Fludarabine inhibits KV1.3 currents in human B lymphocytes

Fludarabine (F-ara-A) is a purine analog commonly used in the treatment of indolent B cell malignancies that interferes with different aspects of DNA and RNA synthesis. KV1.3 K+ channels are membrane proteins involved in the maintenance of K+ homeostasis and the resting potential of the cell, thus controlling signaling events, proliferation and apoptosis in lymphocytes. Here we show that F-ara-A inhibits KV currents in human B lymphocytes. Our data indicate that KV1.3 is expressed in both BL2 and Dana B cell lines, although total KV1.3 levels were higher in BL2 than in Dana cells. However, KV currents in the plasma membrane were similar in both cell lines and were abrogated by the specific KV1.3 channel inhibitor PAP-1, indicating that KV1.3 accounts for most of the KV currents in these cell lines. F-ara-A, at a concentration (3.5 μM) similar to that achieved in the plasma of fludarabine phosphate-treated patients (3 μM), inhibited KV1.3 currents by 61 ± 6.3% and 52.3 ± 6.3% in BL2 and Dana B cells, respectively. The inhibitory effect of F-ara-A was concentration-dependent and showed an IC50 value of 0.36 ± 0.04 μM and a nH value of 1.07 ± 0.15 in BL2 cells and 0.34 ± 0.13 μM (IC50) and 0.77 ± 0.11 (nH) in Dana cells. F-ara-A inhibition of plasma membrane KV1.3 was observed irrespective of its cytotoxic effect on the cells, BL2 cells being sensitive and Dana cells resistant to F-ara-A cytotoxicity. Interestingly, PAP-1, at concentrations as high as 10 μM, did not affect the viability of BL2 and Dana cells, indicating that blockage of KV1.3 in these cells is not toxic. Finally, F-ara-A had no effect on ectopically expressed KV1.3 channels, suggesting an indirect mechanism of current inhibition. In summary, our results describe the inhibitory effect of F-ara-A on the activity of KV1.3 channel. Although KV1.3 inhibition is not sufficient to induce cell death, further research is needed to determine whether it might still contribute to F-ara-A cytotoxicity in sensitive cells or be accountable for some of the clinical side effects of the drug.This study was supported by MINECO (SAF2013-45800-R, SAF2016-75021-R, RD12/0042/0019, CB/11/00222) and ISCIII (PI12/01135 and PI16/00895). The cost of this publication was paid in part by funds from the European Fund for Economic and Regional Development (FEDER). TG is supported by the Ramón y Cajal Program.Peer reviewedPeer Reviewe

Identification of a critical binding site for local anaesthetics in the side pockets of Kv1 channels

© 2021 The Authors.[Background and Purpose]: Local anaesthetics block sodium and a variety of potassium channels. Although previous studies identified a residue in the pore signature sequence together with three residues in the S6 segment as a putative binding site, the precise molecular basis of inhibition of Kv channels by local anaesthetics remained unknown. Crystal structures of Kv channels predict that some of these residues point away from the central cavity and face into a drug binding site called side pockets. Thus, the question arises whether the binding site of local anaesthetics is exclusively located in the central cavity or also involves the side pockets. [Experimental Approach]: A systematic functional alanine mutagenesis approach, scanning 58 mutants, together with in silico docking experiments and molecular dynamics simulations was utilized to elucidate the binding site of bupivacaine and ropivacaine. [Key Results]: Inhibition of Kv1.5 channels by local anaesthetics requires binding to the central cavity and the side pockets, and the latter requires interactions with residues of the S5 and the back of the S6 segments. Mutations in the side pockets remove stereoselectivity of inhibition of Kv1.5 channels by bupivacaine. Although binding to the side pockets is conserved for different local anaesthetics, the binding mode in the central cavity and the side pockets shows considerable variations. [Conclusion and Implications]: Local anaesthetics bind to the central cavity and the side pockets, which provide a crucial key to the molecular understanding of their Kv channel affinity and stereoselectivity, as well as their spectrum of side effects.The study was supported by the Ministerio de Ciencia e Innovación (MICINN, Spain) Grants SAF2016-75021-R and PID2019-104366RB-C21 (to C.V. and T.G.); the European Regional Development Fund (Fondo Europeo de Desarrollo Regional [FEDER]) and the Instituto de Salud Carlos III CIBERCV programme CB/11/00222 (to C.V. and T.G.); the Consejo Superior de Investigaciones Científicas (CSIC) Grants PIE201820E104 and 2019AEP148 (to C.V.); the Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT) 1191133 and the Fondo de Equipamiento Científico y Tecnológico (FONDEQUIP) 160063 grants from ANID (to W.G.); and the Deutsche Forschungsgemeinschaft (DFG) Grant DE1482-4/1 to N.D

