myelin damage by Kv1.3-mediated Ol/myelin injury. Thus, blockade of Kv1.3 as a potential therapeutic strategy for inflammatory CNS disease may partially attribute to the direct protection on OPC proliferation and differentiation other than immunomodulation.

Potassium channel blocking 1,2-bis(aryl)ethane-1,2-diamines active as antiarrhythmic agents

Atrial fibrillation (AF) is a major cause of stroke, heart failure, sudden death and cardiovascular morbidity. The Kv1.5 potassium channel conducts the IKur current and has been demonstrated to be predominantly expressed in atrial versus ventricular tissue. Blockade of Kv1.5 has been proven to be an effective approach to restoring and maintaining sinus rhythm in preclinical models of AF. In the clinical setting, however, the therapeutic value of this approach remains an open question. Herein, we present synthesis and optimization of a novel series of 1,2-bis(aryl)ethane-1,2-diamines with selectivity for Kv1.5 over other potassium ion channels. The effective refractory period in the right atrium (RAERP) in a rabbit PD model was investigated for a selection of potent and selective compounds with balanced DMPK properties. The most advanced compound (10) showed nanomolar potency in blocking Kv1.5 in human atrial myocytes and based on the PD data, the estimated dose to man is 700 mg/day. As previously reported, 10 efficiently converted AF to sinus rhythm in a dog disease model.

Kv1.3 channel blockade with the Vm24 scorpion toxin attenuates the CD4+ effector memory T cell response to TCR stimulation

BackgroundIn T cells, the Kv1.3 and the KCa3.1 potassium channels regulate the membrane potential and calcium homeostasis. Notably, during TEM cell activation, the number of Kv1.3 channels on the cell membrane dramatically increases. Kv1.3 blockade results in inhibition of Ca2+ signaling in TEM cells, thus eliciting an immunomodulatory effect. Among the naturally occurring peptides, the Vm24 toxin from the Mexican scorpion Vaejovis mexicanus is the most potent and selective Kv1.3 channel blocker known, which makes it a promissory candidate for its use in the clinic. We have shown that addition of Vm24 to TCR-activated human T cells inhibits CD25 expression, cell proliferation and reduces delayed-type hypersensitivity reactions in a chronic inflammation model. Here, we used the Vm24 toxin as a tool to investigate the molecular events that follow Kv1.3 blockade specifically on human CD4+ TEM cells as they are actively involved in inflammation and are key mediators of autoimmune diseases.MethodsWe combined cell viability, activation, and multiplex cytokine assays with a proteomic analysis to identify the biological processes affected by Kv1.3 blockade on healthy donors CD4+ TEM cells, following TCR activation in the presence or absence of the Vm24 toxin.ResultsThe peptide completely blocked Kv1.3 channels currents without impairing TEM cell viability, and in response to TCR stimulation, it inhibited the expression of the activation markers CD25 and CD40L (but not that of CD69), as well as the secretion of the pro-inflammatory cytokines IFN-γ and TNF and the anti-inflammatory cytokines IL-4, IL-5, IL-9, IL-10, and IL-13. These results, in combination with data from the proteomic analysis, indicate that the biological processes most affected by the blockade of Kv1.3 channels in a T cell activation context were cytokine-cytokine receptor interaction, mRNA processing via spliceosome, response to unfolded proteins and intracellular vesicle transport, targeting the cell protein synthesis machinery.ConclusionsThe Vm24 toxin, a highly specific inhibitor of Kv1.3 channels allowed us to define downstream functions of the Kv1.3 channels in human CD4+ TEM lymphocytes. Blocking Kv1.3 channels profoundly affects the mRNA synthesis machinery, the unfolded protein response and the intracellular vesicle transport, impairing the synthesis and secretion of cytokines in response to TCR engagement, underscoring the role of Kv1.3 channels in regulating TEM lymphocyte function.

