A novel potassium channel in lymphocyte mitochondria

The margatoxin-sensitive Kv1.3 is the major potassium channel in the plasma membrane of T lymphocytes. Electron microscopy, patch clamp, and immunological studies identified the potassium channel Kv1.3, thought to be localized exclusively in the cell membrane, in the inner mitochondrial membrane of T lymphocytes. Patch clamp of mitoplasts and mitochondrial membrane potential measurements disclose the functional expression of a mitochondrial margatoxin-sensitive potassium channel. To identify unambiguously the mitochondrial localization of Kv1.3, we employed a genetic model and stably transfected CTLL-2 cells, which are genetically deficient for this channel, with Kv1.3. Mitochondria isolated from Kv1.3-reconstituted CTLL-2 expressed the channel protein and displayed an activity, which was identical to that observed in Jurkat mitochondria, whereas mitochondria of mock-transfected cells lacked a channel with the characteristics of Kv1.3. Our data provide the first molecular identification of a mitochondrial potassium conductance

Regulation of Proliferation by a Mitochondrial Potassium Channel in Pancreatic Ductal Adenocarcinoma Cells

Previous results link the mitochondrial potassium channel Kv1.3 (mitoKv1.3) to the regulation of apoptosis. By synthesizing new, mitochondria-targeted derivatives (PAPTP and PCARBTP) of PAP-1, a specific membrane-permeant Kv1.3 inhibitor, we have recently provided evidence that both drugs acting on mitoKv1.3 are able to induce apoptosis and reduce tumor growth in vivo without affecting healthy tissues and cells. In the present article, by exploiting these new drugs, we addressed the question whether mitoKv1.3 contributes to the regulation of cell proliferation as well. When used at low concentrations, which do not compromise cell survival, both drugs slightly increased the percentage of cells in S phase while decreased the population at G0/G1 stage of cells from two different pancreatic ductal adenocarcinoma lines. Our data suggest that the observed modulation is related to ROS levels within the cells, opening the way to link mitochondrial ion channel function to downstream, ROS-related signaling events that might be important for cell cycle progression

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

Small-Molecule Modulators of Mitochondrial Channels as Chemotherapeutic Agents

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Small-molecule modulators of mitochondrial channels as chemotherapeutic agents

Ion channels residing in the inner (IMM) and outer (OMM) mitochondrial membranes are emerging as noteworthy pharmacological targets in oncology. While these aspects have not been investigated for all of them, a role in cancer growth and/or metastasis and/or drug resistance has been shown at least for the IMM-residing Ca2+ uniporter complex and K+- selective mtKV1.3, mtIKCa, mtSKCa and mtTASK-3, and for the OMM Voltage-Dependent Anion Channel (mitochondrial porin). A special case is that of the Mitochondrial Permeability Transition Pore, a large pore which forms in the IMM of severely stressed cells, and which may be exploited to precipitate the death of cancerous cells. Here we briefly discuss the oncological relevance of mitochondria and their channels, and summarize the methods that can be adopted to selectively target these intracellular organelles. We then present an updated list of known mitochondrial channels, and review the pharmacology of those with proven relevance for cancer