Caracterización clínica e histopatológica de la infección por Papiloma Virus humano de muestras de cérvix. Hospital Teodoro Maldonado Carbo “IESS” // Histopathological and clinical characterization of Human Papilloma Virus infection in cervical samples. Teodoro Maldonado Carbo “IESS”

El Virus del Papiloma Humano (HPV), posee una predilección por los tejidos poliestratificados, con persistencia en capas basales, de allí que a partir de ello es el agente etiológico del cáncer de cérvix, principalmente. Este tipo de cáncer es el segundo más frecuente en mujeres, alrededor del mundo. El HPV es único agente infeccioso oncogénico que lo provoca. Se realizó un trabajo para determinar la prevalencia del virus en mujeres, utilizando técnicas de detección de ADN mediante PCR, en tiempo real, a partir de biopsia de cérvix, además de establecer las características histopatológicas y clínicas relacionadas. La prevalencia fue del 30,67%, la presentación histopatológica más frecuentemente infectada fue el Cáncer de cérvix y el in situ.  Mientras más displásica es la histopatología del cérvix, más frecuente es la presentación del virus. Se demostró una asociación entre la exposición a la infección y la presencia de secreción vaginal blanquecina y la dispareunia. ABSTRACT Human Papilloma Virus (HPV) has a predilection for poly-stratified tissues, with persistence in the basal layers, hence mainly is the etiological agent of cervical cancer. Cervical cancer is the second most common cancer in women worldwide. HPV is unique oncogenic infectious agent that causes it. The objective of the study was to determine the prevalence of the virus in women, using DNA detection techniques through PCR, in real-time, from the cervical biopsy. In addition, the related histopathological and clinical characteristics were established. The prevalence was 30.67%, the most frequently infected histopathology presentation was cervical and in situ cancer. The more dysplastic the histopathology of the cervix, the more frequent the presentation of the virus. An association between exposure to infection and the presence of whitish vaginal discharge and dyspareunia was demonstrated

Pathophysiological modulation of Kv1.5 in the cardiovascular system. Role of SIGMA-1 receptor

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de Lectura: 21-03-2022Esta Tesis tiene embargado el acceso al texto completo hasta el 21-09-2023El desarrollo de esta tesis doctoral ha sido posible gracias a los proyectos concedidos por el Ministerio de Ciencia e Innovación – Agencia Estatal de Investigación: PID2019-104366RB-C21 y SAF2016-75021-R, y a la Fundación contra la Hipertensión Pulmona

Fludarabine inhibits Kv1.3 currents in human B lymphocytes

Resumen del trabajo presentado al 5th Symposium on Biomedical Research: "Advances and Perspectives In Pharmacology, Drug Toxicity and Pharmacogenetics", celebrado en Madrid del 15 al 16 de marzo de 2018.[Introduction]:Fludarabine (F-ara-A) is a purine analogue commonly used in the treatment of indolent B cell malignancies that interferes with different aspects of DNA and RNA synthesis. KV1.3 potassium channels are membrane proteins involved in the control of K+ homeostasis and in the maintenance of the resting potential of the cell, thus controlling signalling events, proliferation and apoptosis in lymphocytes. The aim of this study was to determine if F-ara-A modulates KV currents in human B lymphocytes. [Material and methods]: We assessed the expression, the activity and the effect of F-ara-A on the KV1.3 channel in BL2 and Dana B cell lines. Currents were registered by whole-cell patch-clamp. Statistical significance was determined by t-Student test or by nonparametric Mann-Whitney test. [Results]: We show that KV1.3 is expressed in both BL2 and Dana B cell lines, although total KV1.3 levels were higher in BL2 compared to those in Dana cells. However, KV currents in the plasma membrane were similar in both cell lines. These KV currents were abrogated by the specific KV1.3 channel inhibitor PAP-1, indicating that most KV currents in these B cell lines are controlled by KV1.3. F-ara-A (3.5 μM), a concentration similar to that achieved in the plasma of fludarabine phosphate-treated patients (3 μM), inhibited KV1.3 currents by 61±6.3% and 52.3±6.3% in BL2 and Dana B cells, respectively. The inhibitory effect of F-ara-A was concentration dependent and showed an IC50 value of 0.36±0.04 μM and a nH value of 1.07±0.15 in BL2 cells and 0.34±0.13 μM (IC50) and 0.77±0.11 (nH) in Dana cells. This inhibitory effect of F-ara-A on the activity of plasma membrane KV1.3 was observed in these cells irrespective of their cytotoxic effect. F-ara-A had no effect on heterologously expressed KV1.3 channels, suggesting an indirect mechanism of inhibition. [Conclusions]: Fludarabine (F-ara-A), a chemotherapeutic drug extensively used in clinics, strongly inhibits KV1.3 currents in B lymphoma and lymphoblastoid cells. Although this inhibitory activity is not sufficient to induce cell death, it might still contribute to the cytotoxic effect of the drug.This study was supported by MINECO (SAF2013-45800-R, SAF2016-75021-R, RD12/0042/0019, CB/11/00222) and ISCIII (PI12/01135 and PI16/00895).Peer Reviewe