Enhancement of 5-HT2A receptor function and blockade of Kv1.5 by MK801 and ketamine: implications for PCP derivative-induced disease models

MK801 and ketamine, which are phencyclidine (PCP) derivative N-methyl-d-aspartate receptor (NMDAr) blockers, reportedly enhance the function of 5-hydroxytryptamine (HT)-2A receptors (5-HT2ARs). Both are believed to directly affect the pathogenesis of schizophrenia, as well as hypertension. 5-HT2AR signaling involves the inhibition of Kv conductance. This study investigated the interaction of these drugs with Kv1.5, which plays important roles in 5-HT2AR signaling and in regulating the excitability of the cardiovascular and nervous system, and the potential role of this interaction in the enhancement of the 5-HT2AR-mediated response. Using isometric organ bath experiments with arterial rings and conventional whole-cell patch-clamp recording of Chinese hamster ovary (CHO) cells ectopically overexpressing Kv1.5, we examined the effect of ketamine and MK801 on 5-HT2AR-mediated vasocontraction and Kv1.5 channels. Both ketamine and MK801 potentiated 5-HT2AR-mediated vasocontraction. This potentiation of 5-HT2AR function occurred in a membrane potential-dependent manner, indicating the involvement of ion channel(s). Both ketamine and MK801 rapidly and directly inhibited Kv1.5 channels from the extracellular side independently of NMDArs. The potencies of MK801 in facilitating the 5-HT2AR-mediated response and blocking Kv1.5 were higher than those of ketamine. Our data demonstrated the direct inhibition of Kv1.5 channels by MK801/ketamine and indicated that this inhibition may potentiate the functions of 5-HT2ARs. We suggest that 5-HT2AR-Kv1.5 may serve as a receptor-effector module in response to 5-HT and is a promising target in the pathogenesis of MK801-/ketamine-induced disease states such as hypertension and schizophrenia.

Immunomodulation by memantine in therapy of Alzheimer's disease is mediated through inhibition of Kv1.3 channels and T cell responsiveness.

Memantine is approved for the treatment of advanced Alzheimer's disease (AD) and reduces glutamate-mediated neuronal excitotoxicity by antagonism of N-methyl-D-aspartate receptors. In the pathophysiology of AD immune responses deviate and infectious side effects are observed during memantine therapy. However, the particular effects of memantine on human T lymphocytes are unresolved. Here, we provide evidence that memantine blocks Kv1.3 potassium channels, inhibits CD3-antibody- and alloantigen-induced proliferation and suppresses chemokine-induced migration of peripheral blood T cells of healthy donors. Concurrent with the in vitro data, CD4+ T cells from AD patients receiving therapeutic doses of memantine show a transient decline of Kv1.3 channel activity and a long-lasting reduced proliferative response to alloantigens in mixed lymphocyte reactions. Furthermore, memantine treatment provokes a profound depletion of peripheral blood memory CD45RO+ CD4+ T cells. Thus, standard doses of memantine profoundly reduce T cell responses in treated patients through blockade of Kv1.3 channels. This may normalize deviant immunopathology in AD and contribute to the beneficial effects of memantine, but may also account for the enhanced infection rate.

Targeting Kv1.3 channels to reduce white matter pathology after traumatic brain injury

Axonal injury is present in essentially all clinically significant cases of traumatic brain injury (TBI). While no effective treatment has been identified to date, experimental TBI models have shown promising axonal protection using immunosuppressants FK506 and Cyclosporine-A, with treatment benefits attributed to calcineurin inhibition or protection of mitochondrial function. However, growing evidence suggests neuroprotective efficacy of these compounds may also involve direct modulation of ion channels, and in particular Kv1.3. The present study tested whether blockade of Kv1.3 channels, using Clofazimine (CFZ), would alleviate TBI-induced white matter pathology in rodents. Postinjury CFZ administration prevented suppression of compound action potential (CAP) amplitude in the corpus callosum of adult rats following midline fluid percussion TBI, with injury and treatment effects primarily expressed in unmyelinated CAPs. Kv1.3 protein levels in callosal tissue extracts were significantly reduced postinjury, but this loss was prevented by CFZ treatment. In parallel, CFZ also attenuated the injury-induced elevation in pro-inflammatory cytokine IL1-β. The effects of CFZ on glial function were further studied using mixed microglia/astrocyte cell cultures derived from P3-5 mouse corpus callosum. Cultures of callosal glia challenged with lipopolysaccharide exhibited a dramatic increase in IL1-β levels, accompanied by reactive morphological changes in microglia, both of which were attenuated by CFZ treatment. These results support a cell specific role for Kv1.3 signaling in white matter pathology after TBI, and suggest a treatment approach based on the blockade of these channels. This therapeutic strategy may be especially efficacious for normalizing neuro-glial interactions affecting unmyelinated axons after TBI.