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

The voltage-gated potassium channel Kv1.3 plays a crucial role in T-cell activation and is considered a promising target for the treatment of autoimmune diseases. However, the lack of reliable antibodies has prevented its accurate detection and study under endogenous conditions, so that most published studies have been conducted in heterologous systems. To address this limitation, we engineered a Jurkat T-cell line expressing endogenous Kv1.3 channels tagged with a signal peptide to investigate the expression and localization of native Kv1.3 channels, and their role associated to T cell physiological responses. Using the CRISPR-Cas9 tool, we inserted a Flag-Myc peptide at the C terminus of the KCNA3 gene. Basal and activated channel expression were assessed through western blot analysis and imaging techniques. Surprisingly, besides the canonical Kv1.3 channel (54 KDa), we identified two additional isoforms with distinct N termini: a longer isoform (70 KDa) and a truncated isoform (43 KDa). All three isoforms showed upregulation after T-cell activation. Our focus was on characterizing the truncated isoform (short form, SF) as it had not been previously described and could be present in available Kv1.3-/- mouse models. Overexpressing SF in HEK cells generated Kv1.3-like currents with smaller amplitudes, which, unlike 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. While the canonical isoform localized primarily at the plasma membrane, SF remained intracellular, accumulating perinuclearly. Consequently, SF Jurkat cells lacked Kv1.3 currents, exhibited depolarized resting membrane potential (EM), reduced Ca2+ influx, and diminished increases in intracellular calcium ([Ca2+]i) upon stimulation. Functional characterization of these Kv1.3 channel isoforms revealed their differential contributions to signaling pathways involved in immunological synapse formation. In conclusion, alternative translation initiation generates at least three endogenous Kv1.3 channel isoforms in T cells with distinct functional roles. Importantly, some of these functions do not require the formation of functional plasma membrane channels by Kv1.3 proteins.El canal de potasio dependiente de voltaje Kv1.3 juega un papel crucial en la activación de las células T y se considera una buena diana terapéutica para el tratamiento de enfermedades autoinmunes. Sin embargo, la falta de anticuerpos específicos de la proteína ha impedido su detección y estudio precisos en condiciones endógenas, por lo que la mayoría de los estudios publicados se han realizado en sistemas heterólogos. Para abordar esta limitación, diseñamos una línea de células T Jurkat que expresa canales Kv1.3 endógenos marcados con un péptido señal para estudiar su expresión y localización además de su papel fisiológico en los linfocitos T. Usando la herramienta CRISPR-Cas9, insertamos un péptido Flag-Myc en el extremo C del gen KCNA3. La expresión del canal basal y activado se evaluó mediante análisis de transferencia Western y técnicas de imagen. Sorprendentemente, además del canal canónico Kv1.3 (54 KDa), identificamos dos isoformas adicionales con extremos N distintos: una isoforma más larga (70 KDa) y una isoforma truncada (43 KDa). Las tres isoformas mostraron un aumento de su expresión después de la activación de las células T. Nuestro objetivo fue caracterizar la isoforma truncada (forma abreviada, SF) ya que no se había descrito previamente y podría estar presente en los modelos de ratón Kv1.3-/- disponibles. La sobreexpresión de SF en células HEK generó corrientes similares a Kv1.3 con amplitudes más pequeñas que, a diferencia del Kv1.3 canónico, no indujeron la proliferación de HEK. Para explorar el papel de la isoforma SF endógena en un sistema nativo, generamos un clon de Jurkat knockout y un clon que expresa solo la isoforma SF. Mientras que la isoforma canónica se localizó principalmente en la membrana plasmática, el SF permaneció intracelular, acumulándose perinuclearmente. En consecuencia, las células SF Jurkat carecían de las corrientes Kv1.3, presentaban un potencial de membrana en reposo (EM) despolarizado, una entrada de Ca2+ reducida y un aumento disminuido del calcio intracelular ([Ca2+]i) tras la estimulación. La caracterización funcional de estas isoformas del canal Kv1.3 reveló sus contribuciones diferenciales a las vías de señalización involucradas en la formación de sinapsis inmunológicas. En conclusión, el inicio de la traducción alternativa genera al menos tres isoformas endógenas del canal Kv1.3 en las células T con distintas funciones funcionales. Es importante destacar que algunas de estas funciones no requieren la formación de canales de membrana plasmática funcionales por las proteínas Kv1.3.Escuela de DoctoradoDoctorado en Investigación Biomédic

Characterization of endogenous Kv1.3 channel isoforms in T cells

Producción CientíficaVoltage-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.Ministerio de Economía y Competitividad (grant PID 2020‐118517RB‐I00)Junta de Castilla y León (grants VA172P20) and (CLU-2019-02)Funds from Institut Curie, INSERM,Agence Nationale de la Recherche RetroTact (ANR‐20CE15‐0009‐01,ANR‐10‐IDEX‐0001‐02 PSL*, and ANR‐11‐LABX‐0043)Fondation pour la Recherche Médicale FRM (EQU202003010280