Efectos electrofisiólogicos de PC332 Y PC342 sobre los canales Kv4.3 y Kv4.3+KChIP3

Trabajo fin de Máster en Investigación Farmacológica realizado por Diego Alberto Peraza Pérez en la Facultad de Medicina de la Universidad Autónoma de Madrid y el Instituto de Investigaciones Biomédicas "Alberto Sols" (IIBM-CSIC).En el presente Trabajo de Fin de Máster, hemos estudiado los efectos electrofisiológicos de los compuestos PC332 y PC342 sobre canales KV4.3 y KV4.3+KChIP3. Para ello hemos transfectado células CHO con el cDNA codificante de KV4.3 y KChIP3 y hemos registrado las corrientes generadas por dichos canales KV4.3 y Kv4.3+KChIP3 utilizando la configuración de célula entera de la técnica de patch-clamp. En este estudio hemos demostrado que: i) PC332 se une de forma selectiva a KChIP3, mientras que PC342 no presenta selectividad por esta subunidad reguladora, inhibiendo de forma similar los canales KV4.3 y KV4.3+KChIP3 y ii) los efectos de PC332 resultaron ser tiempo- y voltaje- dependientes, de forma consistente con la unión del fármaco preferentemente a un estado cerrado-activo.Peer Reviewe

KV1.5–KV 1.3 Recycling Is PKC-Dependent

KV1.5 channel function is modified by different regulatory subunits. KVβ1.3 subunits assemble with KV1.5 channels and induce a fast and incomplete inactivation. Inhibition of PKC abolishes the KVβ1.3-induced fast inactivation, decreases the amplitude of the current KV1.5–KVβ1.3 and modifies their pharmacology likely due to changes in the traffic of KV1.5–KVβ1.3 channels in a PKC-dependent manner. In order to analyze this hypothesis, HEK293 cells were transfected with KV1.5–KVβ1.3 channels, and currents were recorded by whole-cell configuration of the patch-clamp technique. The presence of KV1.5 in the membrane was analyzed by biotinylation techniques, live cell imaging and confocal microscopy approaches. PKC inhibition resulted in a decrease of 33 ± 7% of channels in the cell surface due to reduced recycling to the plasma membrane, as was confirmed by confocal microscopy. Live cell imaging indicated that PKC inhibition almost abolished the recycling of the KV1.5–KVβ1.3 channels, generating an accumulation of channels into the cytoplasm. All these results suggest that the trafficking regulation of KV1.5–KVβ1.3 channels is dependent on phosphorylation by PKC and, therefore, they could represent a clinically relevant issue, mainly in those diseases that exhibit modifications in PKC activity.This research was funded by Ministerio de Ciencia e Innovación (MICINN) Spain SAF2016-75021-R and PID2019-104366RB-C21 (to C.V. and T.G.), the Instituto de Salud Carlos III CIBERCV program CB/11/00222 (to C.V.), and the Consejo Superior de Investigaciones Científicas grants: PIE 201820E104 and 2019AEP148 (to C.V.). The cost of this publication was paid in part by funds from the European Fund for Economic and Regional Development (FEDER). A.M. holds a postdoctoral contract at CNIC. A.d.l.C. and D.A.P. held CSIC contracts. A.d.B.-B. holds an MICINN predoctoral contract (BES-2017-080184

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

Small Molecules as Dream Modulators: New Avenues for the Search of Drugs for Neurodegenerative Diseases

Trabajo presentado en el 9th drug Design and Medicinal Chemistry, celebrado en Berlín (Alemania) del 05 al 06 de mayo de 2015.Altered neuronal calcium homeostasis and early compensatory changes in transcriptional programs are common features of many neurodegenerative pathologies including Alzheimer¿s disease, Down syndrome and Huntington¿s disease. DREAM (Downstream Regulatory Element Antagonist Modulator), also known as calsenilin or KChIP-3 (potassium channel interacting protein-3), is a multifunctional calcium binding protein that controls the expression level and/or the activity of several proteins related to calcium homeostasis, neuronal excitability and neuronal survival. This protein is widely expressed in the brain and, depending on the cell type and physiological conditions, shows multiple subcellular localizations, in the nucleus, cytosol or cell membrane. The interest in DREAM is based on its key role in the regulation of intracellular calcium levels. As a calcium-dependent transcriptional repressor, DREAM is a master regulator of activity-dependent gene expression and controls genes important for calcium homeostasis such as the sodium/calcium exchanger-3 (NCX3), IP3R and L-type calcium channels. As an auxiliary protein in the plasma membrane, DREAM interacts with and regulates the gating of Kv4 potassium channels, L- and T-type voltage-dependent calcium channels and NMDA receptors. These findings suggest that DREAM could be a novel and versatile target for therapeutic intervention in neurodegeneration and that molecules able to bind to DREAM and block its physiological functions could be candidates for drugs to treat neurodegenerative diseases. Moreover, up to now, only two DREAM-binding molecules have been identified. In this communication we report the rational design and the synthesis of novel DREAM-binding molecules and their effects on the modulation of DREAM/protein interactions