Blockade of Kv1.5 channels by the antidepressant drug sertraline.

Sertraline, a selective serotonin reuptake inhibitor (SSRI), has been reported to lead to cardiac toxicity even at therapeutic doses including sudden cardiac death and ventricular arrhythmia. And in a SSRI-independent manner, sertraline has been known to inhibit various voltage-dependent channels, which play an important role in regulation of cardiovascular system. In the present study, we investigated the action of sertraline on Kv1.5, which is one of cardiac ion channels. The eff ect of sertraline on the cloned neuronal rat Kv1.5 channels stably expressed in Chinese hamster ovary cells was investigated using the whole-cell patch-clamp technique. Sertraline reduced Kv1.5 whole-cell currents in a reversible concentration-dependent manner, with an IC50 value and a Hill coefficient of 0.71 µM and 1.29, respectively. Sertraline accelerated the decay rate of inactivation of Kv1.5 currents without modifying the kinetics of current activation. The inhibition increased steeply between –20 and 0 mV, which corresponded with the voltage range for channel opening. In the voltage range positive to +10 mV, inhibition displayed a weak voltage dependence, consistent with an electrical distance δ of 0.16. Sertraline slowed the deactivation time course, resulting in a tail crossover phenomenon when the tail currents, recorded in the presence and absence of sertraline, were superimposed. Inhibition of Kv1.5 by sertraline was use-dependent. The present results suggest that sertraline acts on Kv1.5 currents as an open-channel blocker.

Blockade of Kv1.5 by paroxetine, an antidepressant drug.

Paroxetine, a selective serotonin reuptake inhibitor (SSRI), has been reported to have an effect on several ion channels including human ether-a-go-go-related gene in a SSRI-independent manner. These results suggest that paroxetine may cause side effects on cardiac system. In this study, we investigated the effect of paroxetine on Kv1.5, which is one of cardiac ion channels. The action of paroxetine on the cloned neuronal rat Kv1.5 channels stably expressed in Chinese hamster ovary cells was investigated using the whole-cell patch-clamp technique. Paroxetine reduced Kv1.5 whole-cell currents in a reversible concentration-dependent manner, with an IC50 value and a Hill coefficient of 4.11 µM and 0.98, respectively. Paroxetine accelerated the decay rate of inactivation of Kv1.5 currents without modifying the kinetics of current activation. The inhibition increased steeply between -30 and 0 mV, which corresponded with the voltage range for channel opening. In the voltage range positive to 0 mV, inhibition displayed a weak voltage dependence, consistent with an electrical distance δ of 0.32. The binding (k+1) and unbinding (k-1) rate constants for paroxetine-induced block of Kv1.5 were 4.9 µM-1s-1 and 16.1 s-1, respectively. The theoretical KD value derived by k-1/k+1 yielded 3.3 µM. Paroxetine slowed the deactivation time course, resulting in a tail crossover phenomenon when the tail currents, recorded in the presence and absence of paroxetine, were superimposed. Inhibition of Kv1.5 by paroxetine was use-dependent. The present results suggest that paroxetine acts on Kv1.5 currents as an open-channel blocker.

Evidence of more ion channels inhibited by celecoxib: KV1.3 and L-type Ca(2+) channels.

