RESUMEN

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.

Asunto(s)

Canal de Potasio Kv1.3 , Factor de Transcripción STAT3 , Transducción de Señal , Proteína p53 Supresora de Tumor , Canal de Potasio Kv1.3/metabolismo , Canal de Potasio Kv1.3/genética , Proteína p53 Supresora de Tumor/metabolismo , Factor de Transcripción STAT3/metabolismo , Humanos , Animales , Ratones , Línea Celular Tumoral , Melanoma/metabolismo , Melanoma/patología , Melanoma/genética , Mitocondrias/metabolismo , Proliferación Celular , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

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.

Asunto(s)

Herpesvirus Humano 8 , Canal de Potasio Kv1.3 , Factores de Transcripción NFATC , Replicación Viral , Herpesvirus Humano 8/fisiología , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/metabolismo , Humanos , Canal de Potasio Kv1.3/metabolismo , Canal de Potasio Kv1.3/genética , Canal de Potasio Kv1.3/antagonistas & inhibidores , Factores de Transcripción NFATC/metabolismo , Factores de Transcripción NFATC/genética , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Inmediatas-Precoces/genética , Transactivadores/metabolismo , Transactivadores/genética , Linfocitos B/virología , Linfocitos B/metabolismo , Calcio/metabolismo , Sarcoma de Kaposi/virología , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/genética

RESUMEN

The voltage-dependent potassium channel Kv1.3 is a promising therapeutic target for the treatment of autoimmune and chronic inflammatory disorders. Kv1.3 blockers are effective in treating multiple sclerosis (fampridine) and psoriasis (dalazatide). However, most Kv1.3 pharmacological antagonists are not specific enough, triggering potential side effects and limiting their therapeutic use. Functional Kv are oligomeric complexes in which the presence of ancillary subunits shapes their function and pharmacology. In leukocytes, Kv1.3 associates with KCNE4, which reduces the surface abundance and enhances the inactivation of the channel. This mechanism exerts profound consequences on Kv1.3-related physiological responses. Because KCNE peptides alter the pharmacology of Kv channels, we studied the effects of KCNE4 on Kv1.3 pharmacology to gain insights into pharmacological approaches. To that end, we used margatoxin, which binds the channel pore from the extracellular space, and Psora-4, which blocks the channel from the intracellular side. While KCNE4 apparently did not alter the affinity of either margatoxin or Psora-4, it slowed the inhibition kinetics of the latter in a stoichiometry-dependent manner. The results suggested changes in the Kv1.3 architecture in the presence of KCNE4. The data indicated that while the outer part of the channel mouth remains unaffected, KCNE4 disturbs the intracellular architecture of the complex. Various leukocyte types expressing different Kv1.3/KCNE4 configurations participate in the immune response. Our data provide evidence that the presence of these variable architectures, which affect both the structure of the complex and their pharmacology, should be considered when developing putative therapeutic approaches.

Asunto(s)

Canal de Potasio Kv1.3 , Canales de Potasio con Entrada de Voltaje , Canal de Potasio Kv1.3/antagonistas & inhibidores , Canal de Potasio Kv1.3/metabolismo , Canal de Potasio Kv1.3/genética , Humanos , Canales de Potasio con Entrada de Voltaje/metabolismo , Canales de Potasio con Entrada de Voltaje/antagonistas & inhibidores , Animales , Bloqueadores de los Canales de Potasio/farmacología , Cricetulus , Células CHO , Células HEK293 , Ficusina , Venenos de Escorpión

RESUMEN

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.

Asunto(s)

Mitocondrias , Neoplasias Pancreáticas , Esfingosina , Humanos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Animales , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Línea Celular Tumoral , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Canal de Potasio Kv1.3/metabolismo , Canal de Potasio Kv1.3/genética , Canal de Potasio Kv1.3/antagonistas & inhibidores , Ratones , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Muerte Celular/efectos de los fármacos , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética

RESUMEN

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.