KV1.3 channel inhibition by indolic compounds in chronic lymphocytic leukemia cells

Resumen del trabajo presentado al 5th Symposium on Biomedical Research: "Advances and Perspectives In Pharmacology, Drug Toxicity and Pharmacogenetics", celebrado en Madrid del 15 al 16 de marzo de 2018.[Introduction]: Chronic lymphocytic leukemia (CLL) is the most common leukemia in western countries and it is based on B cell clonal expansion. This disease has no cure and the appearance of pharmacological resistance is very common. We have previously identified indole-3-carbinol (I3C) and its main metabolite, 3,3’-diindolylmethane (DIM), as active compounds with pharmacological activity against CLL. KV1.3 potassium channels are involved in B and T cells function controlling plasmatic membrane potential, Ca2+ entry and cellular proliferation. Therefore, these channels could be a new therapeutic target against CLL. Here, we have analysed if these indolic compounds can act, in part, by modulating the KV1.3 function.[Material and Methods]: we assessed the activity and effect of the indolic compounds on KV1.3 channels in CLL cells that are either sensitive or resistant to I3C cytotoxicity. Currents were registered by whole-cell patch-clamp. Statistical significance was determined by t-Student test or by nonparametric Mann-Whitney test.[Results]: The KV1.3 current magnitude on CLL cells correlated with their sensitivity to the cytotoxic effect of I3C; I3C resistant CLL (IR-CLL) cells exhibiting ≈2.5-fold higher KV1.3 current amplitude than sensitive (IS-CLL) cells. Both I3C and DIM inhibited KV1.3 current in CLL cells, DIM being ≈4-fold more potent than the parent compound. However, a non-cytotoxic compound, indole-3-carboxylic acid (I3CA), did not inhibit the KV1.3 current.[Conclusions]: IR-CLL cells exhibit greater KV1.3 current magnitude than IS-CLL cells, which can be due to the existence of different canalosomes in these cells. The KV1.3 inhibitory effect of the indolic compounds tested, correlates with their cytotoxic effect on CLL cells. Our results suggest that KV1.3 channel could be involved in CLL cells pharmacological resistance mechanisms and its inhibition could be part of the cytotoxic effect of I3C and DIM, which open new venues to the treatment of this disease.This study was supported by MINECO (SAF2013-45800-R, SAF2016-75021-R, RD12/0042/0019, CB/11/00222) and ISCIII (PI12/01135 and PI16/00895).Peer reviewe

Caracterización clínica e histopatológica de la infección por Papiloma Virus humano de muestras de cérvix. Hospital Teodoro Maldonado Carbo “IESS”

ABSTRACT Human Papilloma Virus (HPV) has a predilection for poly-stratified tissues, with persistence in the basal layers, hence mainly is the etiological agent of cervical cancer. Cervical cancer is the second most common cancer in women worldwide. HPV is unique oncogenic infectious agent that causes it. The objective of the study was to determine the prevalence of the virus in women, using DNA detection techniques through PCR, in real-time, from the cervical biopsy. In addition, the related histopathological and clinical characteristics were established. The prevalence was 30.67%, the most frequently infected histopathology presentation was cervical and in situ cancer. The more dysplastic the histopathology of the cervix, the more frequent the presentation of the virus. An association between exposure to infection and the presence of whitish vaginal discharge and dyspareunia was demonstrated.RESUMEN El Virus del Papiloma Humano (HPV), posee una predilección por los tejidos poliestratificados, con persistencia en capas basales, de allí que a partir de ello es el agente etiológico del cáncer de cérvix, principalmente. Este tipo de cáncer es el segundo más frecuente en mujeres, alrededor del mundo. El HPV es único agente infeccioso oncogénico que lo provoca. Se realizó un trabajo para determinar la prevalencia del virus en mujeres, utilizando técnicas de detección de ADN mediante PCR, en tiempo real, a partir de biopsia de cérvix, además de establecer las características histopatológicas y clínicas relacionadas. La prevalencia fue del 30,67%, la presentación histopatológica más frecuentemente infectada fue el Cáncer de cérvix y el in situ.  Mientras más displásica es la histopatología del cérvix, más frecuente es la presentación del virus. Se demostró una asociación entre la exposición a la infección y la presencia de secreción vaginal blanquecina y la dispareunia.