BackgroundCelecoxib, a selective inhibitor of cyclooxygenase-2, can directly modulate many voltage-activated potassium, sodium and calcium channels and alter functioning of excitable cells. The inhibitory and facilitating effects of celecoxib on ion channels occur at low micromolar concentrations, bordering on therapeutic concentrations achievable in the clinical setting. The experiments described here were performed with the goals (1) to increase the range of ion channels tested, and (2) to examine possible differences in celecoxib’s effects on channels from different species.FindingsThe channels examined in this study using patch-clamp and intracellular recording methods were human KV1.3 channels expressed in CHO cells, L-type Ca2+ channels (LTCC) from guinea pig cardiomyocytes, and LTCCs from Drosophila larval body-wall muscles. Celecoxib inhibited KV1.3 currents with IC50 of 5.0 μM at the end of 200 ms pulses to +20 mV. Celecoxib inhibited peak currents through guinea pig and Drosophila LTCCs with IC50s of 10.6 and 76.0 μM, respectively.ConclusionsAs blockade of KV1.3 channels is associated with suppression of inflammatory immune reactions, the finding that celecoxib can inhibit these channels raises a question of possible contribution of KV1.3 inhibition to the anti-inflammatory effects of celecoxib. On the other hand, the Ca2+ channel results are consistent with previous observations indicating that, in contrast to K+ channels, strength of celecoxib effects on LTCCs strongly varies from species to species.

NMDA-receptor antagonists block B-cell function but foster IL-10 production in BCR/CD40-activated B cells.

BackgroundB cells are important effectors and regulators of adaptive and innate immune responses, inflammation and autoimmunity, for instance in anti-NMDA-receptor (NMDAR) encephalitis. Thus, pharmacological modulation of B-cell function could be an effective regimen in therapeutic strategies. Since the non-competitive NMDAR antagonist memantine is clinically applied to treat advanced Alzheimer`s disease and ketamine is supposed to improve the course of resistant depression, it is important to know how these drugs affect B-cell function.ResultsNon-competitive NMDAR antagonists impaired B-cell receptor (BCR)- and lipopolysaccharide (LPS)-induced B-cell proliferation, reduced B-cell migration towards the chemokines SDF-1α and CCL21 and downregulated IgM and IgG secretion. Mechanistically, these effects were mediated through a blockade of Kv1.3 and KCa3.1 potassium channels and resulted in an attenuated Ca2+-flux and activation of Erk1/2, Akt and NFATc1. Interestingly, NMDAR antagonist treatment increased the frequency of IL-10 producing B cells after BCR/CD40 stimulation.ConclusionsNon-competitive NMDAR antagonists attenuate BCR and Toll-like receptor 4 (TLR4) B-cell signaling and effector function and can foster IL-10 production. Consequently, NMDAR antagonists may be useful to target B cells in autoimmune diseases or pathological systemic inflammation. The drugs’ additional side effects on B cells should be considered in treatments of neuronal disorders with NMDAR antagonists.

Stimulation of glucose uptake in murine soleus muscle and adipocytes by 5-(4-phenoxybutoxy)psoralen (PAP-1) may be mediated by Kv1.5 rather than Kv1.3.

