Search

The voltage-dependent potassium (Kv) channel Kvβ family was the first identified group of modulators of Kv channels. Kvβ regulation of the α-subunits, in addition to their aldoketoreductase activity, has been under extensive study. However, scarce information about their specific α-subunit-independent biology is available. The expression of Kvβs is ubiquitous and, similar to Kv channels, is tightly regulated in leukocytes. Although Kvβ subunits exhibit cytosolic distribution, spatial localization, in close contact with plasma membrane Kv channels, is crucial for a proper immune response. Therefore, Kvβ2.1 is located near cell surface Kv1.3 channels within the immunological synapse during lymphocyte activation. The objective of this study was to analyze the structural elements that participate in the cellular distribution of Kvβs. It was demonstrated that Kvβ peptides, in addition to the cytoplasmic pattern, targeted the cell surface in the absence of Kv channels. Furthermore, Kvβ2.1, but not Kvβ1.1, targeted lipid raft microdomains in an S-acylation-dependent manner, which was concomitant with peptide localization within the immunological synapse. A pair of C-terminal cysteines (C301/C311) was mostly responsible for the specific palmitoylation of Kvβ2.1. Several insults altered Kvβ2.1 membrane localization. Therefore, growth factor-dependent proliferation enhanced surface targeting, whereas PKC activation impaired lipid raft expression. However, PSD95 stabilized Kvβ2.1 in these domains. This data shed light on the molecular mechanism by which Kvβ2.1 clusters into immunological synapses during leukocyte activation.

Transient potential receptor vanilloid 2 (TRPV2) is widely expressed through the nervous system and specifically found in neuronal subpopulations and some glial cells. TRPV2 is known to be sensitized by methionine oxidation, which results from inflammation. Here we aim to characterize the expression and regulation of TRPV2 in myelination pathologies, such as hypomyelination and demyelination. We validated the interaction between TRPV2 and its putative interactor Opalin, an oligodendrocyte marker, in mixed glial cultures under pro-and anti-inflammatory conditions. Then, we characterized TRPV2 time-course expression in experimental animal models of hypomyelination (jimpy mice) and de-/remyelination (cuprizone intoxication and experimental autoimmune encephalomyelitis (EAE)). TRPV2 showed upregulation associated with remyelination, inflammation in cuprizone and EAE models, and downregulation in hypomyelinated jimpy mice. TRPV2 expression was altered in human samples of multiple sclerosis (MS) patients. Additionally, we analyzed the expression of methionine sulfoxide reductase A (MSRA), an enzyme that reduces oxidated methionines in TRPV2, which we found increased in inflammatory conditions. These results suggest that TRPV2 may be a key player in myelination in accordance with the recapitulation hypothesis, and that it may become an interesting clinical target in the treatment of demyelination disorders., The authors acknowledge financial support from the Spanish Government MCIN/AEI/ 10.13039/501100011033 (Project BFU2017-87843-R- to B.C. and A.P.-M.; Project PID2020-120222GB-I00 to A.P.-M.).

[eng] Kv1.3 plays a crucial role in leukocytes, where it regulates activation and proliferation. In these cells, Kv1.3 is coexpressed with modulatory subunit KCNE4. This ancillary peptide acts as a dominant negative regulator of the channel with a dual function. On the one hand, KCNE4 triggers the intracellular retention of Kv1.3 reducing K+ currents. On the other, KCNE4 enhances their C- type inactivation kinetics. Thus, the aim of this thesis was to characterize the molecular mechanisms of Kv1.3/KCNE4 association and modulation in the context of the immune system physiology. To do so, the thesis is structured in three chapters. Chapter I (KCNE4 in the immune system). We confirmed the physiological role of KCNE4 in immune cells like T lymphocytes and dendritic cells (Contribution 1). By manipulating the expression of KCNE4 in these cells, we were the first to describe a relationship between KCNE4 and the leukocyte function. Our results highlight a possible therapeutic role for KCNE4. Chapter II (A protein interaction hub (L69-72) in the C-terminus of KCNE4). We analysed the relationship between Kv1.3, KCNE4 and calmodulin (CaM). We identified a tetraleucine motif in the C-terminus of KCNE4 (L69-72) that determines association to Kv1.3 (Contribution 2). In a nearby position, an ER retention motif (ERRM) triggers intracellular retention of the channel. These results were replicated in a peptide containing just 22 residues of KCNE4, including both clusters. This peptide partially replicated the inhibitory effects of KCNE4 and thus, a potential pharmacological interest. We are also the first to propose a structural model for the Kv1.3/KCNE4 association. On the other hand, L69-72 in KCNE4 also determines CaM binding. The role of Ca2+/CaM is relevant in the context of the immune system physiology, where Kv1.3 potentiates the calcium signalling. Furthermore, we are the first to report dimerization of KCNE4 (Contribution 3), which modulates the traffic of KCNE4. Dimerization was also mediated by the leucine cluster, acting as a protein interaction hub. We propose CaM binding as a mechanism to fine-tune interactions with Kv1.3 and dimerization. Chapter III (Structural relationships of the Kv1.3/KCNE4 complex). We focused on the structural Kv1.3/KCNE4 association. A series of chimaeras between KCNE2 and KCNE4 allowed us to decipher roles for each domain of KCNE4 (Contribution 4). Thus, we confirmed that the C- terminus of KCNE4 mediates association and intracellular retention of Kv1.3. Moreover, for the first time we characterized the molecular determinants of KCNE4-dependent kinetic modulation of Kv1.3. These results were confirmed with Gibbs free energy calculations and the Kv1.3/KCNE4 structural model. Using alanine-scanning mutagenesis, we mapped some residues implicated in this process. Moreover, we found that, similar to other Kv/KCNE associations, Kv1.3/KCNE4 associated in an open stoichiometry with up to four KCNE4 subunits per complex (Contribution 5). The number of KCNE4 peptides in the channelosome was proportional to the effects on activation kinetics and current amplitude. However, the accentuation of Kv1.3 inactivation was independent of stoichiometry. The similarities in stoichiometry and docking to Kv channels suggest a conserved mechanism among KCNE peptides. Finally, because KCNE4 caused structural Kv1.3 changes, we studied the effect of KCNE4 on Kv1.3 pharmacology (Contribution 6). We studied margatoxin and Psora-4, two well-characterized Kv1.3 inhibitors with different binding sites. Our results point out the relevance of considering auxiliary subunits in the development of therapeutic approaches. In conclusion, this thesis deepens the knowledge of the molecular mechanisms of Kv1.3 modulation by KCNE4. We provide a structural insight into Kv channel modulation while considering the physiological context. Our results are of considerably physiological interest due to the multitherapeutic potential of Kv1.3.

