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.

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.

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.

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.

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.

A Fluorescent Peptide Toxin for Selective Visualization of the Voltage-Gated Potassium Channel KV1.3.

Upregulation of the voltage-gated potassium channel KV1.3 is implicated in a range of autoimmune and neuroinflammatory diseases, including rheumatoid arthritis, psoriasis, multiple sclerosis, and type I diabetes. Understanding the expression, localization, and trafficking of KV1.3 in normal and disease states is key to developing targeted immunomodulatory therapies. HsTX1[R14A], an analogue of a 34-residue peptide toxin from the scorpion Heterometrus spinifer, binds KV1.3 with high affinity (IC50 of 45 pM) and selectivity (2000-fold for KV1.3 over KV1.1). We have synthesized a fluorescent analogue of HsTX1[R14A] by N-terminal conjugation of a Cy5 tag. Electrophysiology assays show that Cy5-HsTX1[R14A] retains activity against KV1.3 (IC50 ∼ 0.9 nM) and selectivity over a range of other potassium channels (KV1.2, KV1.4, KV1.5, KV1.6, KCa1.1 and KCa3.1), as well as selectivity against heteromeric channels assembled from KV1.3/KV1.5 tandem dimers. Live imaging of CHO cells expressing green fluorescent protein-tagged KV1.3 shows co-localization of Cy5-HsTX1[R14A] and KV1.3 fluorescence signals at the cell membrane. Moreover, flow cytometry demonstrated that Cy5-HsTX1[R14A] can detect KV1.3-expressing CHO cells. Stimulation of mouse microglia by lipopolysaccharide, which enhances membrane expression of KV1.3, was associated with increased staining by Cy5-HsTX1[R14A], demonstrating that it can be used to identify KV1.3 in disease-relevant models of inflammation. Furthermore, the biodistribution of Cy5-HsTX1[R14A] could be monitored using ex vivo fluorescence imaging of organs in mice dosed subcutaneously with the peptide. These results illustrate the utility of Cy5-HsTX1[R14A] as a tool for visualizing KV1.3, with broad applicability in fundamental investigations of KV1.3 biology, and the validation of novel disease indications where KV1.3 inhibition may be of therapeutic value.

Kv1.3 Channel Is Involved In Ox-LDL-induced Macrophage Inflammation Via ERK/NF-κB signaling pathway.

Macrophage inflammatory response is crucial for the initiation and progression of atherosclerosis. The voltage-gated potassium channel Kv1.3 plays an important role in the modulation of macrophage function. The aim of this study was to investigate the effect and possible mechanism of Kv1.3 on inflammation in oxidized low-density lipoprotein (ox-LDL)-induced RAW264.7 macrophages. Treatment with Kv1.3-siRNA attenuated the expression of IL-6 and TNF-α and reduced the phosphorylation of ERK1/2 and NF-κB in ox-LDL-induced macrophages. In contrast, overexpression of Kv1.3 with Lv-Kv1.3 promoted the expression of IL-6 and TNF-α, and increased ERK1/2 and NF-κB phosphorylation in macrophages. PD-98059, a specific inhibitor of ERK, reversed the expression of IL-6 and TNF-α in ox-LDL-treated macrophages. Kv1.3-siRNA did not inhibit inflammation any further when cells were treated with PD-98059. This suggests that ERK acts as a downstream regulator of the Kv1.3 channel. In conclusion, Kv1.3 may be an indispensable membrane protein in ox-LDL-induced RAW264.7 macrophage inflammation in atherosclerosis through the ERK/NF-κB pathway.

Bergapten attenuates microglia-mediated neuroinflammation and ischemic brain injury by targeting Kv1.3 and Carbonyl reductase 1.

Microglia-mediated neuroinflammation plays a vital role in the pathogenesis of ischemic stroke, which serves as a prime target for developing novel therapeutic agent. However, feasible and effective agents for controlling neuroinflammation are scarce. Bergapten were acknowledged to hold therapeutic potential in restricting inflammation in multiple diseases, including peripheral neuropathy, migraine headaches and osteoarthritis. Here, we aimed to investigate the impact of bergapten on microglia-mediated neuroinflammation and its therapeutic potential in ischemic stroke. Our study demonstrated that bergapten significantly reduced the expression of pro-inflammatory cytokines and the activation of NF-κB signaling pathway in LPS-stimulated primary microglia. Mechanistically, bergapten suppressed cellular potassium ion efflux by inhibiting Kv1.3 channel and inhibits the degradation of Carbonyl reductase 1 induced by LPS, which might contribute to the anti-inflammatory effect of bergapten. Furthermore, bergapten suppressed microglial activation and post-stroke neuroinflammation in an experimental stroke model, leading to reduced infarct size and improved functional recovery. Thus, our study identified that bergapten might be a potential therapeutic compound for the treatment of ischemic stroke.