Kv1 channels are shaker-related potassium channels that influence insulin sensitivity. Kv1.3−/− mice are protected from diet-induced insulin resistance and some studies suggest that Kv1.3 inhibitors provide similar protection. However, it is unclear whether blockade of Kv1.3 in adipocytes or skeletal muscle increases glucose uptake. There is no evidence that the related channel Kv1.5 has any influence on insulin sensitivity and its expression in adipose tissue has not been reported. PAP-1 is a selective inhibitor of Kv1.3, with 23-fold, 32-fold and 125-fold lower potencies as an inhibitor of Kv1.5, Kv1.1 and Kv1.2 respectively. Soleus muscles from wild-type and genetically obese ob/ob mice were incubated with 2-deoxy[1-14C]-glucose for 45 min and formation of 2-deoxy[1-14C]-glucose-6-phosphate was measured. White adipocytes were incubated with D-[U-14C]-glucose for 1 h. TNFα and Il-6 secretion from white adipose tissue pieces were measured by enzyme-linked-immunoassay. In the absence of insulin, a high concentration (3 µM) of PAP-1 stimulated 2-deoxy[1-14C]-glucose uptake in soleus muscle of wild-type and obese mice by 30% and 40% respectively, and in adipocytes by 20% and 50% respectively. PAP-1 also stimulated glucose uptake by adipocytes at the lower concentration of 1 µM, but at 300 nM, which is still 150-fold higher than its EC50 value for inhibition of the Kv1.3 channel, it had no effect. In the presence of insulin, PAP-1 (3 µM) had a significant effect only in adipocytes from obese mice. PAP-1 (3 µM) reduced the secretion of TNFα by adipose tissue but had no effect on the secretion of IL-6. Expression of Kv1.1, Kv1.2, Kv1.3 and Kv1.5 was determined by RT-PCR. Kv1.3 and Kv1.5 mRNA were detected in liver, gastrocnemius muscle, soleus muscle and white adipose tissue from wild-type and ob/ob mice, except that Kv1.3 could not be detected in gastrocnemius muscle, nor Kv1.5 in liver, of wild-type mice. Expression of both genes was generally higher in liver and muscle of ob/ob mice compared to wild-type mice. Kv1.5 appeared to be expressed more highly than Kv1.3 in soleus muscle, adipose tissue and adipocytes of wild-type mice. Expression of Kv1.2 appeared to be similar to that of Kv1.3 in soleus muscle and adipose tissue, but Kv1.2 was undetectable in adipocytes. Kv1.1 could not be detected in soleus muscle, adipose tissue or adipocytes. We conclude that inhibition of Kv1 channels by PAP-1 stimulates glucose uptake by adipocytes and soleus muscle of wild-type and ob/ob mice, and reduces the secretion of TNFα by adipose tissue. However, these effects are more likely due to inhibition of Kv1.5 than to inhibition of Kv1.3 channels.

Kv1.3 channels modulate human vascular smooth muscle cells proliferation independently of mTOR signaling pathway

Phenotypic modulation (PM) of vascular smooth muscle cells (VSMCs) is central to the process of intimal hyperplasia which constitutes a common pathological lesion in occlusive vascular diseases. Changes in the functional expression of Kv1.5 and Kv1.3 currents upon PM in mice VSMCs have been found to contribute to cell migration and proliferation. Using human VSMCs from vessels in which unwanted remodeling is a relevant clinical complication, we explored the contribution of the Kv1.5 to Kv1.3 switch to PM. Changes in the expression and the functional contribution of Kv1.3 and Kv1.5 channels were studied in contractile and proliferating VSMCs obtained from human donors. Both a Kv1.5 to Kv1.3 switch upon PM and an anti-proliferative effect of Kv1.3 blockers on PDGF-induced proliferation were observed in all vascular beds studied. When investigating the signaling pathways modulated by the blockade of Kv1.3 channels, we found that anti-proliferative effects of Kv1.3 blockers on human coronary artery VSMCs were occluded by selective inhibition of MEK/ERK and PLCγ signaling pathways, but were unaffected upon blockade of PI3K/mTOR pathway. The temporal course of the anti-proliferative effects of Kv1.3 blockers indicates that they have a role in the late signaling events essential for the mitogenic response to growth factors. These findings establish the involvement of Kv1.3 channels in the PM of human VSMCs. Moreover, as current therapies to prevent restenosis rely on mTOR blockers, our results provide the basis for the development of novel, more specific therapies.

P0111 Voltage-gated K+ channels as potential therapeutic targets for lung cancer therapy

Background Voltage-gated K + (kV) channels are considered to be a regulator of membrane potential and cell excitability. Recently, evidence has indicated that several kV channel subtypes contribute to the control of cell proliferation in various types of cancer cells and are worthy of investigation as molecular targets for cancer therapy. Methods We investigated the effects of the dendrotoxin-κ (DTX-κ) and margatoxin (MgTX) which are Kv1.1 and Kv1.3 specific blockers, respectively, on tumour formation using a xenograft model induced by A549 and H460 cells. Findings The mRNA and protein of Kv1.1 and Kv1.3 was expressed in A549 and H460 cells and the blockade of Kv1.1 by DTX-κ or Kv1.3 by MgTX reduced the formation of tumours in nude mice. Furthermore, treatment with DTX-κ significantly increased the protein expression of p21 Waf 1/ Cip 1 , p27 Kip 1 , and p15 INK 4 B , and significantly decreased the protein expression of cyclin D3 in tumour tissues compared to control. In addition, selective inhibition of Kv1.3 significantly increased expression level of p21 Waf 1/ Cip 1 and significantly decreased the expression level of Cdk4 and cyclin D3. Moreover, we found that the effect of DTX- κ and gefitinib was synergistic in H460 and A549 cells. Interpretation These results suggest that certain kV channels may serve as novel therapeutic targets for lung cancer therapy and could be one of multiple molecular targets to overcome tyrosine kinase inhibitor resistance. This work is supported by National Research Foundation of Korea (2012R1A2A2A01047151).