Programa de Doctorat: Bioinformàtica Membrane proteins are a large group of proteins that play an essential role in the cell. This group includes receptors, ion channels, transporters, and enzymes, which account for 25% of the proteins in the human genome. About 50% of membrane proteins are pharmacological targets for various diseases. In addition, there are pathogenic mutations that affect their folding, stability, and function for 90% of membrane proteins. Research on membrane proteins has grown in recent years. along with the development of computational tools to process sequence data, both structural and functional, have become essential to understanding all available information about these proteins. The main goal of this thesis is to develop bioinformatics tools to model and analyze membrane proteins. Les proteïnes de membrana són un gran grup de proteïnes que tenen un paper essencial a la cèl·lula. Aquest grup inclou receptors, canals iònics, transportadors i enzims, que representen el 25% de les proteïnes del genoma humà. Al voltant del 50% de les proteïnes de membrana són dianes farmacològiques per a diverses malalties. A més, hi ha mutacions patògenes que afecten el seu plegament, estabilitat i funció per al 90% de les proteïnes de membrana. La investigació sobre proteïnes de membrana ha crescut al llarg dels darrers anys. juntament amb el desenvolupament d'eines computacionals per tractar dades de seqüències, estructurals i funcionals, s'han convertit en essencials per entendre tota la informació disponible sobre aquestes proteïnes. L'objectiu principal d'aquesta tesi és desenvolupar eines bioinformàtiques per modelar i analitzar proteïnes de membrana.

Volume-regulated anion channel (VRAC), constituted by leucine-rich repeat-containing 8 (LRRC8) heteromers, is crucial for volume homeostasis in vertebrate cells. This widely expressed channel has been associated with membrane potential modulation, proliferation, migration, apoptosis, and glutamate release. VRAC is activated by cell swelling and by low cytoplasmic ionic strength or intracellular guanosine 5′-O-(3-thiotriphosphate) (GTP-γS) in isotonic conditions. Despite the substantial number of studies that characterized the biophysical properties of VRAC, its mechanism of activation remains a mystery. Different evidence suggests a possible effect of caveolins in modulating VRAC activity: (1) Caveolin 1 (Cav1)-deficient cells display insignificant swelling-induced Cl– currents mediated by VRAC, which can be restored by Cav1 expression; (2) Caveolin 3 (Cav3) knockout mice display reduced VRAC currents; and (3) Interaction between LRRC8A, the essential subunit for VRAC, and Cav3 has been found in transfected human embryonic kidney 293 (HEK 293) cells. In this study, we demonstrate a physical interaction between endogenous LRRC8A and Cav1 proteins, that is enhanced by hypotonic stimulation, suggesting that this will increase the availability of the channel to Cav1. In addition, LRRC8A targets plasma membrane regions outside caveolae of HEK 293 cells where it associates with non-caveolar Cav1. We propose that a rise in cell membrane tension by hypotonicity would flatten caveolae, as described previously, increasing the amount of Cav1 outside of caveolar structures interacting with VRAC. Besides, the expression of Cav1 in HEK Cav1- cells increases VRAC current density without changing the main biophysical properties of the channel. The present study provides further evidence on the relevance of Cav1 on the activation of endothelial VRAC through a functional molecular interaction.

Protons reaching the eyeball from exogenous acidic substances or released from damaged cells during inflammation, immune cells, after tissue injury or during chronic ophthalmic conditions, activate or modulate ion channels present in sensory nerve fibers that innervate the ocular anterior surface. Their identification as well as their role during disease is critical for the understanding of sensory ocular pathophysiology. They are likely to mediate some of the discomfort sensations accompanying several ophthalmic formulations and may represent novel targets for the development of new therapeutics for ocular pathologies. Among the ion channels expressed in trigeminal nociceptors innervating the anterior surface of the eye (cornea and conjunctiva) and annex ocular structures (eyelids), members of the TRP and ASIC families play a critical role in ocular acidic pain. Low pH (pH 6) activates TRPV1, a polymodal ion channel also activated by heat, capsaicin and hyperosmolar conditions. ASIC1, ASIC3 and heteromeric ASIC1/ASIC3 channels present in ocular nerve terminals are activated at pH 7.2–6.5, inducing pain by moderate acidifications of the ocular surface. These channels, together with TRPA1, are involved in acute ocular pain, as well as in painful sensations during allergic keratoconjunctivitis or other ophthalmic conditions, as blocking or reducing channel expression ameliorates ocular pain. TRPV1, TRPA1 and other ion channels are also present in corneal and conjunctival cells, promoting inflammation of the ocular surface after injury. In addition to the above-mentioned ion channels, members of the K2P and P2X ion channel families are also expressed in trigeminal neurons, however, their role in ocular pain remains unclear to date. In this report, these and other ion channels and receptors involved in acid sensing during ocular pathologies and pain are reviewed.

