Kv1.3-induced hyperpolarization is required for efficient Kaposi's sarcoma-associated herpesvirus lytic replication.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus that is linked directly to the development of Kaposi's sarcoma. KSHV establishes a latent infection in B cells, which can be reactivated to initiate lytic replication, producing infectious virions. Using pharmacological and genetic silencing approaches, we showed that the voltage-gated K+ channel Kv1.3 in B cells enhanced KSHV lytic replication. The KSHV replication and transcription activator (RTA) protein increased the abundance of Kv1.3 and led to enhanced K+ channel activity and hyperpolarization of the B cell membrane. Enhanced Kv1.3 activity promoted intracellular Ca2+ influx, leading to the Ca2+-driven nuclear localization of KSHV RTA and host nuclear factor of activated T cells (NFAT) proteins and subsequently increased the expression of NFAT1 target genes. KSHV lytic replication and infectious virion production were inhibited by Kv1.3 blockers or silencing. These findings highlight Kv1.3 as a druggable host factor that is key to the successful completion of KSHV lytic replication.

Differential potassium channel inhibitory activities of a novel thermostable degradation peptide BmKcug1a-P1 from scorpion medicinal material and its N-terminal truncated/restored peptides.

Thermally stable full-length scorpion toxin peptides and partially degraded peptides with complete disulfide bond pairing are valuable natural peptide resources in traditional Chinese scorpion medicinal material. However, their pharmacological activities are largely unknown. This study discovered BmKcug1a-P1, a novel N-terminal degraded peptide, in this medicinal material. BmKcug1a-P1 inhibited hKv1.2 and hKv1.3 potassium channels with IC50 values of 2.12 ± 0.27 µM and 1.54 ± 0.28 µM, respectively. To investigate the influence of N-terminal amino acid loss on the potassium channel inhibiting activities, three analogs (i.e., full-length BmKcug1a, BmKcug1a-P1-D2 and BmKcug1a-P1-D4) of BmKcug1a-P1 were prepared, and their potassium channel inhibiting activities on hKv1.3 channel were verified by whole-cell patch clamp technique. Interestingly, the potassium channel inhibiting activity of full-length BmKcug1a on the hKv1.3 channel was significantly improved compared to its N-terminal degraded form (BmKcug1a-P1), while the activities of two truncated analogs (i.e., BmKcug1a-P1-D2 and BmKcug1a-P1-D4) were similar to that of BmKcug1a-P1. Extensive alanine-scanning experiments identified the bonding interface (including two key functional residues, Asn30 and Arg34) of BmKcug1a-P1. Structural and functional dissection further elucidated whether N-terminal residues of the peptide are located at the bonding interface is important in determining whether the N-terminus significantly influences the potassium channel inhibiting activity of the peptide. Altogether, this research identified a novel N-terminal degraded active peptide, BmKcug1a-P1, from traditional Chinese scorpion medicinal material and elucidated how the N-terminus of peptides influences their potassium channel inhibiting activity, contributing to the functional identification and molecular truncation optimization of full-length and degraded peptides from traditional Chinese scorpion medicinal material Buthus martensii Karsch.

Sphingosine is involved in PAPTP-induced death of pancreas cancer cells by interfering with mitochondrial functions.

Pancreas ductal adenocarcinoma belongs to the most common cancers, but also to the tumors with the poorest prognosis. Here, we pharmacologically targeted a mitochondrial potassium channel, namely mitochondrial Kv1.3, and investigated the role of sphingolipids and mutated Kirsten Rat Sarcoma Virus (KRAS) in Kv1.3-mediated cell death. We demonstrate that inhibition of Kv1.3 using the Kv1.3-inhibitor PAPTP results in an increase of sphingosine and superoxide in membranes and/or membranes associated with mitochondria, which is enhanced by KRAS mutation. The effect of PAPTP on sphingosine and mitochondrial superoxide formation as well as cell death is prevented by sh-RNA-mediated downregulation of Kv1.3. Induction of sphingosine in human pancreas cancer cells by PAPTP is mediated by activation of sphingosine-1-phosphate phosphatase and prevented by an inhibitor of sphingosine-1-phosphate phosphatase. A rapid depolarization of isolated mitochondria is triggered by binding of sphingosine to cardiolipin, which is neutralized by addition of exogenous cardiolipin. The significance of these findings is indicated by treatment of mutated KRAS-harboring metastasized pancreas cancer with PAPTP in combination with ABC294640, a blocker of sphingosine kinases. This treatment results in increased formation of sphingosine and death of pancreas cancer cells in vitro and, most importantly, prolongs in vivo survival of mice challenged with metastatic pancreas cancer. KEY MESSAGES: Pancreatic ductal adenocarcinoma (PDAC) is a common tumor with poor prognosis. The mitochondrial Kv1.3 ion channel blocker induced mitochondrial sphingosine. Sphingosine binds to cardiolipin thereby mediating mitochondrial depolarization. Sphingosine is formed by a PAPTP-mediated activation of S1P-Phosphatase. Inhibition of sphingosine-consumption amplifies PAPTP effects on PDAC in vivo.