Asunto(s)

Canal de Potasio Kv1.3 , Bloqueadores de los Canales de Potasio , Proteínas Recombinantes de Fusión , Animales , Humanos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/antagonistas & inhibidores , Sitios de Unión , Canal de Potasio Kv1.3/metabolismo , Canal de Potasio Kv1.3/antagonistas & inhibidores , Canal de Potasio Kv1.3/genética , Canal de Potasio Kv1.3/química , Ligandos , Biblioteca de Péptidos , Bloqueadores de los Canales de Potasio/química , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/metabolismo , Canales de Potasio/química , Canales de Potasio/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Línea Celular

RESUMEN

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.

Asunto(s)

Índice de Masa Corporal , Canal de Potasio Kv1.3 , Trastornos del Olfato , Adulto , Anciano , Humanos , Odorantes , Polimorfismo Genético , Umbral Sensorial/fisiología , Olfato/genética , Canal de Potasio Kv1.3/genética

RESUMEN

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.

Asunto(s)

Trastorno del Espectro Autista , Epilepsia Generalizada , Epilepsia , Animales , Humanos , Fluoxetina , Epilepsia/tratamiento farmacológico , Epilepsia/genética , Epilepsia/complicaciones , Mutación Missense/genética , Mamíferos , Canal de Potasio Kv1.3/genética

RESUMEN

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.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Melanoma , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Canal de Potasio Kv1.3/genética , Inhibidores de Proteasoma/farmacología , Inhibidores de Proteasoma/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Resistencia a Antineoplásicos , Línea Celular Tumoral , Neoplasias Pulmonares/tratamiento farmacológico , Melanoma/patología , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Reguladoras de la Apoptosis/metabolismo , Mutación

RESUMEN

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.

Asunto(s)

Escherichia coli , Péptidos , Animales , Secuencia de Aminoácidos , Unión Proteica/fisiología , Escherichia coli/metabolismo , Ligandos , Péptidos/farmacología , Péptidos/metabolismo , Bloqueadores de los Canales de Potasio/química , Canal de Potasio Kv1.3/genética , Canal de Potasio Kv1.3/metabolismo , Mamíferos/metabolismo

RESUMEN

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.

Asunto(s)

Canal de Potasio Kv1.3 , Animales , Humanos , Ratones , Línea Celular , Membrana Celular/metabolismo , Células Jurkat , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Canal de Potasio Kv1.3/genética , Canal de Potasio Kv1.3/metabolismo

RESUMEN

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.

Asunto(s)

Artritis Reumatoide , Probióticos , Ratas , Humanos , Animales , Canal de Potasio Kv1.3/genética , Canal de Potasio Kv1.3/metabolismo , Péptidos/metabolismo , Artritis Reumatoide/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Probióticos/uso terapéutico , Bloqueadores de los Canales de Potasio/farmacología , Bloqueadores de los Canales de Potasio/uso terapéutico

RESUMEN

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.

Asunto(s)

Canales de Potasio con Entrada de Voltaje , Canales de Potasio con Entrada de Voltaje/genética , Canales de Potasio con Entrada de Voltaje/metabolismo , Secuencia de Aminoácidos , Bloqueadores de los Canales de Potasio/química , Péptidos/química , Ligandos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Canal de Potasio Kv1.3/genética , Canal de Potasio Kv1.3/metabolismo , Canal de Potasio Kv.1.1/metabolismo , Canal de Potasio Kv.1.2/metabolismo , Canal de Potasio Kv1.5/metabolismo

RESUMEN

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.

Asunto(s)

Canal de Potasio Kv1.3/química , Linfocitos T , Humanos , Inmunoglobulinas/metabolismo , Canal de Potasio Kv1.3/genética , Canal de Potasio Kv1.3/metabolismo , Lisina , Linfocitos T/química

RESUMEN

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.

Asunto(s)

Venenos de Escorpión , Secuencia de Aminoácidos , Aminoácidos , Animales , Simulación por Computador , Canal de Potasio Kv1.3/química , Canal de Potasio Kv1.3/genética , Canal de Potasio Kv1.3/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Péptidos/química , Bloqueadores de los Canales de Potasio/química , Bloqueadores de los Canales de Potasio/farmacología , Venenos de Escorpión/química , Venenos de Escorpión/metabolismo , Venenos de Escorpión/farmacología , Escorpiones/química , Escorpiones/genética , Escorpiones/metabolismo

RESUMEN

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.