[cat] TRESK (K2P18.1) és un canal de potassi de fuga que s’expressa en algunes subpoblacions de neurones sensorials, on modula el potencial de membrana en repòs, la descàrrega de potencial d’acció i l’excitabilitat neuronal. Un subconjunt d’aquestes neurones sensorials, que expressen receptors acoblats a proteïnes G com els Mrgprs, són activats pels pruritògens i medien les sensacions de picor. Considerant que TRESK participa en la somatosensació i la percepció del dolor, hem avaluat la contribució d’aquest canal en la sensibilitat a la picor i el seu potencial com a diana terapèutica per al tractament de patologies que cursen amb picor crònica on s’inclouen la insuficiència renal o hepàtica, el limfoma de Hodgkin i diferents tipus de dermatitis. Els resultats dels registres de calci intracel·lular mostres que l'absència de TRESK no modifica el percentatge de neurones que responen a diferents pruritògens histaminèrgics i no histaminèrgics, indicant que TRESK participaria en la regulació de la excitabilitat neuronal, però no en la regulació dels mecanismes de detecció dels estímuls de picor. En els experiments in vivo hem observat l’absència del canal provoca un increment de les respostes de rascat induïdes per cloroquina, agonista del receptor MRGPRA3. Així doncs, suggerim que TRESK contribueix en la modulació de la sensibilitat a la picor aguda no histaminèrgica mediada per la subpoblació neuronal no peptidèrgica (NP2) que presenta una expressió característica del receptor MRGPRA3, mentre que el seu paper en altres subpoblacions (NP3) no és important. A més, en diferents models de picor crònica (dermatitis al·lèrgica de contacte, pell seca i psoriasis), aquesta està incrementada per l'absència del canal TRESK, cosa que indica la participació de les subpoblacions que expressen aquest canal en la fisiopatologia de la picor crònica d'origen dermatològic. Curiosament, la participació de TRESK és més rellevant en les primeres fases del desenvolupament de la picor crònica, mentre que la seva participació es més lleu en fases més tardanes de la patologia, quan la lesió dermatològica està més establerta. En resum, els resultats d’aquest estudi indiquen que TRESK és una diana farmacològica interessant per al tractament de la picor d'origen no histaminèrgic mitjançant el desenvolupament d'activadors selectius per aquest canal.

Actin polymerization and assembly into stress fibers (SFs) is central to many cellular processes. However, how SFs form in response to the mechanical interaction of cells with their environment is not fully understood. Here we have identified Piezo2 mechanosensitive cationic channel as a transducer of environmental physical cues into mechanobiological responses. Piezo2 is needed by brain metastatic cells from breast cancer (MDA-MB-231-BrM2) to probe their physical environment as they anchor and pull on their surroundings or when confronted with confined migration through narrow pores. Piezo2-mediated Ca2+ influx activates RhoA to control the formation and orientation of SFs and focal adhesions (FAs). A possible mechanism for the Piezo2-mediated activation of RhoA involves the recruitment of the Fyn kinase to the cell leading edge as well as calpain activation. Knockdown of Piezo2 in BrM2 cells alters SFs, FAs, and nuclear translocation of YAP; a phenotype rescued by overexpression of dominant-positive RhoA or its downstream effector, mDia1. Consequently, hallmarks of cancer invasion and metastasis related to RhoA, actin cytoskeleton, and/or force transmission, such as migration, extracellular matrix degradation, and Serpin B2 secretion, were reduced in cells lacking Piezo2.

Brugada syndrome (BrS) is defined as a cardiac disorder associated with high risk of sudden cardiac death and ventricular fibrillation. It is responsible of 4% of all sudden deaths and 20% of sudden cardiac deaths in patients without structural abnormalities. BrS is caused by dysfunction of ion channels involved in generation of the cardiac action potential. Approximately 20-25% of BrS patients have mutations in the coding region of the SCN5A gene, which encodes the α-subunit of the cardiac voltage-gated sodium channel. This work is included in the “Regulome-Seq” project, which aims to study non-coding DNA variants within transcription factor (TF) binding sites nearby BrS-associated genes. To characterize genetic variation in these regions, a set of pre-selected 1,293 loci was sequenced in a cohort of 89 diagnosed-BrS cases. To evaluate the consequences of the variants identified that overlap TF CTCF binding sites, the DeepBind algorithm was used to predict the effect of each variant in CTCF binding. 34 of these variants overlapping CTCF binding sites were cloned into a luciferase reporter vector to evaluate their effect in cell-based assays. After co-transfection in H9c2 cells, the luciferase activity obtained was measured to examine the CTCF binding level for each sequence. The results obtained indicate that DeepBind algorithm and luciferase assays results have a similar trend to evaluate the effect of the variants on CTCF binding. However, the results did not show a significant correlation between both methods. This suggests that the use of other DeepBind algorithms based on Machine Learning to predict BrS effects should be further explored. The experimental part of this work was complemented by its communication to the public and by innovative steps in general Science dissemination, with the conception of a Molecular Biology workshop on possible mutations in DNA. Communicating genome and DNA-related concepts is a great opportunity to improve scientific culture of society and to increase Science awareness among young students. Most people have a small idea about what BrS is, because they think is “something” related to mutations in their body hearts. This study seeks to explain the effect and influence of mutations in genetic diseases, to contribute to the open scientific attitude in the improvement of the educational process of Science in secondary education students and society