Regulation of T Lymphocyte Functions through Calcium Signaling Modulation by Nootkatone.

Recent advancements in understanding the intricate molecular mechanisms underlying immunological responses have underscored the critical involvement of ion channels in regulating calcium influx, particularly in inflammation. Nootkatone, a natural sesquiterpenoid found in Alpinia oxyphylla and various citrus species, has gained attention for its diverse pharmacological properties, including anti-inflammatory effects. This study aimed to elucidate the potential of nootkatone in modulating ion channels associated with calcium signaling, particularly CRAC, KV1.3, and KCa3.1 channels, which play pivotal roles in immune cell activation and proliferation. Using electrophysiological techniques, we demonstrated the inhibitory effects of nootkatone on CRAC, KV1.3, and KCa3.1 channels in HEK293T cells overexpressing respective channel proteins. Nootkatone exhibited dose-dependent inhibition of channel currents, with IC50 values determined for each channel. Nootkatone treatment did not significantly affect cell viability, indicating its potential safety for therapeutic applications. Furthermore, we observed that nootkatone treatment attenuated calcium influx through activated CRAC channels and showed anti-proliferative effects, suggesting its role in regulating inflammatory T cell activation. These findings highlight the potential of nootkatone as a natural compound for modulating calcium signaling pathways by targeting related key ion channels and it holds promise as a novel therapeutic agent for inflammatory disorders.

Voltage-gated potassium channel 1.3: A promising molecular target in multiple disease therapy.

Voltage-gated potassium channel 1.3 (Kv1.3) has emerged as a pivotal player in numerous biological processes and pathological conditions, sparking considerable interest as a potential therapeutic target across various diseases. In this review, we present a comprehensive examination of Kv1.3 channels, highlighting their fundamental characteristics and recent advancements in utilizing Kv1.3 inhibitors for treating autoimmune disorders, neuroinflammation, and cancers. Notably, Kv1.3 is prominently expressed in immune cells and implicated in immune responses and inflammation associated with autoimmune diseases and chronic inflammatory conditions. Moreover, its aberrant expression in certain tumors underscores its role in cancer progression. While preclinical studies have demonstrated the efficacy of Kv1.3 inhibitors, their clinical translation remains pending. Molecular imaging techniques offer promising avenues for tracking Kv1.3 inhibitors and assessing their therapeutic efficacy, thereby facilitating their development and clinical application. Challenges and future directions in Kv1.3 inhibitor research are also discussed, emphasizing the significant potential of targeting Kv1.3 as a promising therapeutic strategy across a spectrum of diseases.

Effect of the rs2821557 Polymorphism of the Human Kv1.3 Gene on Olfactory Function and BMI in Different Age Groups.

The sense of smell plays an important role in influencing the eating habits of individuals and consequently, their body weight, and its impairment has been associated with modified eating behaviors and malnutrition problems. The inter-individual variability of olfactory function depends on several factors, including genetic and physiological ones. In this study, we evaluated the role of the Kv1.3 channel genotype and age, as well as their mutual relationships, on the olfactory function and BMI of individuals divided into young, adult and elderly groups. We assessed olfactory performance in 112 healthy individuals (young n = 39, adult n = 36, elderly n = 37) based on their TDI olfactory score obtained through the Sniffin' Sticks test and their BMI. Participants were genotyped for the rs2821557 polymorphism of the human gene encoding Kv1.3 channels, the minor C allele of which was associated with a decreased sense of smell and higher BMIs compared to the major T allele. The results show that TT homozygous subjects obtained higher TDI olfactory scores and showed lower BMIs than CC homozygous subjects, in all age groups considered. Furthermore, the positive effect of the T allele on olfactory function and BMI decreased with increasing age. The contribution of the genetic factor is less evident with advancing age, while the importance of the age factor is compensated for by genetics. These results show that genetic and physiological factors such as age act to balance each other.

