Structures of the T cell potassium channel Kv1.3 with immunoglobulin modulators.
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ABSTRACT:
SUBMITTER: Selvakumar P
PROVIDER: S-EPMC9253088 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Kv1.3 is a multifunctional potassium channel implicated in multiple pathologies, including cancer. However, how it is involved in disease progression is not fully clear. We interrogated the interactome of Kv1.3 in intact cells using BioID proximity labeling, revealing that Kv1.3 interacts with STAT3- and p53-linked pathways. To prove the relevance of Kv1.3 and of its interactome in the context of tumorigenesis, we generated stable melanoma clones, in which ablation of Kv1.3 remodeled gene expression, reduced proliferation and colony formation, yielded fourfold smaller tumors, and decreased metastasis in vivo in comparison to WT cells. Kv1.3 deletion or pharmacological inhibition of mitochondrial Kv1.3 increased mitochondrial Reactive Oxygen Species release, decreased STAT3 phosphorylation, stabilized the p53 tumor suppressor, promoted metabolic switch, and altered the expression of several BioID-identified Kv1.3-networking proteins in tumor tissues. Collectively, our work revealed the tumor-promoting Kv1.3-interactome landscape, thus opening the way to target Kv1.3 not only as an ion-conducting entity but also as a signaling hub.
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Project description:The potassium channel Kv1.3 has recently been located to the inner mitochondrial membrane of lymphocytes. Here, we show that mouse and human cells either genetically deficient in Kv1.3 or transfected with siRNA to suppress Kv1.3-expression resisted apoptosis induced by several stimuli, including Bax over-expression [corrected]. Retransfection of either Kv1.3 or a mitochondrial-targeted Kv1.3 restored cell death . Bax interacted with and functionally inhibited mitochondrial Kv1.3. Incubation of isolated Kv1.3-positive mitochondria with recombinant Bax, t-Bid, or toxins that bind to and inhibit Kv1.3 successively triggered hyperpolarization, formation of reactive oxygen species, release of cytochrome c, and marked depolarization. Kv1.3-deficient mitochondria were resistant to Bax, t-Bid, and the toxins. Mutation of Bax at K128, which corresponds to a conserved lysine in Kv1.3-inhibiting toxins, abrogated its effects on both Kv1.3 and mitochondria. These findings suggest that Bax mediates cytochrome c release and mitochondrial depolarization in lymphocytes, at least in part, via its interaction with mitochondrial Kv1.3.
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Project description:The voltage-gated potassium channel subfamily A member 3 (Kv1.3) dominantly expresses on T cells and neurons. Recently, the interaction between Kv1.3 and NavBeta1 subunits has been explored through ionic current measurements, but the molecular mechanism has not been elucidated yet. We explored the functional interaction between Kv1.3 and NavBeta1 through gating current measurements using the Cut-open Oocyte Voltage Clamp (COVC) technique. We showed that the N-terminal 1-52 sequence of hKv1.3 disrupts the channel expression on the Xenopus oocyte membrane, suggesting a potential role as regulator of hKv1.3 expression in neurons and lymphocytes. Our gating currents measurements showed that NavBeta1 interacts with the voltage sensing domain (VSD) of Kv1.3 through W172 in the transmembrane segment and modifies the gating operation. The comparison between G-V and Q-V with/without NavBeta1 indicates that NavBeta1 may strengthen the coupling between hKv1.3-VSD movement and pore opening, inducing the modification of kinetics in ionic activation and deactivation.
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Project description:It is widely known that ion channels are expressed in the plasma membrane. However, a few studies have suggested that several ion channels including voltage-gated K(+) (Kv) channels also exist in intracellular organelles where they are involved in the biochemical events associated with cell signaling. In the present study, Western blot analysis using fractionated protein clearly indicates that Kv1.3 channels are expressed in the nuclei of MCF7, A549, and SNU-484 cancer cells and human brain tissues. In addition, Kv1.3 is located in the plasma membrane and the nucleus of Jurkat T cells. Nuclear membrane hyperpolarization after treatment with margatoxin (MgTX), a specific blocker of Kv1.3 channels, provides evidence for functional channels at the nuclear membrane of A549 cells. MgTX-induced hyperpolarization is abolished in the nuclei of Kv1.3 silenced cells, and the effects of MgTX are dependent on the magnitude of the K(+) gradient across the nuclear membrane. Selective Kv1.3 blockers induce the phosphorylation of cAMP response element-binding protein (CREB) and c-Fos activation. Moreover, Kv1.3 is shown to form a complex with the upstream binding factor 1 in the nucleus. Chromatin immunoprecipitation assay reveals that Sp1 transcription factor is directly bound to the promoter region of the Kv1.3 gene, and the Sp1 regulates Kv1.3 expression in the nucleus of A549 cells. These results demonstrate that Kv1.3 channels are primarily localized in the nucleus of several types of cancer cells and human brain tissues where they are capable of regulating nuclear membrane potential and activation of transcription factors, such as phosphorylated CREB and c-Fos.