The SCN5A gene encodes the alpha subunit of the cardiac sodium channel (NaV1.5), which is responsible for the influx of sodium ions through membrane of cardiomyocytes. Different evidences suggest that an aberrant expression of the SCN5A gene may cause cardiac arrhythmias. However, the mechanisms that control SCN5A expression regulation are largely unknown. This thesis proposes a new mechanism of SCN5A transcriptional regulation in the adult human heart: transcription factors GATA4 and GATA5 synergize in the activation of the SCN5A expression. In addition, it has been proposed that GATA4 activity on the SCN5A is regulated by acetylation/deacetylation via the acetyltransferase p300 and the deacetylase HDAC2. It has been identified three lysines of GATA4 that are targets of p300 and HDAC2. In summary, this study contributes to further understand the molecular basis of the cardiac arrhythmias associated with alteration of sodium currents, El gen SCN5A codifica per la subunitat alfa del canal de sodi cardíac dependent de voltatge (NaV1.5), el qual permet l’entrada de ions sodi a través de la membrana dels cardiomiòcits. Diverses evidències suggereixen que una expressió anòmala del gen SCN5A pot donar lloc a arítmies cardíaques. Malauradament, els mecanismes que regulen l’expressió d’SCN5A són molt poc coneguts. Aquesta tesi proposa un nou mecanisme de regulació transcripcional del gen SCN5A en el cor humà adult: els factors de transcripció GATA4 i GATA5 activen sinèrgicament l’expressió del gen SCN5A. També s’ha proposat que l’activitat de GATA4 sobre SCN5A està regulada per un mecanisme d’acetilació/desacetilació a on hi participen l’acetiltransferasa p300 i la desacetilasa HDAC2. S’han identificat tres residus de lisines de GATA4 que són dianes de p300 i HDAC2. En resum, aquest estudi permet entendre millor les bases moleculars de les arítmies cardíaques associades amb alteracions del corrent de sodi

Current theories and mechanisms of nerve impulse propagation inside neurons do not give a satisfactory explanation to the nanoscale processes that occur inside the membrane. Propagation at the dendritic zones, and at the myelinated and unmyelinated axons, presents effects and events that, despite numerical calculations of current models fit with biological results obtained with macroscopic measurements, are far from giving an explanation to the real operation of biological mechanisms and to the effects and behaviors that the involved biological elements support. Current biological knowledge on the structure and behavior of biological membranes, as well as on the structure and function of voltage-regulated ion channels, allows us to consider alternatives to existing propagation mechanisms, giving explanation to the effects and behavior from both nanoscale and macroscopic level. This theoretical study proposes intra-membrane electric field propagation as a feasible propagation mechanism capable to explain both macroscopic and nanometric effects. With current theories and mechanisms, generation, propagation and regeneration of the nerve impulse is based on the action potential propagation effect. The action potential can be defined as the temporal imbalance that affects the membrane potential during nerve impulse propagation, as a consequence of ion channel opening. Progress on the neural membrane biological structure knowledge has allowed to propose a new hypothesis on the action potential operation. Besides the membrane potential variation, the electric charge that crosses the membrane generates an electric field that propagates inside the cell membrane, activating the closer ionic channels. This mechanism gives an explanation compatible with existing biological components and with other effects hard to explain with the standard current mechanisms. In addition, the proposed intra-membrane electric field propagation mechanism allows for a different explanation of the action potential evolution, as well as ion traffic level more in line with the capabilities of the biological reality, both on the ion traffic level and energy consumption. The proposed mechanism allows to explain the so-called saltatory action potential propagation on myelinated axons, and to apply the saltatory propagation to unmyelinated axons. In the latter, as short-distance steps; in the former, as long-distance jumps, also giving a coherent answer to the constant-time propagation independent of the distance between nodes of Ranvier. The proposed mechanism suggests that the effects the action potential presents are more the result of a charge displacement inside sodium and potassium channels than a current that crosses ion channels. An important factor to consider in this mechanism is the energy consumption minimization, a fundamental biological premise for maximum optimization of process operation., Les actuals teories i mecanismes de propagació de l'impuls nerviós per l'interior de les neurones no donen una explicació suficientment satisfactòria ni als principis físics als quals estan subjectes, ni als processos nanomètrics que es produeixen al seu interior. La propagació a les zones dendrítiques i als axons mielinitzats i no mielinitzats presenten efectes i esdeveniments que si bé el càlcul numèric dels actuals models concorda amb els resultats biològics obtinguts amb mesures macroscòpiques, estan lluny de donar una explicació al funcionament real dels mecanismes biològics i als efectes i comportaments que presenten la resta d'elements biològics participants que hi donen suport. Els actuals coneixements sobre l'estructura i comportament de les membranes biològiques, així com el coneixement sobre l'estructura i el funcionament dels canals iònics regulats per voltatge, ens permet plantejar una opció alternativa als mecanismes existents de propagació, donant explicació als efectes i comportaments tant des d'una escala nanomètrica com des d'una escala macroscòpica. En aquest treball teòric es proposa la propagació de camps elèctrics intra-membrana com un mecanisme possible de propagació amb la capacitat de donar explicació tant als efectes macroscòpics com als nanomètrics. Amb les teories i mecanismes actuals, la generació, la propagació i la reemissió de l'impuls nerviós es fonamenta en un efecte anomenat potencial d'acció. El potencial d'acció el podem definir com el desequilibri temporal que pateix el potencial de membrana, durant la propagació de l'impuls nerviós, i que és conseqüència de l'obertura dels canals iònics. El progrés en el coneixement sobre les estructures biològiques existents a les membranes neuronals ha permès plantejar una nova hipòtesi sobre el funcionament del potencial d'acció. A més de la variació del potencial de membrana, la càrrega elèctrica que travessa la membrana genera un camp elèctric que es propaga per l'interior de la membrana cel·lular, activant els canals iònics propers. Aquest mecanisme aporta una explicació consistent, compatible amb els components biològics existents i amb altres efectes difícilment explicables amb els mecanismes estàndard actuals. A més, el mecanisme de propagació per camp elèctric intra-membrana proposat permet donar una explicació diferent de l'evolució dels potencials d'acció, així com un nivell de tràfic iònic més en consonància amb les capacitats de la realitat biològica, tant en el nivell de tràfic existent com en el consum energètic. El mecanisme proposat permet explicar l'anomenada propagació saltatòria del potencial d'acció dels axons mielinitzats, i també aplicar la propagació saltatòria als axons no mielinitzats. En aquest cas com una propagació saltatòria de salts curts; per als axons mielinitzats, com una propagació saltatòria de salts llargs, donant també una resposta coherent a la propagació en temps constant independent de la distància entre nodes de Ranvier. El mecanisme proposat permet plantejar que els efectes que presenta el potencial d'acció són més el resultat d'un desplaçament de càrrega a l'interior dels canals de sodi i de potassi, que d'un corrent que realment travessa els canals iònics. Un factor important a tenir en compte en el mecanisme proposat és la minimització del cost energètic, premissa fonamental de la biologia per a la màxima optimització en el funcionament de processos.