KcsA-Kv1.x chimeras with complete ligand-binding sites provide improved predictivity for screening selective Kv1.x blockers.

Despite significant advances in the development of therapeutic interventions targeting autoimmune diseases and chronic inflammatory conditions, lack of effective treatment still poses a high unmet need. Modulating chronically activated T cells through the blockade of the Kv1.3 potassium channel is a promising therapeutic approach; however, developing selective Kv1.3 inhibitors is still an arduous task. Phage display-based high throughput peptide library screening is a rapid and robust approach to develop promising drug candidates; however, it requires solid-phase immobilization of target proteins with their binding site preserved. Historically, the KcsA bacterial channel chimera harboring only the turret region of the human Kv1.3 channel was used for screening campaigns. Nevertheless, literature data suggest that binding to this type of chimera does not correlate well with blocking potency on the native Kv1.3 channels. Therefore, we designed and successfully produced advanced KcsA-Kv1.3, KcsA-Kv1.1, and KcsA-Kv1.2 chimeric proteins in which both the turret and part of the filter regions of the human Kv1.x channels were transferred. These T+F (turret-filter) chimeras showed superior peptide ligand-binding predictivity compared to their T-only versions in novel phage ELISA assays. Phage ELISA binding and competition results supported with electrophysiological data confirmed that the filter region of KcsA-Kv1.x is essential for establishing adequate relative affinity order among selected peptide toxins (Vm24 toxin, Hongotoxin-1, Kaliotoxin-1, Maurotoxin, Stichodactyla toxin) and consequently obtaining more reliable selectivity data. These new findings provide a better screening tool for future drug development efforts and offer insight into the target-ligand interactions of these therapeutically relevant ion channels.

Kv1.3 in the spotlight for treating immune diseases.

INTRODUCTION: Kv1.3 is the main voltage-gated potassium channel of leukocytes from both the innate and adaptive immune systems. Channel function is required for common processes such as Ca2+ signaling but also for cell-specific events. In this context, alterations in Kv1.3 are associated with multiple immune disorders. Excessive channel activity correlates with numerous autoimmune diseases, while reduced currents result in increased cancer prevalence and immunodeficiencies. AREAS COVERED: This review offers a general view of the role of Kv1.3 in every type of leukocyte. Moreover, diseases stemming from dysregulations of the channel are detailed, as well as current advances in their therapeutic research. EXPERT OPINION: Kv1.3 arises as a potential immune target in a variety of diseases. Several lines of research focused on channel modulation have yielded positive results. However, among the great variety of specific channel blockers, only one has reached clinical trials. Future investigations should focus on developing simpler administration routes for channel inhibitors to facilitate their entrance into clinical trials. Prospective Kv1.3-based treatments will ensure powerful therapies while minimizing undesired side effects.

Activity of Potassium Channels in CD8+ T Lymphocytes: Diagnostic and Prognostic Biomarker in Ovarian Cancer?

CD8+ T cells play a role in the suppression of tumor growth and immunotherapy. Ion channels control the Ca2+-dependent function of CD8+ lymphocytes such as cytokine/granzyme production and tumor killing. Kv1.3 and KCa3.1 K+ channels stabilize the negative membrane potential of T cells to maintain Ca2+ influx through CRAC channels. We assessed the expression of Kv1.3, KCa3.1 and CRAC in CD8+ cells from ovarian cancer (OC) patients (n = 7). We found that the expression level of Kv1.3 was higher in patients with malignant tumors than in control or benign tumor groups while the KCa3.1 activity was lower in the malignant tumor group as compared to the others. We demonstrated that the Ca2+ response in malignant tumor patients is higher compared to control groups. We propose that altered Kv1.3 and KCa3.1 expression in CD8+ cells in OC could be a reporter and may serve as a biomarker in diagnostics and that increased Ca2+ response through CRAC may contribute to the impaired CD8+ function.