Asunto(s)

Canal de Potasio Kv1.3/metabolismo , Linfocitos T , Actinas/metabolismo , Humanos , Canal de Potasio Kv1.3/genética , Potenciales de la Membrana , Sinapsis/metabolismo , Linfocitos T/metabolismo

RESUMEN

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.

Asunto(s)

Enfermedades Autoinmunes , Venenos de Escorpión , Perfilación de la Expresión Génica , Humanos , Canal de Potasio Kv1.3/genética , Canal de Potasio Kv1.3/metabolismo , Péptidos/metabolismo , Péptidos/farmacología , Bloqueadores de los Canales de Potasio/química , Bloqueadores de los Canales de Potasio/farmacología , Venenos de Escorpión/química , Venenos de Escorpión/farmacología , Linfocitos T/metabolismo

RESUMEN

BACKGROUND: Hypertension and proteinuria are common bevacizumab-induced toxicities. No validated biomarkers are available for identifying patients at risk of these toxicities. METHODS: A genome-wide association study (GWAS) meta-analysis was performed in 1039 bevacizumab-treated patients of European ancestry in four clinical trials (CALGB 40502, 40503, 80303, 90401). Grade ≥2 hypertension and proteinuria were recorded (CTCAE v.3.0). Single-nucleotide polymorphism (SNP)-toxicity associations were determined using a cause-specific Cox model adjusting for age and sex. RESULTS: The most significant SNP associated with hypertension with concordant effect in three out of the four studies (p-value <0.05 for each study) was rs6770663 (A > G) in KCNAB1, with the G allele increasing the risk of hypertension (p-value = 4.16 × 10-6). The effect of the G allele was replicated in ECOG-ACRIN E5103 in 582 patients (p-value = 0.005). The meta-analysis of all five studies for rs6770663 led to p-value = 7.73 × 10-8, close to genome-wide significance. The most significant SNP associated with proteinuria was rs339947 (C > A, between DNAH5 and TRIO), with the A allele increasing the risk of proteinuria (p-value = 1.58 × 10-7). CONCLUSIONS: The results from the largest study of bevacizumab toxicity provide new markers of drug safety for further evaluations. SNP in KCNAB1 validated in an independent dataset provides evidence toward its clinical applicability to predict bevacizumab-induced hypertension. ClinicalTrials.gov Identifier: NCT00785291 (CALGB 40502); NCT00601900 (CALGB 40503); NCT00088894 (CALGB 80303) and NCT00110214 (CALGB 90401).

Asunto(s)

Bevacizumab/efectos adversos , Estudio de Asociación del Genoma Completo/métodos , Hipertensión/patología , Canal de Potasio Kv1.3/genética , Neoplasias/tratamiento farmacológico , Polimorfismo de Nucleótido Simple , Proteinuria/patología , Anciano , Inhibidores de la Angiogénesis/efectos adversos , Femenino , Humanos , Hipertensión/inducido químicamente , Hipertensión/genética , Masculino , Persona de Mediana Edad , Neoplasias/patología , Proteinuria/inducido químicamente , Proteinuria/genética

RESUMEN

Calcium signaling plays a vital role in the regulation of various cellular processes, including activation, proliferation, and differentiation of T-lymphocytes, which is mediated by ORAI1 and potassium (K+) channels. These channels have also been identified as highly attractive therapeutic targets for immune-related diseases. Licochalcone A is a licorice-derived chalconoid known for its multifaceted beneficial effects in pharmacological treatments, including its anti-inflammatory, anti-asthmatic, antioxidant, antimicrobial, and antitumorigenic properties. However, its anti-inflammatory effects involving ion channels in lymphocytes remain unclear. Thus, the present study aimed to investigate whether licochalcone A inhibits ORAI1 and K+ channels in T-lymphocytes. Our results indicated that licochalcone A suppressed all three channels (ORAI1, Kv1.3, and KCa3.1) in a concentration-dependent matter, with IC50 values of 2.97 ± 1.217 µM, 0.83 ± 1.222 µM, and 11.21 ± 1.07 µM, respectively. Of note, licochalcone A exerted its suppressive effects on the IL-2 secretion and proliferation in CD3 and CD28 antibody-induced T-cells. These results indicate that the use of licochalcone A may provide an effective treatment strategy for inflammation-related immune diseases.