L’objectiu de la present tesi doctoral consisteix en desenvolupar molècules policícliques de baix pes molecular que tinguin com a diana tres canals iònics específics: la viroporina A/M2 del virus de la grip, el receptor purinèrgic dependent de lligand P2X7 i el receptor glutamatèrgic dependent de lligand N-metil-D-Aspartat, per tal de modular-los d’una manera tal que n’evitin o millorin el desenvolupament dels processos patogènics associats a aquestes proteïnes i/o que permetin ser emprades com a eines de recerca. En el capítol 1 es presenta una introducció general del paper dels canals iònics, així com una descripció més específica de cadascun dels 3 canals objecte d’estudi en aquesta Tesi. Els objectius d’aquest treball es detallen en el capítol 2, després del qual, en el capítol 3 es presenten un seguit d’anàlegs per contracció d’anell del fàrmac comercial Amantadina, resultant-ne els primers inhibidors duals del canal M2 del virus de la Influenza A descrits en la bibliografia, actius sobre la soca salvatge (wt) i sobre el mutant resistent a fàrmacs V27A (Rey-Carrizo, M. et al. J. Med. Chem. 2013, 56, 9265). Seguidament en la part A del capítol 4 es gràcies a estudis in silico, s’observa com un dels inhibidors triples presenta un mode d’unió diferent sobre els canals wt i V27A, que podria estar directament relacionat amb el mecanisme de resistència a fàrmacs del mutant V27A (Rey-Carrizo, M., et al. J. Med. Chem. 2014, 57(13), 5738). En el capítol 5 es completa el treball presentant per primera vegada la proba experimental d’una possible explicació del mecanisme de resistència a fàrmacs del mutant V27A. (Barniol-Xicota, M., et al. manuscrit acceptat al J. Med. Chem.). Com a continuació en el capítol 6 s’explora una diana diferent de la viroporina M2: l’hemaglutinina (HA) (Leiva, R., et al. manuscrit pendent d’enviar a J. Med. Chem.). El capítol 7 representa el primer treball que marca l’inici de la línia de recerca d’antagonistes del receptor P2X7 en el grup. (Barniol-Xicota, M., et al. manuscrit en revisió al Bioorg. Med. Chem. Lett.). Finalment, en el capítol 8, es presenten els primers exemples descrits a la bibliografia de multi-target directed ligands (MTDL) amb els receptors NMDA i P2X7 com a dianes. (Karou, O., et al. manuscrit pendent d’enviar a ACS Med. Chem. Lett.). Deixant de banda la recerca de compostos bioactius en la part B del capítol 4 es descriu un estudi teòric i experimental d’un alquè altament piramidalitzat, derivat de l’estructura d’un compost bioactiu detallat en la part A del mateix capítol. (Rey-Carrizo, M. et al. Angew. Chem. Int. Ed. 2014, 53, 8195).

The Spanish Ion Channel Initiative consortium (SICI, http://sici.umh.es/default.htm) started in 2008 merging the research interests of 26 Spanish multidisciplinary groups focused on ion channel investigations from the pathophysiology to drug discovery. This initiative, partially supported by public Spanish government funds, was also the promoter of the Spanish Network on Ion Channels (RECI), a multidisciplinary network that includes more than 50 Spanish groups interested on ion channels (www.reci-ionchannel.org). In the present Special Issue, some SICI members, as well as RECI colleagues, wanted to contribute with the most update knowledge of representative ion channel research in Spain for the last 10 years.