De novo variants in KCNA3 cause developmental and epileptic encephalopathy.

OBJECTIVE: Variants in several potassium channel genes, including KCNA1 and KCNA2, cause Developmental and Epileptic Encephalopathies (DEEs). We investigated whether variants in KCNA3, another mammalian homologue of the Drosophila shaker family and encoding for Kv1.3 subunits, can cause DEE. METHODS: Genetic analysis of study individuals was performed by routine exome or genome sequencing, usually of parent-offspring trios. Phenotyping was performed via a standard clinical questionnaire. Currents from wild-type and/or mutant Kv1.3 subunits were investigated by whole-cell patch-clamp upon their heterologous expression. RESULTS: Fourteen individuals, each carrying a de novo heterozygous missense variant in KCNA3, were identified. Most (12/14; 86%) had DEE with marked speech delay with or without motor delay, intellectual disability, epilepsy, and autism spectrum disorder. Functional analysis of Kv1.3 channels carrying each variant revealed heterogeneous functional changes, ranging from "pure" loss-of-function (LoF) effects due to faster inactivation kinetics, depolarized voltage-dependence of activation, slower activation kinetics, increased current inactivation, reduced or absent currents with or without dominant-negative effects, to "mixed" loss- and gain-of-function (GoF) effects. Compared to controls, Kv1.3 currents in lymphoblasts from 1 of the proband displayed functional changes similar to those observed upon heterologous expression of channels carrying the same variant. The antidepressant drug fluoxetine inhibited with similar potency the currents from wild-type and 1 of the Kv1.3 GoF variant. INTERPRETATION: We describe a novel association of de novo missense variants in KCNA3 with a human DEE, and provide evidence that fluoxetine might represent a potential targeted treatment for individuals carrying variants with significant GoF effects. ANN NEUROL 2024;95:365-376.

Proteasome inhibition potentiates Kv1.3 potassium channel expression as therapeutic target in drug-sensitive and -resistant human melanoma cells.

Primary and acquired therapy resistance is a major problem in patients with BRAF-mutant melanomas being treated with BRAF and MEK inhibitors (BRAFI, MEKi). Therefore, development of alternative therapy regimes is still required. In this regard, new drug combinations targeting different pathways to induce apoptosis could offer promising alternative approaches. Here, we investigated the combination of proteasome and Kv1.3 potassium channel inhibition on chemo-resistant, BRAF inhibitor-resistant as well as sensitive human melanoma cells. Our experiments demonstrated that all analyzed melanoma cell lines were sensitive to proteasome inhibitor treatment at concentrations that are not toxic to primary human fibroblasts. To further reduce proteasome inhibitor-associated side effects, and to foster apoptosis, potassium channels, which are other targets to induce pro-apoptotic effects in cancer cells, were blocked. In support, combined exposure of melanoma cells to proteasome and Kv1.3 channel inhibitor resulted in synergistic effects and significantly reduced cell viability. On the molecular level, enhanced apoptosis correlated with an increase of intracellular Kv1.3 channels and pro-apoptotic proteins such as Noxa and Bak and a reduction of anti-apoptotic proteins. Thus, use of combined therapeutic strategies triggering different apoptotic pathways may efficiently prevent the outgrowth of drug-resistant and -sensitive BRAF-mutant melanoma cells. In addition, this could be the basis for an alternative approach to treat other tumors expressing mutated BRAF such as non-small-cell lung cancer.

Kv1.3 mediates ox-LDL-induced vascular smooth muscle cell proliferation through JAK2/STAT3 signaling pathway.