Blockade of Kv1.3 channels ameliorates radiation-induced brain injury

Tumors affecting the head, neck, and brain account for significant morbidity and mortality. The curative efficacy of radiotherapy for these tumors is well established, but radiation carries a significant risk of neurologic injury. So far, neuroprotective therapies for radiation-induced brain injury are still limited. In this study we demonstrate that Stichodactyla helianthus (ShK)-170, a specific inhibitor of the voltage-gated potassium (Kv)1.3 channel, protected mice from radiation-induced brain injury. Mice were treated with ShK-170 for 3 days immediately after brain irradiation. Radiation-induced brain injury was assessed by MRI scans and a Morris water maze. Pathophysiological change of the brain was measured by immunofluorescence. Gene and protein expressions of Kv1.3 and inflammatory factors were measured by quantitative real-time PCR, reverse transcription PCR, ELISA assay, and western blot analyses. Kv currents were recorded in the whole-cell configuration of the patch-clamp technique. Radiation increased Kv1.3 mRNA and protein expression in microglia. Genetic silencing of Kv1.3 by specific short interference RNAs or pharmacological blockade with ShK-170 suppressed radiation-induced production of the proinflammatory factors interleukin-6, cyclooxygenase-2, and tumor necrosis factor-α by microglia. ShK-170 also inhibited neurotoxicity mediated by radiation-activated microglia and promoted neurogenesis by increasing the proliferation of neural progenitor cells. The therapeutic effect of ShK-170 is mediated by suppression of microglial activation and microglia-mediated neurotoxicity and enhanced neurorestoration by promoting proliferation of neural progenitor cells.

Blockade of Kv1.3 potassium channels inhibits differentiation and granzyme B secretion of human CD8+ T effector memory lymphocytes.

Increased expression of the voltage-gated potassium channel Kν1.3 on activated effector memory T cells (TEM) is associated with pathology in multiple sclerosis (MS). To date, most studies of Kν1.3 channels in MS have focused on CD4+ TEM cells. Much less is known about the functional relevance of Kv1.3 on CD8+ TEM cells. Herein, we examined the effects of Kν1.3 blockade on CD8+ T cell proliferation, differentiation into cytotoxic effector cells, and release of granzyme B (GrB), a key effector of CD8+ T cell-mediated cytotoxicity. We confirmed the expression of Kv1.3 channels on activated human CD8+ T lymphocytes by immunofluorescent staining. To test the functional relevance of the Kv1.3 channel in CD8+ T cells, we inhibited this channel via pharmacological blockers or a lentiviral-dominant negative (Kv1.xDN) approach and determined the effects of the blockade on critical pathogenic parameters of CD8+ T cells. We found that blockade of Kv1.3 with both lentivirus and pharmacologic agents effectively inhibited cytotoxic effector memory cells’ proliferation, secretion of GrB, and their ability to kill neural progenitor cells. Intriguingly, the KvDN transduced T cells exhibited arrested differentiation from central memory (TCM) to effector memory (TEM) states. Transduction of cells that had already differentiated into TEM with KvDN led to their conversion into TCM. CD8+ TEM have a critical role in MS and other autoimmune diseases. Our present results indicate a critical role for Kv1.3 in the conversion of CD8+ T cells into potential pathogenic effector cells with cytotoxic function.