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

Identification of IQM-266, a Novel DREAM Ligand That Modulates KV4 Currents

Downstream Regulatory Element Antagonist Modulator (DREAM)/KChIP3/calsenilin is a neuronal calcium sensor (NCS) with multiple functions, including the regulation of A-type outward potassium currents (IA). This effect is mediated by the interaction between DREAM and KV4 potassium channels and it has been shown that small molecules that bind to DREAM modify channel function. A-type outward potassium current (IA) is responsible of the fast repolarization of neuron action potentials and frequency of firing. Using surface plasmon resonance (SPR) assays and electrophysiological recordings of KV4.3/DREAM channels, we have identified IQM-266 as a DREAM ligand. IQM-266 inhibited the KV4.3/DREAM current in a concentration-, voltage-, and time-dependent-manner. By decreasing the peak current and slowing the inactivation kinetics, IQM-266 led to an increase in the transmembrane charge (QKV4.3/DREAM) at a certain range of concentrations. The slowing of the recovery process and the increase of the inactivation from the closed-state inactivation degree are consistent with a preferential binding of IQM-266 to a pre-activated closed state of KV4.3/DREAM channels. Finally, in rat dorsal root ganglion neurons, IQM-266 inhibited the peak amplitude and slowed the inactivation of IA. Overall, the results presented here identify IQM-266 as a new chemical tool that might allow a better understanding of DREAM physiological role as well as modulation of neuronal IA in pathological processes

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Effects of n-3, n-6 PUFAs and their derivatives on KV7.1/KCNE1 channels

Resumen del trabajo presentado al VI Meeting de la Red Española de Canales Ióniocs (RECI), celebrado en Santiago de Compostela del 6 al 8 de septiembre de 2017.Voltage-dependent potassium channels are spanning-membrane integral proteins involved in several and diverse physiological and pathological processes such as immune response or cardiovascular function. KV7.1 channel with the KCNE1 regulatory subunit form one of the most important current of the repolarization phase of the cardiac action potential (IKs). We analyzed, using the patch-clamp technique, the acute and chronic exposition of polyunsaturated fatty acids (PUFAs), DHA and AA, in KV7.1/KCNE1 channels expressed in COS-7 cells. Acute DHA and AA exposition increased the magnitude of KV7.1/KCNE1 currents. DHA but not AA slowed the activation kinetics. However, both PUFAs accelerated the deactivation kinetics. Chronic exposition to these compounds did not modify the magnitude of the currents. However, DHA shifted the activation curve toward more negative membrane potentials and accelerated the activation and deactivation kinetics of the channels. On the contrary, AA slowed the activation whereas it accelerated the deactivation process. PUFAs effects on KV7.1/KCNE1 channels depends on both the compound’s chemical nature, n-3 or n-6 PUFAs, as well as the time of exposure, which might be acute or chronic. It has been described that enriched PUFAs diet has anti-inflammatory properties. Moreover, n-3 or n-6 PUFAs lipidderived mediators such as lipoxins and resolvins (which levels are increased by the consumption of aspirin), have been emerged as potent anti-inflammatory agents that promote the resolution phase of the inflammation process. Ion channels, and specially potassium ion channels, play an essential role in macrophages physiology. Accordingly, we also studied the lipid-derived mediators effects on both bone marrow derived macrophages (BMDM), which LPSdependent activation are related with the increased of KV1.3/KV1.5 expression, and in KV7.1/KCNE1 channels where the PUFAs effects have been studied. Lipoxins, but not resolvins, could reverse the LPS-activated macrophages. However, resolvins KV7.1/KCNE1 block were much more potent.Funded by SAF2013-45800-R, SAF2016-75021-R, BFU2014-54928-R and CIBERCV (FIS CB/11/00222).Peer reviewe

Remodeling of mice macrophages induced by trabectedin

Resumen del trabajo presentado al VII Congreso Red Española Canales Iónicos, celebrado en Cáceres del 15 al 17 de mayo de 2019.Immune cells have an important role in the tumor-microenvironment. Macrophages may tune the immune response toward inflammatory or tolerance pathways. Tumor associated macrophages (TAM) have immunosuppressive functions and they are considered a therapeutic target in cancer. The aim of this study was to evaluate the effects of trabectedin, a new class of antitumor agent, on the tumor-microenvironment through the study of electrophysiological and molecular phenotype of macrophages. Experiments were performed using the whole-cell patchclamp configuration of the patch-clamp technique in resident peritoneal mouse macrophages under different types of polarization. Trabectedin decreased macrophages viability and increased ROS production. Trabectedin does not directly interact with KV1.5 and KV1.3 channels, but treatment (16h) of macrophages with sub-cytotoxic concentrations (0.1-5 nM) increased their KV current in a concentration-dependent manner due to an upregulation of KV1.3 channels. In vitro generated TAM (TAMiv), by a co-culture of ID8 cells and macrophages, exhibited a M2 phenotype. TAMiv generated a small KV current, similarly to M2 polarized macrophages, and expressed high levels of M2 markers. In this study, we demonstrated that TAMiv polarization could be re-educated by using sub-cytotoxic concentration of trabectedin. TAMiv treated with sub-cytotoxic concentrations of trabectedin exhibited an upregulation of KV1.3 channels and their M2 phenotype changed towards M1 pro-inflammatory one.Funded by PharmaMar, CSIC 201820E104, CIBERCV and SAF2016-75021-R.Peer reviewe

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