Molecular Determinants of Kv1.3 Potassium Channels-induced Proliferation

Producción CientíficaChanges in voltage-dependent potassium channels (Kv channels) associate to proliferation in many cell types, including transfected HEK293 cells. In this system Kv1.5 overexpression decreases proliferation, whereas Kv1.3 expression increases it independently of K+ fluxes. To identify Kv1.3 domains involved in a proliferation-associated signaling mechanism(s), we constructed chimeric Kv1.3-Kv1.5 channels and point-mutant Kv1.3 channels, which were expressed as GFP- or cherry-fusion proteins. We studied their trafficking and functional expression, combining immunocytochemical and electrophysiological methods, and their impact on cell proliferation. We found that the C terminus is necessary for Kv1.3-induced proliferation. We distinguished two residues (Tyr-447 and Ser-459) whose mutation to alanine abolished proliferation. The insertion into Kv1.5 of a sequence comprising these two residues increased proliferation rate. Moreover, Kv1.3 voltage-dependent transitions from closed to open conformation induced MEK-ERK1/2-dependent Tyr-447 phosphorylation. We conclude that the mechanisms for Kv1.3-induced proliferation involve the accessibility of key docking sites at the C terminus. For one of these sites (Tyr-447) we demonstrated the contribution of MEK/ERK-dependent phosphorylation, which is regulated by voltage-induced conformational changes.Ministerio de Economía y Competitividad (MINECO), Instituto de Salud Carlos III y Programa Estatal de Investigación , Fundación Ramón Areces y Consejería de Sanidad de la Junta de Castilla y León

The Phosphorylation of Kv1.3: A Modulatory Mechanism for a Multifunctional Ion Channel

The voltage-gated potassium channel Kv1.3 plays a pivotal role in a myriad of biological processes, including cell proliferation, differentiation, and apoptosis. Kv1.3 undergoes fine-tuned regulation, and its altered expression or function correlates with tumorigenesis and cancer progression. Moreover, posttranslational modifications (PTMs), such as phosphorylation, have evolved as rapid switch-like moieties that tightly modulate channel activity. In addition, kinases are promising targets in anticancer therapies. The diverse serine/threonine and tyrosine kinases function on Kv1.3 and the effects of its phosphorylation vary depending on multiple factors. For instance, Kv1.3 regulatory subunits (KCNE4 and Kvβ) can be phosphorylated, increasing the complexity of channel modulation. Scaffold proteins allow the Kv1.3 channelosome and kinase to form protein complexes, thereby favoring the attachment of phosphate groups. This review compiles the network triggers and signaling pathways that culminate in Kv1.3 phosphorylation. Alterations to Kv1.3 expression and its phosphorylation are detailed, emphasizing the importance of this channel as an anticancer target. Overall, further research on Kv1.3 kinase-dependent effects should be addressed to develop effective antineoplastic drugs while minimizing side effects. This promising field encourages basic cancer research while inspiring new therapy development

Kv1.3 channels can modulate cell proliferation during phenotypic switch by an ion-flux independent mechanism

Producción CientíficaObjective: Phenotypic modulation of vascular smooth muscle cells has been associated with a decreased expression of all voltage-dependent potassium channel (Kv)1 channel encoding genes but Kcna3 (which encodes Kv1.3 channels). In fact, upregulation of Kv1.3 currents seems to be important to modulate proliferation of mice femoral vascular smooth muscle cells in culture. This study was designed to explore if these changes in Kv1 expression pattern constituted a landmark of phenotypic modulation across vascular beds and to investigate the mechanisms involved in the proproliferative function of Kv1.3 channels. Methods and Results: Changes in Kv1.3 and Kv1.5 channel expression were reproduced in mesenteric and aortic vascular smooth muscle cells, and their correlate with protein expression was electrophysiologicaly confirmed using selective blockers. Heterologous expression of Kv1.3 and Kv1.5 channels in HEK cells has opposite effects on the proliferation rate. The proproliferative effect of Kv1.3 channels was reproduced by “poreless” mutants but disappeared when voltagedependence of gating was suppressed. Conclusion: These findings suggest that the signaling cascade linking Kv1.3 functional expression to cell proliferation is activated by the voltage-dependent conformational change of the channels without needing ion conduction. Additionally, the conserved upregulation of Kv1.3 on phenotypic modulation in several vascular beds makes this channel a good target to control unwanted vascular remodeling.Instituto de Salud Carlos III (grant R006/009)Ministerio de Ciencia, Innovación y Universidades (grant BFU2010-15898)Junta de Castilla y León (grant VA094A11-2

Mitochondrial Kv1.3: a New Target in Cancer Biology?