The aim of the development of these investigation’s projects in Almirall is to find new molecules for a particular disease and it can get started in two different ways: the selection of a phenotype or of a protein targeting (GPCRs, enzymes, ion channels, etc.) whose modulation could become a benefit to a pathology of interest. One of the proteins targeting that is currently raising more interest in the therapeutic investigation are the ion channels. The reason is that they play a critical role in the monitoring of many physiological processes. These processes form the molecular base of the nervous system, they also adjust the selective transport of the ions and macromolecules, they have a vital role in the synthesis of the mediators, they take part in the homeostasis and in the cellular communication. This project focus in the (VGSC). Nine sodium channels in humans have been identified and found nowadays that perform vital functions in the central nervous system and in the peripheral nervous system, as well. The dysfunction of these channels can create diseases which are called channelopathies such as the Brugada syndrome, childhood epilepsy or chronic pain. Due to the required need in the discover of new drugs, the main objective of the study that was carried out for Almirall focused in the set-up of a new primary high-efficiency assay and minor costs than the electrophysiology using the platform FLIPR Tetra and monitoring the changes in the membrane potential, that could allow to characterize the power of new inhibitors of the sodium channel Nav1x. Once the set-up of the FLIPR Tetra was done, twenty-four inhibitors that were previously tested with the electrophysiology IonWorks quatro were characterized. The results obtained with the FLIPR Tetra were in truth successful, the 87,5% being the most detected compounds in the same potency order than the electrophysiology. Moreover, the 75% of the detected compounds with a different potency order were more powerful than in the electrophysiology assay and the remaining 25% only seven times less powerful. This could assure its detection with the set-up assay. The controlling assay of the membrane potential using the FLIPR Tetra could be considered acceptable as a primary assay of characterization and narrowed down for specific inhibitors of the sodium channel Nav1.x, putting the electrophysiology as a activityconfirmation assay and characterization of inhibitors

Sudden cardiac death poses a unique challenge to clinicians because it may be the only symptom of an inherited heart condition. Indeed, inherited heart diseases can cause sudden cardiac death in older and younger individuals. Two groups of familial diseases are responsible for sudden cardiac death: cardiomyopathies (mainly hypertrophic cardiomyopathy, dilated cardiomyopathy, and arrhythmogenic cardiomyopathy) and channelopathies (mainly long QT syndrome, Brugada syndrome, short QT syndrome, and catecholaminergic polymorphic ventricular tachycardia). This review focuses on cardiac channelopathies, which are characterized by lethal arrhythmias in the structurally normal heart, incomplete penetrance, and variable expressivity. Arrhythmias in these diseases result from pathogenic variants in genes encoding cardiac ion channels or associated proteins. Due to a lack of gross structural changes in the heart, channelopathies are often considered as potential causes of death in otherwise unexplained forensic autopsies. The asymptomatic nature of channelopathies is cause for concern in family members who may be carrying genetic risk factors, making the identification of these genetic factors of significant clinical importance.

The excitable properties of the neural cell membrane is the driving mechanism of the neural pulses. Coordinated ionic fluxes across Na and K channels are the devices responsible of this function. Here we present a simple microscopic physical scenario which accounts for this phenomenology. The main elements are ions and channel doors that obey the standard formulation of statistical physics (overdamped Langevin equations) with appropriate nonlinear interacting potentials. From these equations we obtain the ionic flux and the dynamics of the membrane potential. We show that the excitable properties of the membrane are present in a single and simple Na channel. From this framework, additional microscopic information can be obtained, such as statistics of single-channels dynamics or the energetics of action potential events.