Sigma-1 receptor modulation fine-tunes KV1.5 channels and impacts pulmonary vascular function

KV1.5 channels are key players in the regulation of vascular tone and atrial excitability and their impairment is associated with cardiovascular diseases including pulmonary arterial hypertension (PAH) and atrial fibrillation (AF). Unfortunately, pharmacological strategies to improve KV1.5 channel function are missing. Herein, we aimed to study whether the chaperone sigma-1 receptor (S1R) is able to regulate these channels and represent a new strategy to enhance their function. By using different electrophysiological and molecular techniques in X. laevis oocytes and HEK293 cells, we demonstrate that S1R physically interacts with KV1.5 channels and regulate their expression and function. S1R induced a bimodal regulation of KV1.5 channel expression/activity, increasing it at low concentrations and decreasing it at high concentrations. Of note, S1R agonists (PRE084 and SKF10047) increased, whereas the S1R antagonist BD1047 decreased, KV1.5 expression and activity. Moreover, PRE084 markedly increased KV1.5 currents in pulmonary artery smooth muscle cells and attenuated vasoconstriction and proliferation in pulmonary arteries. We also show that both KV1.5 channels and S1R, at mRNA and protein levels, are clearly downregulated in samples from PAH and AF patients. Moreover, the expression of both genes showed a positive correlation. Finally, the ability of PRE084 to increase KV1.5 function was preserved under sustained hypoxic conditions, as an in vitro PAH model. Our study provides insight into the key role of S1R in modulating the expression and activity of KV1.5 channels and highlights the potential role of this chaperone as a novel pharmacological target for pathological conditions associated with KV1.5 channel dysfunction

Running title: Novel loss of function KCNA5 variants in PAH

Reduced expression and/or activity of Kv1.5 channels (encoded by KCNA5) is a common hallmark in human or experimental pulmonary arterial hypertension (PAH). Likewise, genetic variants in KCNA5 have been found in PAH patients, but their functional consequences and potential impact on the disease are largely unknown. Herein, we aimed to characterize the functional consequences of 7 KCNA5 variants found in a cohort of PAH patients. Potassium currents were recorded by patch-clamp technique in HEK293 cells transfected with WT or mutant Kv1.5 cDNA. Flow cytometry, western blot and confocal microscopy techniques were used for measuring protein expression and cell apoptosis in HEK293 and human pulmonary artery smooth muscle cells (hPASMC). KCNA5 variants found in PAH patients (namely, p.Arg184Pro and p.Gly384Arg) resulted in a clear loss of potassium channel function as assessed by electrophysiological and molecular modelling analyses. The p.Arg184Pro variant also resulted in a pronounced reduction of Kv1.5 expression. Transfection with p.Arg184Pro or p.Gly384Arg variants decreased apoptosis of hPASMCs compared with the WT, demonstrating that KCNA5 dysfunction in both variants affects cell viability. Thus, in addition to affecting channel activity, both variants were associated with impaired apoptosis, a crucial process linked to the disease. The estimated prevalence of dysfunctional KCNA5 variants in the PAH population analyzed was around 1 %. Our data indicate that some KCNA5 variants found PAH patients have critical consequences for channel function supporting the idea that KCNA5 pathogenic variants may be a causative or contributing factor for PAH.This work was supported by Fundación Contra la Hipertensión Pulmonar (FCHP); Ministerio de Ciencia e Innovación [PID2020-117939RB-I00 to AC, PID2019-104366RB-C21 to TG, PID2019-107363RB-I00 to FPV]; Comunidad de Madrid [B2017/BMD-3727 to AC] and Instituto de Salud Carlos III [PI18/01233, PI21/01593] with funds from the European Union (Fondo Europeo de Desarrollo Regional FEDER); and by an annual grant by the FEDER foundation (Federación Española de Enfermedades Raras).Peer reviewe