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Scorpion Toxins from <em>Buthus martensii</em> Karsch (BmK) as Potential Therapeutic Agents for Neurological Disorders: State of the Art and Beyond

Scorpions are fascinating creatures which became residents of the planet well before human beings dwelled on Earth. Scorpions are always considered as a figure of fear, causing notable pain or mortality throughout the world. Their venoms are cocktails of bioactive molecules, called toxins, which are responsible for their toxicity. Fortunately, medical researchers have turned the life-threatening toxins into life-saving therapeutics. From Song Dynasty in ancient China, scorpions and their venoms have been applied in traditional medicine for treating neurological disorders, such as pain, stroke, and epilepsy. Neurotoxins purified from Chinese scorpion Buthus Martensii Karsch (BmK) are considered as the main active ingredients, which act on membrane ion channels. Long-chain toxins of BmK, composed of 58–76 amino acids, could specifically recognize voltage-gated sodium channels (VGSCs). Short-chain BmK toxins, containing 28–40 amino acids, are found to modulate the potassium or chloride channels. These components draw attention as useful scaffolds for drug-design in order to tackle the emerging global medical threats. In this chapter, we aim to summarize the most promising candidates that have been isolated from BmK venoms for drug development

Implication of Voltage-Gated Potassium Channels in Neoplastic Cell Proliferation

Altres ajuts: The work carried out by the Molecular Physiology Laboratory was funded by Fondo Europeo de Desarrollo Regional (FEDER). CSN and JC hold fellowships from MINECO and the Fundación Tatiana Pérez de Guzmán el Bueno, respectively.Voltage-gated potassium channels (Kv) are the largest group of ion channels. Kv are involved in controlling the resting potential and action potential duration in the heart and brain. Additionally, these proteins participate in cell cycle progression as well as in several other important features in mammalian cell physiology, such as activation, differentiation, apoptosis, and cell volume control. Therefore, Kv remarkably participate in the cell function by balancing responses. The implication of Kv in physiological and pathophysiological cell growth is the subject of study, as Kv are proposed as therapeutic targets for tumor regression. Though it is widely accepted that Kv channels control proliferation by allowing cell cycle progression, their role is controversial. Kv expression is altered in many cancers, and their participation, as well as their use as tumor markers, is worthy of effort. There is an ever-growing list of Kv that remodel during tumorigenesis. This review focuses on the actual knowledge of Kv channel expression and their relationship with neoplastic proliferation. In this work, we provide an update of what is currently known about these proteins, thereby paving the way for a more precise understanding of the participation of Kv during cancer development

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

Implication of voltage-gated potassium channels in neoplastic cell proliferation

Voltage-gated potassium channels (Kv) are the largest group of ion channels. Kv are involved in controlling the resting potential and action potential duration in the heart and brain. Additionally, these proteins participate in cell cycle progression as well as in several other important features in mammalian cell physiology, such as activation, differentiation, apoptosis, and cell volume control. Therefore, Kv remarkably participate in the cell function by balancing responses. The implication of Kv in physiological and pathophysiological cell growth is the subject of study, as Kv are proposed as therapeutic targets for tumor regression. Though it is widely accepted that Kv channels control proliferation by allowing cell cycle progression, their role is controversial. Kv expression is altered in many cancers, and their participation, as well as their use as tumor markers, is worthy of effort. There is an ever-growing list of Kv that remodel during tumorigenesis. This review focuses on the actual knowledge of Kv channel expression and their relationship with neoplastic proliferation. In this work, we provide an update of what is currently known about these proteins, thereby paving the way for a more precise understanding of the participation of Kv during cancer development