En el presente trabajo, utilizamos técnicas de microfabricación, simulaciones numéricas, experimentos de electrofisiología para explorar la viabilidad en me- jorar la interface ordenador-neurona a través de microcanales, y la biofísica para la generación de señales en los dispositivos con microcanales. También demos- tramos que los microcanales pueden ser usados como una técnica prometedora con alto rendimiento en el muestreo automático de canales iónicos a nivel subce- lular. Finalmente, se ha diseñado, fabricado y probado el micropozo-microcanal como modificación adicional a los arreglos de multielectrodos, permitiendo una alta ganancia en la relación señal/ ruido (en inglés Signal to Noise Ratio SNR), y el registro de múltiples-lugares en poblaciones de baja densidad de redes neu- ronales del hipocampo in vitro. Primero, demostramos que son de alto rendimiento los microcanales de bajo costo con interface neurona-electrodo, para el registro extracelular de la activi- dad neuronal con baja complexidad, por periodos estables de larga duración y con alta ganancia SNR. En seguida, se realiza un estudio mediante experimentos y simulaciones nu- méricas de la biofísica para la generación de las señales obtenidas de los dispositi- vos con microcanales. Basados en los resultados, racionalizamos y demostramos como es que la longitud del canal (siendo 200 μm) y la sección transversal del microcanal (siendo 12 μm2) canaliza a los potenciales de acción para estar dentro del rango de milivolts. A pesar del bajo grado de complexidad envuelto en la fabricación y aplicación, los dispositivos con microcanales otorgan una sola media de valor SNR de 101 76, lo cual es favorablemente comparable con la SNR que se obtiene de desarrollos recientes que emplean electrodos curados con CNT y Si-NWFETs. Más aún, nosotros demostramos que el microcanal es una técnica promete- dora para el alto rendimiento del muestro automático de canales iónicos a nivel subcelular: (1) Información experimental y simulaciones numéricas sugieren que las señales registradas sólo afectan los parches membranales localizados dentro del microcanal o alrededor de 100 μm de las entradas del microcanal. (2) La transferencia de masa de los componentes químicos en los microcanales fue ana- lizada por experimentos y simulaciones FEM. Los resultados muestran que los microcanales que contienen glía y tejido neuronal pueden funcionar como barre- ra de fluido/química. Los componentes químicos pueden ser solamente aplicados a diferentes compartimentos a nivel subcelular. Finalmente, basado en simulaciones numéricas y resultados experimentales, se propone que del micropozo-microcanal, obtenido de la modificación de MEA (MWMC-MEA), la longitud óptima del canal debe ser 0,3 mm y la posición 1 óptima del electrodo intracanal, hacia la entrada más cercana del microcanal, debe ser 0,1 mm. Nosotros fabricamos un prototipo de MWMC-MEA, cuyo hoyo pasante sobre las películas de Polydimethylsiloxane (PDMS) fue microtrabajado a través de la técnica de grabados reactivos de plasma de iones. La baja densidad del cultivo (57 neuronas /mm2) en el MWMC-MEAs permitió que las neuronas vivieran al menos 14 días, con lo que la señal neuronal con la máxima SNR obtenida fue de 142. 2, In this present work, we used microfabrication techniques, numerical simulations, electrophysiological experiments to explore the feasibility of enhancing neuron-computer interfaces with microchannels and the biophysics of the signal generation in microchannel devices. We also demonstrate the microchannel can be used as a promising technique for high-throughput automatic ion-channel screening at subcellular level. Finally, a microwell-microchannel enhanced multielectrode array allowing high signal-to-noise ratio (SNR), multi-site recording from the low-density hippocampal neural network in vitro was designed, fabricated and tested. First, we demonstrate using microchannels as a low-cost neuron-electrode interface to support low-complexity, long-term-stable, high SNR extracellular recording of neural activity, with high-throughput potential. Next, the biophysics of the signal generation of microchannel devices was studied by experiments and numerical simulations. Based on the results, we demonstrate and rationalize how channels with a length of 200 μm and channel cross section of 12 μm2 yielded spike sizes in the millivolt range. Despite the low degree of complexity involved in their fabrication and use, microchannel devices provided a single-unit mean SNR of 101 76, which compares favourably with the SNR obtained from recent developments employing CNT-coated electrodes and Si-NWFETs. Moreover, we further demonstrate that the microchannel is a promising technique for high-throughput automatic ion-channel screening at subcellular level: (1) Experimental data and numerical simulations suggest that the recorded signals are only affected by the membrane patches located inside the microchannel or within 100 μm to the microchannel entrances. (2) The mass transfer of chemical compounds in microchannels was analyzed by experiments and FEM simulations. The results show that the microchannel threaded by glial and neural tissue can function as fluid/chemical barrier. Thus chemical compounds can be applied to different subcellular compartments exclusively. Finally, a microwell-microchannel enhanced MEA (MWMC-MEA), with the optimal channel length of 0.3 mm and the optimal intrachannel electrode position of 0.1 mm to the nearest channel entrance, was proposed based on numerical simulation and experiment results. We fabricated a prototype of the MWMCMEA, whose through-hole feature of Polydimethylsiloxane film (PDMS) was micromachined by reactive-ion etching. The low-density culture (57 neurons/mm2) were survived on the MWMC-MEAs for at least 14 days, from which the neuronal signal with the maximum SNR of 142 was obtained.

In contrast to the highly-selective channels of neurophysiology employing mostly the exclusion mechanism, different factors account for the selectivity of large channels. Elucidation of these factors is essential for understanding the permeation mechanisms in ion channels and their regulation in vivo. The interaction between divalent cations and a protein channel, the bacterial porin OmpF, has been investigated paying attention to the channel selectivity and its dependence on the solution pH. Unlike the experiments performed in salts of monovalent cations, the channel is now practically insensitive to pH, being anion selective all over the pH range considered. Electrostatic calculations based on the available structural data suggest that the binding of divalent cations has two main effects: (i) the pK(a) values of key ionizable groups differ significantly from those of the isolated groups in solution and (ii) the cation binding has a decisive impact on the effective electric charge regulating the channel selectivity. A simple molecular model based on statistical thermodynamics provides additional qualitative explanations to the experimental findings that could also be useful for other related systems like synthetic nanopores, ion exchange membranes, and polyelectrolyte multilayers.

Trp(Nps)-Lys-NH(2) derivatives, bearing alkyl or guanidine groups either at the N-terminus or on the Lys side-chain or at both positions were conveniently prepared on solid-phase and evaluated as TRPV1 channel antagonists.

Esta tesis plantea por primera vez la existencia de canalopatías autoinmunes en el sistema nervioso central, aportando los resultados obtenidos de dos trabajos importantes. Se conocía la existencia de autoanticuerpos contra canales iónicos dependientes de voltaje en síndromes neurológicos periféricos. Dada la asociación entre síndromes que afectan al SNP y síndromes neurológicos centrales, se quiso estudiar si los autoanticuerpos contra canales iónicos dependientes de voltaje presentes en los síndromes neurológicos periféricos, podían estar presentes en síndromes neurológicos centrales. Esto se quiso estudiar en los anticuerpos contra canales de calcio dependientes de voltaje (VGCC) presentes en el síndrome miasténico de Eaton-Lambert (SMEL). Dada la conocida asociación entre el SMEL y la degeneración cerebelosa paraneoplásica subaguda (DCP), quedaba por determinar si la DCP podía estar asociada o no los anticuerpos anti-VGCC. Posteriormente, se amplió el estudio de los anticuerpos antineuronales contra canales iónicos en asociación con síndromes neurológicos del SNC, a los canales de potasio dependientes de voltaje (VGKC). Se conocía la presencia de anticuerpos anti-VGKC en la neuromiotonía. Surgió la posibilidad de una asociación entre la encefalitis límbica y otros anticuerpos o trastornos. Además, un 40% de los pacientes con encefalitis límbica quedaban por determinar séricamente, por la ausencia de anticuerpos (seronegativos) o la presencia de anticuerpos no caracterizados. Se estudió de la presencia de anticuerpos anti-VGKC en pacientes con una encefalitis límbica, de origen paraneoplásico o idiopático.Con los trabajos realizados, se ha abierto la posibilidad a la existencia de canalopatías autoinmunes en el SNC, como la ataxia causada por la degeneración cerebelosa asociada al cáncer de pulmón de célula pequeña y la encefalitis límbica. La relación causal del anticuerpo con el síndrome neurológico no se ha dilucidado, dado que es difícil demostrar si los anticuerpos son capaces de cruzar la barrera hematoencefálica o si son sintetizados en el propio SNC, causando la posterior disfunción neuronal.Los anticuerpos descritos hasta ahora en el SNC eran utilizados como marcadores de síndromes paraneoplásicos y por lo tanto, no eran patogénicos y su diana era intracelular. En cambio, los anticuerpos que se han descrito en esta tesis como causantes de las canalopatías autoinmunes son patogénicos y en principio tienen dianas extracelulares: los canales iónicos neuronales. Esta diferencia plantea interesantes preguntas sobre la relación entre el SNC y el sistema inmune. Aunque a nivel práctico, nos permite tener una herramienta diagnóstico-terapéutica frente a pacientes que presenten estos síndromes, pues aquellos pacientes que presentan anticuerpos, especialmente si tienen una encefalitis límbica, responden muy bien a tratamiento inmunosupresor. Por lo tanto, los anticuerpos descritos en esta tesis permiten ser utilizados como un marcador diagnóstico y de control de mejoría clínico-inmunológica. Esto es un avance para manejo de estos pacientes neurológicos.