KCa3.1 Blockers Inhibit Cell Proliferation in Chronic Lymphocytic Leukemia

AbstractAbstract 3887Potassium (K)-channels play an important role in regulating cell proliferation by maintenance of the membrane potential and subsequent Ca2+ signaling. Out of 80 known human K-channel genes only the voltage gated K-channel Kv1.3 and the calcium-gated K-channel KCa3.1 are expressed in lymphocytes, with expression levels varying greatly depending on lymphocyte maturation and activation status (for review see Cahalan MD, Chandy KG, Immunol Rev. 2009). Accordingly, proliferation of various lymphocyte subtypes can be inhibited by blockade of the respective predominant K-channel. As the modulation of K-channel expression on malignantly transformed lymphocytes and their potential as therapeutic targets has been largely overlooked, we characterized the expression and function of Kv1.3 and KCa3.1 in Chronic Lymphocytic Leukemia (CLL).Primary cells from unselected CLL-patients were isolated from peripheral blood mononuclear cells (PBMCs). Comparison of Kv1.3 and KCa3.1 levels on unstimulated CLL-cells versus PMA/ionomycin-activated CLL-cells revealed a significant reduction in the Kv1.3/KCa3.1 ratio (n=31, 1.526 vs. 0.9054, p=0.0005), as evidenced on mRNA and protein levels by RT-PCR and patch clamp analysis, respectively. Stimulation of CLL-cells with enriched and activated autologous CD4+ T-cells resulted in higher CLL-cell activation as measured by CD80/86 expression, and an even more pronounced reduction of the Kv1.3/KCa3.1 ratio. This stimulation protocol also effectively induced CLL-cell proliferation as verified by Ki-67 expression and CFSE dilution via flow cytometric measurement. Highly activated and/or proliferating CLL-cells consistently up-regulated KCa3.1 (RT-PCR: Ki-67 n=5, p=0.0013; CFSE n=5, p=0.0436; immunofluorescence (IF) staining - n=4, p=0.0121), whereas Kv1.3 was fairly low. Consistent with our in vitro data, CLL-cells in lymph node and bone marrow, believed to be primary sites of CLL-proliferation in vivo, were highly positive for KCa3.1 channels in contrast to CLL-cells from the peripheral blood as revealed by IF staining on paraffin-embedded tissue sections.In light of the significantly increased KCa3.1-expression on activated and proliferating CLL-cells, we investigated whether specific blockade of KCa3.1 could inhibit CLL-cell proliferation. CLL-cells in PBMCs were pre-stimulated by co-culture with CD40L expressing (murine) fibroblasts for 24 hours, and were treated with highly specific blockers for KCa3.1 (TRAM-34 or Clotrimazole) or Kv1.3 (PAP-1 or Psora-4) prior to addition of α-CD3/CD28 beads to activate autologous T-cells. KCa3.1 blockade could effectively diminish CLL-cell cycle entry in all samples investigated (TRAM-34: n=12, p<0.0001; Clotrimazole: n=5, p=0.0625) with a mean relative reduction in Ki-67+ cells of 52% (SD=1.56) for TRAM-34 (see Figure left) and 53% (SD=1.11) for Clotrimazole. This antagonizing effect of TRAM-34 was clearly dose-dependent (n=10) without affecting CLL-cell viability (see Figure right) nor activation. Viability, activation and proliferation of T-cells and fibroblasts present in the co-culture system were not affected by TRAM-34. Blockade of Kv1.3 did not reduce proliferation in CLL-cells.In summary, we showed that CLL-cells exhibit significant changes in their K-channel constitution following activation and proliferation, analogous to healthy B-cells. Notably, in vitro CLL-cell proliferation was effectively inhibited by highly specific KCa3.1 blockers. Thus far in vitro testing of potential CLL-drugs has been primarily performed on G0-arrested CLL-cells, sometimes in co-culture with stromal cells to enhance their viability. Our approach attempts to specifically target proliferating CLL-cells, presumably the most relevant CLL-cell fraction contributing to disease progression. Given their low toxicity profile, KCa3.1 blockers could represent a promising therapeutic option in CLL. [Display omitted] Disclosures:No relevant conflicts of interest to declare.