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Project description:AimsNeutrophil trafficking within the vasculature strongly relies on intracellular calcium signalling. Sustained Ca2+ influx into the cell requires a compensatory efflux of potassium to maintain membrane potential. Here, we aimed to investigate whether the voltage-gated potassium channel KV1.3 regulates neutrophil function during the acute inflammatory process by affecting sustained Ca2+ signalling.Methods and resultsUsing in vitro assays and electrophysiological techniques, we show that KV1.3 is functionally expressed in human neutrophils regulating sustained store-operated Ca2+ entry through membrane potential stabilizing K+ efflux. Inhibition of KV1.3 on neutrophils by the specific inhibitor 5-(4-Phenoxybutoxy)psoralen (PAP-1) impaired intracellular Ca2+ signalling, thereby preventing cellular spreading, adhesion strengthening, and appropriate crawling under flow conditions in vitro. Using intravital microscopy, we show that pharmacological blockade or genetic deletion of KV1.3 in mice decreased neutrophil adhesion in a blood flow dependent fashion in inflamed cremaster muscle venules. Furthermore, we identified KV1.3 as a critical component for neutrophil extravasation into the inflamed peritoneal cavity. Finally, we also revealed impaired phagocytosis of Escherichia coli particles by neutrophils in the absence of KV1.3.ConclusionWe show that the voltage-gated potassium channel KV1.3 is critical for Ca2+ signalling and neutrophil trafficking during acute inflammatory processes. Our findings do not only provide evidence for a role of KV1.3 for sustained calcium signalling in neutrophils affecting key functions of these cells, they also open up new therapeutic approaches to treat inflammatory disorders characterized by overwhelming neutrophil infiltration.
| S-EPMC8953450 | biostudies-literature
Project description: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.
| S-EPMC9740890 | biostudies-literature
Project description:Microglia are resident immune cells of the brain and regulate its inflammatory state. In neurodegenerative diseases, microglia transition from a homeostatic state to a state referred to as disease associated microglia (DAM). DAM express higher levels of proinflammatory signaling molecules, like STAT1 and TLR2, and show transitions in mitochondrial activity toward a more glycolytic response. Inhibition of Kv1.3 decreases the proinflammatory signature of DAM, though how Kv1.3 influences the response is unknown. Our goal was to establish the potential proteins interacting with Kv1.3 during transition to DAM. We utilized TurboID, a biotin ligase, fused to Kv1.3 to evaluate the potential interacting proteins with Kv1.3 via mass spectrometry in BV-2 microglia following TLR4-mediated activation. Electrophysiology, western blotting, and flow cytometry were used to evaluate Kv1.3 channel presence and TurboID biotinylation activity. We hypothesized that Kv1.3 contains domain-specific interactors that vary during a TLR4-induced inflammatory response, some of which are dependent on the PDZ-binding domain on the C-terminus. We determined that the N-terminus of Kv1.3 is responsible for trafficking Kv1.3 to the cell surface and mitochondria (e.g. NUNDC, TIMM50). Whereas, the C-terminus interacts with immune signaling proteins in an LPS-induced inflammatory response (e.g. STAT1, TLR2, and C3). There are 70 proteins that rely on the C-terminal PDZ-binding domain to interact with Kv1.3 (e.g. ND3, Snx3, and Sun1). Overall, we highlight that the Kv1.3 potassium channel functions beyond conducting the outward flux of potassium ions in an inflammatory context and that KV1.3 modulates activity of key immune signaling proteins, such as STAT1 and C3
2024-05-06 | PXD049433 | Pride
Project description:Recent genetic studies suggest a central role for innate immunity in Alzheimer's disease (AD) pathogenesis, wherein microglia orchestrate neuroinflammation. Kv1.3, a voltage-gated potassium channel of therapeutic relevance in autoimmunity, is upregulated by activated microglia and mediates amyloid-mediated microglial priming and reactive oxygen species production in vitro. We hypothesized that Kv1.3 channel expression is increased in human AD brain tissue. In a blinded postmortem immunohistochemical semi-quantitative analysis performed on ten AD patients and ten non-disease controls, we observed a significantly higher Kv1.3 staining intensity (p = 0.03) and Kv1.3-positive cell density (p = 0.03) in the frontal cortex of AD brains, compared to controls. This paralleled an increased number of Iba1-positive microglia in AD brains. Kv1.3-positive cells had microglial morphology and were associated with amyloid-? plaques. In immunofluorescence studies, Kv1.3 channels co-localized primarily with Iba1 but not with astrocyte marker GFAP, confirming that elevated Kv1.3 expression is limited to microglia. Higher Kv1.3 expression in AD brains was also confirmed by western blot analysis. Our findings support that Kv1.3 channels are biologically relevant and microglia-specific targets in human AD.