Artificial pore blocker acts specifically on voltage-gated potassium channel isoform KV1.6.

Among voltage-gated potassium channel (KV) isoforms, KV1.6 is one of the most widespread in the nervous system. However, there are little data concerning its physiological significance, in part due to the scarcity of specific ligands. The known high-affinity ligands of KV1.6 lack selectivity, and conversely, its selective ligands show low affinity. Here, we present a designer peptide with both high affinity and selectivity to KV1.6. Previously, we have demonstrated that KV isoform-selective peptides can be constructed based on the simplistic α-hairpinin scaffold, and we obtained a number of artificial Tk-hefu peptides showing selective blockage of KV1.3 in the submicromolar range. We have now proposed amino acid substitutions to enhance their activity. As a result, we have been able to produce Tk-hefu-11 that shows an EC50 of ≈70 nM against KV1.3. Quite surprisingly, Tk-hefu-11 turns out to block KV1.6 with even higher potency, presenting an EC50 of ≈10 nM. Furthermore, we have solved the peptide structure and used molecular dynamics to investigate the determinants of selective interactions between artificial α-hairpinins and KV channels to explain the dramatic increase in KV1.6 affinity. Since KV1.3 is not highly expressed in the nervous system, we hope that Tk-hefu-11 will be useful in studies of KV1.6 and its functions.

Structures of the T cell potassium channel Kv1.3 with immunoglobulin modulators.

The Kv1.3 potassium channel is expressed abundantly on activated T cells and mediates the cellular immune response. This role has made the channel a target for therapeutic immunomodulation to block its activity and suppress T cell activation. Here, we report structures of human Kv1.3 alone, with a nanobody inhibitor, and with an antibody-toxin fusion blocker. Rather than block the channel directly, four copies of the nanobody bind the tetramer's voltage sensing domains and the pore domain to induce an inactive pore conformation. In contrast, the antibody-toxin fusion docks its toxin domain at the extracellular mouth of the channel to insert a critical lysine into the pore. The lysine stabilizes an active conformation of the pore yet blocks ion permeation. This study visualizes Kv1.3 pore dynamics, defines two distinct mechanisms to suppress Kv1.3 channel activity with exogenous inhibitors, and provides a framework to aid development of emerging T cell immunotherapies.

Mechanisms Underlying the Inhibition of KV1.3 Channel by Scorpion Toxin ImKTX58.

Voltage-gated KV1.3 channel has been reported to be a drug target for the treatment of autoimmune diseases, and specific inhibitors of Kv1.3 are potential therapeutic drugs for multiple diseases. The scorpions could produce various bioactive peptides that could inhibit KV1.3 channel. Here, we identified a new scorpion toxin polypeptide gene ImKTX58 from the venom gland cDNA library of the Chinese scorpion Isometrus maculatus Sequence alignment revealed high similarities between ImKTX58 mature peptide and previously reported KV1.3 channel blockers-LmKTX10 and ImKTX88-suggesting that ImKTX58 peptide might also be a KV1.3 channel blocker. By using electrophysiological recordings, we showed that recombinant ImKTX58 prepared by genetic engineering technologies had a highly selective inhibiting effect on KV1.3 channel. Further alanine scanning mutagenesis and computer simulation identified four amino acid residues in ImKTX58 peptide as key binding sites to KV1.3 channel by forming hydrogen bonds, salt bonds, and hydrophobic interactions. Among these four residues, 28th lysine of the ImKTX58 mature peptide was found to be the most critical amino acid residue for blocking KV1.3 channel. SIGNIFICANCE STATEMENT: In this study, we discovered a scorpion toxin gene ImKTX58 that has not been reported before in Hainan Isometrus maculatus and successfully used the prokaryotic expression system to express and purify the polypeptides encoded by this gene. Electrophysiological experiments on ImKTX58 showed that ImKTX58 has a highly selective blocking effect on KV1.3 channel over Kv1.1, Kv1.2, Kv1.5, SK2, SK3, and BK channels. These findings provide a theoretical basis for designing highly effective KV1.3 blockers to treat autoimmune and other diseases.