Kv1.3 channel has been shown to participate in regulating inflammatory activation, proliferation and apoptosis in several cell types. However, most of those existing studies focused on the ion-conducting properties of Kv1.3 in maintaining the resting potential and regulating Ca2+ influx. The aim of our study was to explore whether the Kv1.3-JAK2/STAT3 signaling pathway was involved in oxidized low density lipoprotein (ox-LDL) induced vascular smooth muscle cell (VSMC) proliferation. VSMCs from mouse aorta were cultured and treated with ox-LDL (25 µg/mL). The cell counting kit-8 was used to assess cell proliferation, and western blotting was performed to detect expression levels of Kv1.3, JAK2/STAT3, phosphorylated JAK2/STAT3, cyclin B1 and cyclin D1 in treated VSMCs. VSMCs were transfected with Kv1.3 small interfering RNA (Kv1.3-siRNA) or infected with a Kv1.3 lentiviral expression vector (Lv-Kv1.3) and treated with a JAK2 inhibitor LY2784544 to assess the role of Kv1.3 and JAK2/STAT3 signaling in mediating VSMC proliferation induced by ox-LDL. Ox-LDL induced cell proliferation and upregulated the expression of Kv1.3 in mouse VSMCs. In VSMCs transfected with Kv1.3-siRNA, ox-LDL was not efficient in inducing cell proliferation or the levels of proliferation associated proteins, cyclin B1 and cyclin D1. However, cell proliferation, cyclin B1 and cyclin D1 levels increased in VSMCs infected with Lv-Kv1.3. Levels of phosphorylated JAK2 and STAT3 were increased in ox-LDL-treated VSMCs, and this increase was prevented in VSMCs transfected with Kv1.3-siRNA. Treatment with the JAK2 inhibitor LY2784544 also prevented the increase in VSMCs proliferation treated with ox-LDL. Our findings demonstrated that Kv1.3 promoted proliferation of VSMCs treated with ox-LDL, and that this effect might be mediated through activation of the JAK2/STAT3 signaling pathway.

Interaction of the Inhibitory Peptides ShK and HmK with the Voltage-Gated Potassium Channel KV1.3: Role of Conformational Dynamics.

Peptide toxins that adopt the ShK fold can inhibit the voltage-gated potassium channel KV1.3 with IC50 values in the pM range and are therefore potential leads for drugs targeting autoimmune and neuroinflammatory diseases. Nuclear magnetic resonance (NMR) relaxation measurements and pressure-dependent NMR have shown that, despite being cross-linked by disulfide bonds, ShK itself is flexible in solution. This flexibility affects the local structure around the pharmacophore for the KV1.3 channel blockade and, in particular, the relative orientation of the key Lys and Tyr side chains (Lys22 and Tyr23 in ShK) and has implications for the design of KV1.3 inhibitors. In this study, we have performed molecular dynamics (MD) simulations on ShK and a close homologue, HmK, to probe the conformational space occupied by the Lys and Tyr residues, and docked the different conformations with a recently determined cryo-EM structure of the KV1.3 channel. Although ShK and HmK have 60% sequence identity, their dynamic behaviors are quite different, with ShK sampling a broad range of conformations over the course of a 5 µs MD simulation, while HmK is relatively rigid. We also investigated the importance of conformational dynamics, in particular the distance between the side chains of the key dyad Lys22 and Tyr23, for binding to KV1.3. Although these peptides have quite different dynamics, the dyad in both adopts a similar configuration upon binding, revealing a conformational selection upon binding to KV1.3 in the case of ShK. Both peptides bind to KV1.3 with Lys22 occupying the pore of the channel. Intriguingly, the more flexible peptide, ShK, binds with significantly higher affinity than HmK.

AgTx2-GFP, Fluorescent Blocker Targeting Pharmacologically Important Kv1.x (x = 1, 3, 6) Channels.

The growing interest in potassium channels as pharmacological targets has stimulated the development of their fluorescent ligands (including genetically encoded peptide toxins fused with fluorescent proteins) for analytical and imaging applications. We report on the properties of agitoxin 2 C-terminally fused with enhanced GFP (AgTx2-GFP) as one of the most active genetically encoded fluorescent ligands of potassium voltage-gated Kv1.x (x = 1, 3, 6) channels. AgTx2-GFP possesses subnanomolar affinities for hybrid KcsA-Kv1.x (x = 3, 6) channels and a low nanomolar affinity to KcsA-Kv1.1 with moderate dependence on pH in the 7.0-8.0 range. Electrophysiological studies on oocytes showed a pore-blocking activity of AgTx2-GFP at low nanomolar concentrations for Kv1.x (x = 1, 3, 6) channels and at micromolar concentrations for Kv1.2. AgTx2-GFP bound to Kv1.3 at the membranes of mammalian cells with a dissociation constant of 3.4 ± 0.8 nM, providing fluorescent imaging of the channel membranous distribution, and this binding depended weakly on the channel state (open or closed). AgTx2-GFP can be used in combination with hybrid KcsA-Kv1.x (x = 1, 3, 6) channels on the membranes of E. coli spheroplasts or with Kv1.3 channels on the membranes of mammalian cells for the search and study of nonlabeled peptide pore blockers, including measurement of their affinity.