| S-EPMC4402159 | biostudies-literature
Project description:ObjectiveThe voltage-gated potassium channel Kv1.3, which is expressed on activated, disease-associated microglia and memory T cells, constitutes an attractive target for immunocytoprotection after endovascular thrombectomy (EVT). Using young male mice and rats we previously demonstrated that the Kv1.3 blocker PAP-1 when started 12 h after reperfusion dose-dependently reduces infarction and improves neurological deficit on day 8. However, these proof-of-concept findings are of limited translational value because the majority of strokes occur in patients over 65 and, when considering overall lifetime risk, in females. Here, we therefore tested whether Kv1.3 deletion or delayed pharmacological therapy would be beneficial in females and aged animals.MethodsTransient middle cerebral artery occlusion (tMCAO, 60 min) was induced in 16-week-old and 80-week-old male and female wild-type C57BL/6J and Kv1.3-/- mice. Stroke outcomes were assessed daily with the 14-score tactile and proprioceptive limp placing test and on day 8 before sacrifice by T2-weighted MRI. Young and old female mice were treated twice daily with 40 mg/kg PAP-1 starting 12 h after reperfusion. Microglia/macrophage activation and T-cell infiltration were evaluated in whole slide scans.ResultsKv1.3 deletion provided no significant benefit in young females but improved outcomes in young males, old males, and old females compared with wild-type controls of the same sex. Delayed PAP-1 treatment improved outcomes in both young and old females. In old females, Kv1.3 deletion and PAP-1 treatment significantly reduced Iba-1 and CD3 staining intensity in the ipsilateral hemisphere.InterpretationOur preclinical studies using aged and female mice further validate Kv1.3 inhibitors as potential adjunctive treatments for reperfusion therapy in stroke by providing both genetic and pharmacological verification.
| S-EPMC8528456 | biostudies-literature
Project description:The Kv1.3 potassium channel plays an essential role in effector memory T cells and has been implicated in several important autoimmune diseases including multiple sclerosis, psoriasis and type 1 diabetes. A number of potent small molecule inhibitors of Kv1.3 channel have been reported, some of which were found to be effective in various animal models of autoimmune diseases. We report herein the identification of clofazimine, a known anti-mycobacterial drug, as a novel inhibitor of human Kv1.3. Clofazimine was initially identified as an inhibitor of intracellular T cell receptor-mediated signaling leading to the transcriptional activation of human interleukin-2 gene in T cells from a screen of the Johns Hopkins Drug Library. A systematic mechanistic deconvolution revealed that clofazimine selectively blocked the Kv1.3 channel activity, perturbing the oscillation frequency of the calcium-release activated calcium channel, which in turn led to the inhibition of the calcineurin-NFAT signaling pathway. These effects of clofazimine provide the first line of experimental evidence in support of a causal relationship between Kv1.3 and calcium oscillation in human T cells. Furthermore, clofazimine was found to be effective in blocking human T cell-mediated skin graft rejection in an animal model in vivo. Together, these results suggest that clofazimine is a promising immunomodulatory drug candidate for treating a variety of autoimmune disorders.
| S-EPMC2602975 | biostudies-literature