Co-Application of Statin and Flavonoids as an Effective Strategy to Reduce the Activity of Voltage-Gated Potassium Channels Kv1.3 and Induce Apoptosis in Human Leukemic T Cell Line Jurkat.

Voltage-gated potassium channels of the Kv1.3 type are considered a potential new molecular target in several pathologies, including some cancer disorders and COVID-19. Lipophilic non-toxic organic inhibitors of Kv1.3 channels, such as statins and flavonoids, may have clinical applications in supporting the therapy of some cancer diseases, such as breast, pancreas, and lung cancer; melanoma; or chronic lymphocytic leukemia. This study focuses on the influence of the co-application of statins-simvastatin (SIM) or mevastatin (MEV)-with flavonoids 8-prenylnaringenin (8-PN), 6-prenylnarigenin (6-PN), xanthohumol (XANT), acacetin (ACAC), or chrysin on the activity of Kv1.3 channels, viability, and the apoptosis of cancer cells in the human T cell line Jurkat. We showed that the inhibitory effect of co-application of the statins with flavonoids was significantly more potent than the effects exerted by each compound applied alone. Combinations of simvastatin with chrysin, as well as mevastatin with 8-prenylnaringenin, seem to be the most promising. We also found that these results correlate with an increased ability of the statin-flavonoid combination to reduce viability and induce apoptosis in cancer cells compared to single compounds. Our findings suggest that the co-application of statins and flavonoids at low concentrations may increase the effectiveness and safety of cancer therapy. Thus, the simultaneous application of statins and flavonoids may be a new and promising anticancer strategy.

sVmKTx, a transcriptome analysis-based synthetic peptide analogue of Vm24, inhibits Kv1.3 channels of human T cells with improved selectivity.

Kv1.3 K+ channels play a central role in the regulation of T cell activation and Ca2+ signaling under physiological and pathophysiological conditions. Peptide toxins targeting Kv1.3 have a significant therapeutic potential in the treatment of autoimmune diseases; thus, the discovery of new toxins is highly motivated. Based on the transcriptome analysis of the venom gland of V. mexicanus smithi a novel synthetic peptide, sVmKTx was generated, containing 36 amino acid residues. sVmKTx shows high sequence similarity to Vm24, a previously characterized peptide from the same species, but contains a Glu at position 32 as opposed to Lys32 in Vm24. Vm24 inhibits Kv1.3 with high affinity (Kd = 2.9 pM). However, it has limited selectivity (~1,500-fold) for Kv1.3 over hKv1.2, hKCa3.1, and mKv1.1. sVmKTx displays reduced Kv1.3 affinity (Kd = 770 pM) but increased selectivity for Kv1.3 over hKv1.2 (~9,000-fold) as compared to Vm24, other channels tested in the panel (hKCa3.1, hKv1.1, hKv1.4, hKv1.5, rKv2.1, hKv11.1, hKCa1.1, hNav1.5) were practically insensitive to the toxin at 2.5 µM. Molecular dynamics simulations showed that introduction of a Glu instead of Lys at position 32 led to a decreased structural fluctuation of the N-terminal segment of sVmKTx, which may explain its increased selectivity for Kv1.3. sVmKTx at 100 nM concentration decreased the expression level of the Ca2+ -dependent T cell activation marker, CD40 ligand. The high affinity block of Kv1.3 and increased selectivity over the natural peptide makes sVmKTx a potential candidate for Kv1.3 blockade-mediated treatment of autoimmune diseases.

Role of C-Terminal Domain and Membrane Potential in the Mobility of Kv1.3 Channels in Immune Synapse Forming T Cells.