Characterization of endogenous Kv1.3 channel isoforms in T cells.

Voltage-dependent potassium channel Kv1.3 plays a key role on T-cell activation; however, lack of reliable antibodies has prevented its accurate detection under endogenous circumstances. To overcome this limitation, we created a Jurkat T-cell line with endogenous Kv1.3 channel tagged, to determine the expression, location, and changes upon activation of the native Kv1.3 channels. CRISPR-Cas9 technique was used to insert a Flag-Myc peptide at the C terminus of the KCNA3 gene. Basal or activated channel expression was studied using western blot analysis and imaging techniques. We identified two isoforms of Kv1.3 other than the canonical channel (54 KDa) differing on their N terminus: a longer isoform (70 KDa) and a truncated isoform (43 KDa). All three isoforms were upregulated after T-cell activation. We focused on the functional characterization of the truncated isoform (short form, SF), because it has not been previously described and could be present in the available Kv1.3-/- mice models. Overexpression of SF in HEK cells elicited small amplitude Kv1.3-like currents, which, contrary to canonical Kv1.3, did not induce HEK proliferation. To explore the role of endogenous SF isoform in a native system, we generated both a knockout Jurkat clone and a clone expressing only the SF isoform. Although the canonical isoform (long form) localizes mainly at the plasma membrane, SF remains intracellular, accumulating perinuclearly. Accordingly, SF Jurkat cells did not show Kv1.3 currents and exhibited depolarized resting membrane potential (VM ), decreased Ca2+ influx, and a reduction in the [Ca2+ ]i increase upon stimulation. Functional characterization of these Kv1.3 channel isoforms showed their differential contribution to signaling pathways involved in formation of the immunological synapse. We conclude that alternative translation initiation generates at least three endogenous Kv1.3 channel isoforms in T cells that exhibit different functional roles. For some of these functions, Kv1.3 proteins do not need to form functional plasma membrane channels.

A bioengineered probiotic for the oral delivery of a peptide Kv1.3 channel blocker to treat rheumatoid arthritis.

Engineered microbes for the delivery of biologics are a promising avenue for the treatment of various conditions such as chronic inflammatory disorders and metabolic disease. In this study, we developed a genetically engineered probiotic delivery system that delivers a peptide to the intestinal tract with high efficacy. We constructed an inducible system in the probiotic Lactobacillus reuteri to secrete the Kv1.3 potassium blocker ShK-235 (LrS235). We show that LrS235 culture supernatants block Kv1.3 currents and preferentially inhibit human T effector memory (TEM) lymphocyte proliferation in vitro. A single oral gavage of healthy rats with LrS235 resulted in sufficient functional ShK-235 in the circulation to reduce inflammation in a delayed-type hypersensitivity model of atopic dermatitis mediated by TEM cells. Furthermore, the daily oral gavage of LrS235 dramatically reduced clinical signs of disease and joint inflammation in rats with a model of rheumatoid arthritis without eliciting immunogenicity against ShK-235. This work demonstrates the efficacy of using the probiotic L. reuteri as a novel oral delivery platform for the peptide ShK-235 and provides an efficacious strategy to deliver other biologics with great translational potential.

Spinal voltage-gated potassium channel Kv1.3 contributes to neuropathic pain via the promotion of microglial M1 polarization and activation of the NLRP3 inflammasome.