Asunto(s)

Antiinflamatorios/farmacología , Chalconas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Canal de Potasio Kv1.3/antagonistas & inhibidores , Proteína ORAI1/antagonistas & inhibidores , Linfocitos T/efectos de los fármacos , Calcio/metabolismo , Señalización del Calcio , Células HEK293 , Humanos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Células Jurkat , Canal de Potasio Kv1.3/genética , Canal de Potasio Kv1.3/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo

RESUMEN

The voltage-gated potassium channel Kv1.3 plays an apparent dual physiological role by participating in activation and proliferation of leukocytes as well as promoting apoptosis in several types of tumor cells. Therefore, Kv1.3 is considered a potential pharmacological target for immunodeficiency and cancer. Different cellular locations of Kv1.3, at the plasma membrane or the mitochondria, could be responsible for such duality. While plasma membrane Kv1.3 facilitates proliferation, the mitochondrial channel modulates apoptotic signaling. Several molecular determinants of Kv1.3 drive the channel to the cell surface, but no information is available about its mitochondrial targeting. Caveolins, which are able to modulate cell survival, participate in the plasma membrane targeting of Kv1.3. The channel, via a caveolin-binding domain (CDB), associates with caveolin 1 (Cav1), which localizes Kv1.3 to lipid raft membrane microdomains. The aim of our study was to understand the role of such interactions not only for channel targeting but also for cell survival in mammalian cells. By using a caveolin association-deficient channel (Kv1.3 CDBless), we demonstrate here that while the Kv1.3-Cav1 interaction is responsible for the channel localization in the plasma membrane, a lack of such interaction accumulates Kv1.3 in the mitochondria. Kv1.3 CDBless severely affects mitochondrial physiology and cell survival, indicating that a functional link of Kv1.3 with Cav1 within the mitochondria modulates the pro-apoptotic effects of the channel. Therefore, the balance exerted by these two complementary mechanisms fine-tune the physiological role of Kv1.3 during cell survival or apoptosis. Our data highlight an unexpected role for the mitochondrial caveolin-Kv1.3 axis during cell survival and apoptosis.

Asunto(s)

Apoptosis/genética , Caveolina 1/genética , Supervivencia Celular/genética , Canal de Potasio Kv1.3/genética , Caveolina 1/metabolismo , Células HEK293 , Humanos , Canal de Potasio Kv1.3/metabolismo , Mitocondrias/metabolismo

RESUMEN

The population of regulatory T cells (Tregs) is critical for immunological self-tolerance and homeostasis. Proper ion regulation contributes to Treg lineage identity, regulation, and effector function. Identified ion channels include Ca2+ release-activated Ca2+, transient receptor potential, P2X, volume-regulated anion and K+ channels Kv1.3 and KCa3.1. Ion channel modulation represents a promising therapeutic approach for the treatment of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. This review summarizes studies with gene-targeted mice and pharmacological modulators affecting Treg number and function. Furthermore, participation of ion channels is illustrated and the power of future research possibilities is discussed.

Asunto(s)

Artritis Reumatoide/tratamiento farmacológico , Calcio/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Moduladores del Transporte de Membrana/farmacología , Esclerosis Múltiple/tratamiento farmacológico , Linfocitos T Reguladores/efectos de los fármacos , Animales , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Calcio/inmunología , Canales de Calcio Activados por la Liberación de Calcio/genética , Canales de Calcio Activados por la Liberación de Calcio/inmunología , Señalización del Calcio , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/inmunología , Humanos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/inmunología , Canal de Potasio Kv1.3/genética , Canal de Potasio Kv1.3/inmunología , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Moduladores del Transporte de Membrana/química , Ratones , Esclerosis Múltiple/genética , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/metabolismo , Receptores Purinérgicos P2X/genética , Receptores Purinérgicos P2X/inmunología , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología , Canales de Potencial de Receptor Transitorio/genética , Canales de Potencial de Receptor Transitorio/inmunología