Voltage-gated Kv1.3 potassium channels are essential for maintaining negative membrane potential during T-cell activation. They interact with membrane-associated guanylate kinases (MAGUK-s) via their C-terminus and with TCR/CD3, leading to enrichment at the immunological synapse (IS). Molecular interactions and mobility may impact each other and the function of these proteins. We aimed to identify molecular determinants of Kv1.3 mobility, applying fluorescence correlation spectroscopy on human Jurkat T-cells expressing WT, C-terminally truncated (ΔC), and non-conducting mutants of mGFP-Kv1.3. ΔC cannot interact with MAGUK-s and is not enriched at the IS, whereas cells expressing the non-conducting mutant are depolarized. Here, we found that in standalone cells, mobility of ΔC increased relative to the WT, likely due to abrogation of interactions, whereas mobility of the non-conducting mutant decreased, similar to our previous observations on other membrane proteins in depolarized cells. At the IS formed with Raji B-cells, mobility of WT and non-conducting channels, unlike ΔC, was lower than outside the IS. The Kv1.3 variants possessing an intact C-terminus had lower mobility in standalone cells than in IS-engaged cells. This may be related to the observed segregation of F-actin into a ring-like structure at the periphery of the IS, leaving much of the cell almost void of F-actin. Upon depolarizing treatment, mobility of WT and ΔC channels decreased both in standalone and IS-engaged cells, contrary to non-conducting channels, which themselves caused depolarization. Our results support that Kv1.3 is enriched at the IS via its C-terminal region regardless of conductivity, and that depolarization decreases channel mobility.

Pharmacological modulation of Kv1.3 potassium channel selectively triggers pathological B lymphocyte apoptosis in vivo in a genetic CLL model.

BACKGROUND: Ion channels are emerging as promising oncological targets. The potassium channels Kv1.3 and IKCa are highly expressed in the plasma membrane and mitochondria of human chronic lymphocytic leukemia (CLL) cells, compared to healthy lymphocytes. In vitro, inhibition of mitoKv1.3 by PAPTP was shown to kill ex vivo primary human CLL cells, while targeting IKCa with TRAM-34 decreased CLL cell proliferation. METHODS: Here we evaluated the effect of the above drugs in CLL cells from ibrutinib-resistant patients and in combination with Venetoclax, two drugs used in the clinical practice. The effects of the drugs were tested also in the Eµ-TCL1 genetic CLL murine model, characterized by a lympho-proliferative disease reminiscent of aggressive human CLL. Eµ-TCL1 mice showing overt disease state were treated with intraperitoneal injections of non-toxic 5 nmol/g PAPTP or 10 nmol/g TRAM-34 once a day and the number and percentage of pathological B cells (CD19+CD5+) in different, pathologically relevant body districts were determined. RESULTS: We show that Kv1.3 expression correlates with sensitivity of the human and mouse neoplastic cells to PAPTP. Primary CLL cells from ibrutinib-resistant patients could be killed with PAPTP and this drug enhanced the effect of Venetoclax, by acting on mitoKv1.3 of the inner mitochondrial membrane and triggering rapid mitochondrial changes and cytochrome c release. In vivo, after 2 week- therapy of Eµ-TCL1 mice harboring distinct CLL clones, leukemia burden was reduced by more than 85%: the number and percentage of CLL B cells fall in the spleen and peritoneal cavity and in the peripheral blood, without signs of toxicity. Notably, CLL infiltration into liver and spleen and splenomegaly were also drastically reduced upon PAPTP treatment. In contrast, TRAM-34 did not exert any beneficial effect when administered in vivo to Eµ-TCL1 mice at non-toxic concentration. CONCLUSION: Altogether, by comparing vehicle versus compound effect in different Eµ-TCL1 animals bearing unique clones similarly to CLL patients, we conclude that PAPTP significantly reduced leukemia burden in CLL-relevant districts, even in animals with advanced stage of the disease. Our results thus identify PAPTP as a very promising drug for CLL treatment, even for the chemoresistant forms of the disease.

GFP-Margatoxin, a Genetically Encoded Fluorescent Ligand to Probe Affinity of Kv1.3 Channel Blockers.

Peptide pore blockers and their fluorescent derivatives are useful molecular probes to study the structure and functions of the voltage-gated potassium Kv1.3 channel, which is considered as a pharmacological target in the treatment of autoimmune and neurological disorders. We present Kv1.3 fluorescent ligand, GFP-MgTx, constructed on the basis of green fluorescent protein (GFP) and margatoxin (MgTx), the peptide, which is widely used in physiological studies of Kv1.3. Expression of the fluorescent ligand in E. coli cells resulted in correctly folded and functionally active GFP-MgTx with a yield of 30 mg per 1 L of culture. Complex of GFP-MgTx with the Kv1.3 binding site is reported to have the dissociation constant of 11 ± 2 nM. GFP-MgTx as a component of an analytical system based on the hybrid KcsA-Kv1.3 channel is shown to be applicable to recognize Kv1.3 pore blockers of peptide origin and to evaluate their affinities to Kv1.3. GFP-MgTx can be used in screening and pre-selection of Kv1.3 channel blockers as potential drug candidates.