BACKGROUD: Studies have shown that the activation of microglia is the main mechanism of neuropathic pain. Kv1.3 channel is a novel therapeutic target for treating neuroinflammatory disorders due to its crucial role in subsets of microglial cells. As such, it may be involved in the processes of neuropathic pain, however, whether Kv1.3 plays a role in neuroinflammation following peripheral nerve injury is unclear. METHOD: The spared nerve injury model (SNI) was used to establish neuropathic pain. Western blot and immunofluorescence were used to examine the effect of Kv1.3 in the SNI rats. PAP-1, a Kv1.3 specific blocker was administered to alleviate neuropathic pain in the SNI rats. RESULTS: Neuropathic pain and allodynia occurred after SNI, the levels of M1 (CD68, iNos) and M2 (CD206, Arg-1) phenotypes were up-regulated in the spinal cord, and the protein levels of NLRP3, caspase-1 and IL-1ß were also increased. Pharmacological blocking of Kv1.3 with PAP-1 alleviated hyperpathia induced by SNI. Meanwhile, intrathecal injection of PAP-1 reduced M1 polarization and decreased NLRP3, caspase-1 and IL-1ß expressions of protein levels. CONCLUSION: Our research indicates that the Kv1.3 channel in the spinal cord contributes to neuropathic pain by promoting microglial M1 polarization and activating the NLRP3 inflammasome.

Kv1.3 Blockade Alleviates White Matter Injury through Reshaping M1/M2 Phenotypes via the NF-κB Signaling Pathway after Intracerebral Hemorrhage.

BACKGROUND: White matter injury (WMI) in basal ganglia usually induces long-term disability post intracerebral hemorrhage (ICH). Kv1.3 is an ion channel expressed in microglia and induces neuroinflammation after ICH. Here, we investigated the functions and roles of Kv1.3 activation-induced inflammatory response in WMI and the Kv1.3 blockade effect on microglia polarization after ICH. METHODS: Mice ICH model was constructed by autologous blood injection. The expression of Kv1.3 was measured using immunoblot, real-time quantitative polymerase chain reaction (RT-qPCR), and immunostaining assays. Then, the effect of administration of 5-(4-Phenoxybutoxy) psoralen (PAP-1), a selectively pharmacological Kv1.3 blocker, was investigated using open field test (OFT) and basso mouse score (BMS). RT-qPCR, immunoblot, and enzyme-linked immunosorbent assay (ELISA) were taken to elucidate the expression of pro-inflammatory or anti-inflammatory factors around hematoma. PAP-1's function in regulating microglia polarization was investigated using immunoblot, RT-qPCR, and immunostaining assays. The downstream PAP-1 signaling pathway was determined by RT-qPCR and immunoblot. RESULTS: Kv1.3 expression was increased in microglia around the hematoma significantly after ICH. PAP-1 markedly improved neurological outcomes and the WMI by reducing pro-inflammatory cytokine accumulation and upregulating anti-inflammatory factors. Mechanistically, PAP-1 reduces NF-κB p65 and p50 activation, thus facilitating microglia polarization into M2-like microglia, which exerts this beneficial effect. CONCLUSIONS: PAP-1 reduced pro-inflammatory cytokines accumulation and increased anti-inflammatory factors by facilitating M2-like microglia polarization via the NF-κB signaling pathway. Thus, the current study shows that the Kv1.3 blockade is capable of ameliorating WMI by facilitating M2-like phenotype microglia polarization after ICH.

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.

Combining mKate2-Kv1.3 Channel and Atto488-Hongotoxin for the Studies of Peptide Pore Blockers on Living Eukaryotic Cells.

The voltage-gated potassium Kv1.3 channel is an essential component of vital cellular processes which is also involved in the pathogenesis of some autoimmune, neuroinflammatory and oncological diseases. Pore blockers of the Kv1.3 channel are considered as potential drugs and are used to study Kv1 channels' structure and functions. Screening and study of the blockers require the assessment of their ability to bind the channel. Expanding the variety of methods used for this, we report on the development of the fluorescent competitive binding assay for measuring affinities of pore blockers to Kv1.3 at the membrane of mammalian cells. The assay constituents are hongotoxin 1 conjugated with Atto488, fluorescent mKate2-tagged Kv1.3 channel, which was designed to improve membrane expression of the channel in mammalian cells, confocal microscopy, and a special protocol of image processing. The assay is implemented in the "mix and measure", format and allows the screening of Kv1.3 blockers, such as peptide toxins, that bind to the extracellular vestibule of the K+-conducting pore, and analyzing their affinity.