[Objective]: Cellular cardiomyoplasty using skeletal myoblasts is a promising therapy for myocardial infarct repair. Once transplanted, myoblasts grow, differentiate and adapt their electrophysiological properties towards more cardiac-like phenotypes. Voltage-dependent Na+ channels (Nav) are the main proteins involved in the propagation of the cardiac action potential, and their phenotype affects cardiac performance. Therefore, we examined the expression of Nav during proliferation and differentiation in skeletal myocytes. [Methods and results]: We used the rat neonatal skeletal myocyte cell line L6E9. Proliferation of L6E9 cells induced Nav1.4 and Nav1.5, although neither protein has an apparent role in cell growth. During myogenesis, Nav1.5 was largely induced. Electrophysiological and pharmacological properties, as well as mRNA expression, indicate that cardiac-type Nav1.5 accounts for almost 90% of the Na+ current in myotubes. Unlike in proliferation, this protein plays a pivotal role in myogenesis. The adoption of a cardiac-like phenotype is further supported by the increase in Nav1.5 colocalization in caveolae. Finally, we demonstrate that the treatment of myoblasts with neuregulin further increased Nav1.5 in skeletal myocytes. [Conclusion]: Our results indicate that skeletal myotubes adopt a cardiac-like phenotype in cell culture conditions and that the expression of Nav1.5 acts as an underlying molecular mechanism. © 2007 Elsevier B.V. All rights reserved., Supported by the Ministerio de Educación y Ciencia (MEC), Spain (BFI2002-00764 and BFU2005-00695 to AF; SAF2005-00489 to AG; SAF2004-06856, CAM GR/SAL/0854/2004 and FIS RD06/0014/0006 to CV). RMM, RS and NV hold fellowships from the MEC and MRF from the Generalitat de Catalunya.

The presence of ATP within cells is well established. However, ATP also operates as an intercellular signal via specific purinoceptors. Furthermore, nonsecretory cells can release ATP under certain experimental conditions. To measure ATP release and membrane currents from a single cell simultaneously, we used Xenopus oocytes. We simultaneously recorded membrane currents and luminescence. Here, we show that ATP release can be triggered in Xenopus oocytes by hyperpolarizing pulses. ATP release (3.2 +/- 0.3 pmol/oocyte) generated a slow inward current (2.3 +/- 0.1 microA). During hyperpolarizing pulses, the permeability for ATP(4-) was more than 4000 times higher than that for Cl(-). The sensitivity to GdCl(3) (0. 2 mm) of hyperpolarization-induced ionic current, ATP release and E-ATPase activity suggests their dependence on stretch-activated ion channels. The pharmacological profile of the current inhibition coincides with the inhibition of ecto-ATPase activity. This enzyme is highly conserved among species, and in humans, it has been cloned and characterized as CD39. The translation, in Xenopus oocytes, of human CD39 mRNA encoding enhances the ATP-supported current, indicating that CD39 is directly or indirectly responsible for the electrodiffusion of ATP.

Introduction The cell membranes are formed of lipids and proteins. Because of their hydrophobic nature, phospholipids separate the intracellular from the extracellular medium. Development The extracellular medium is a solution enriched with sodium ions, whilst the intracellular medium is rich in potassium ions. In accordance with the laws of chemistry, the asymmetric distribution of ions causes a small potential difference between the inside and outside of the cell. All the cells of the human body show this small difference in potential of about -60 mV. Another component of the cell membranes are proteins. Some of these--the ion pumps or ATPases--maintain the asymmetric distribution of the ions. Other membrane proteins--the ion channels--permit the selective passage of ions between the intracellular and extracellular media. Electrophysiological techniques (patch clamp) applied at a cellular level allow us to measure the passage of ions across a single molecule. Each ion channel permits the passage of a single ionic species. The cells contain ion channels which are transiently permeable to the ions of sodium, potassium, calcium and chlorine. One of the characteristic properties of neurons is that they are excitable cells. Neuronal excitability depends on the ion channels. The neurons show different electrical behavior during the different phases of life. The changes in the electrical activity of the neurons are determined by the activity of the ion channels and their interaction with the liquid surrounding them. Conclusions Investigations are being done to relate the changes in behavior of the ion channels with the process of aging of the neurone membranes.