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Nucleoside transport systems and their regulation in human B-lymphocytes have been characterized using the cell lines Raji and Bare lymphoma syndrome-1 (BLS-1) as experimental models. These cells express at least three different nucleoside transport systems as follows: a nitrobenzylthioinosine-sensitive equilibrative transport system of the es-type, which appears to be associated with hENT1 expression, and two Na+-dependent transport systems that may correspond to N1 and to the recently characterized N5-type, which is nitrobenzylthioinosine-sensitive and guanosine-preferring. B cell activators such as phorbol 12-myristate 13-acetate and lipopolysaccharide (LPS) up-regulate both concentrative transport systems but down-regulate the equilibrative es-type transporter, which correlates with lower hENT1 mRNA levels. These effects are dependent on protein kinase C activity. Phorbol 12-myristate 13-acetate and LPS also induce an increase in tumor necrosis factor-alpha (TNF-alpha) mRNA levels, which suggest that this cytokine may mediate some of the effects triggered by these agents, since addition of TNF-alpha alone can increase N1 and N5 transport activities by a mechanism that also depends on protein kinase C activation. Interestingly, TNF-alpha down-regulates es activity, but this effect cannot be abolished by inhibiting protein kinase C. This study reveals differential regulation of nucleoside transport systems following activation of human B-lymphocyte cell lines by agents of physiological relevance such as TNF-alpha and LPS. Moreover, it indicates that the recently characterized N5 transport system can also be regulated following B cell activation, which may be relevant to lymphocyte physiology and to the treatment of lymphocyte malignancies.

El transport de bona part d'aminoàcids neutres des de la sang, on són majoritaris, cap a 1'interior de la pràctica totalitat de cèl·lules de mamífer, depèn essencialment d'un sistema de transport conegut com a sistema A. Com la majoria de sistemes de captació de soluts orgànics i inorgànics, l'A ha estat caracteritzat com una entitat cinètica, és a dir, es defineix segons la seva funcionalitat biològica, la qual implica tot un seguit de característiques pròpies pel que fa al tipus de substrat que pot captar, 1'afinitat envers aquests substrats, 1'existència o no d'inhibidors específics, la dependència de fluxos acoblats (dependència de Na+, per exemple), la susceptibilitat a ser regulat bé sigui per mecanismes a curt o a llarg termini i moltes altres 'qualitats' funcionals que permeten assumir que una determinada activitat biològica probablement es correspongui amb un producte gènic determinat.

[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.

[eng] Voltage gated K+ channels (Kv) are transmembrane proteins that allow the pass-thorugh of potassium ions, regulating the electrochemical gradient of the cell membrane. This way, they modulate several physiological processes, such as proliferation, migration or cell volume. Of particular interest in this dissertation is their role in excitable cells, were they control several key functions. The relevance of this ion channels is evidenced when mutations or alterations in the proper functioning of Kv channels causes severe pathologies, including cardiovascular or neuronal diseases, autoimmune affectations or cancer. Kv channels are tetramers of 4 α subunits with 6 transmembrane segments each one, that associate to form the pore and generate a functional channel. The wide functional diversity of currents is due to a vast number of modulations: heterotetramerization of α subunits, splicing variants, post-translational modifications or the association with regulatory subunits. The last ones include KCNE family, which co-assemble with the channel and modulate its electrophysiological, pharmacological or physiological properties. Kv7.1 associates with KCNE1 in cardiomyocytes to generate IKs cardiac repolarizing currents, in charge of finishing the cardiac action potential. Their assembly and traffic to the plasma membrane have been subject of discussion over the last years, with two opposite schools claiming an association early in the biogenesis versus a independent traffic to the plasma membrane, were both proteins would diffuse to assemble. We aimed in the present work to shed a light to this controversial topic. Kv channels have also been described in vascular smooth muscle, were they set the resting membrane potential and, therefore, control vascular tone. Kv7.1, Kv7.4 and Kv7.5 have been detected in different veins and arteries, were aberrations in their expression promote physiological alterations, but the specific role of each subunit remains unknown. In this scenario, the proposed objectives for the current PhD dissertation included the study of Kv7.1-KCNE1 complex, its assembly and traffic mechanisms. We hypothesized an unconventional secretion for the complex and suggest ER-PM junctions as the potential trafficking system. Therefore, we aim to characterize this structures and their implication in Kv7.1 membrane targeting. Finally, due to its implication in proliferation, their importance in cardiovascular system and their known role in some cancers, we studied the changes in the expression of Kv channels in endothelial-derived vascular tumors. We have been able to solve the traffic controversy of Kv7.1-KCNE1 complexes as they are not assembled early in their biogenesis. While KCNE1 is using the conventional secretion pathway, Kv7.1 takes an unconventional route that skips Golgi. Upon co-assembly, Kv7.1 redirects KCNE1 to this unconventional pathway. Moreover, we have proved that this non-conventional route are indeed ER-PM junctions, which also host the assembly of the complex. The molecular interactors of the channel during its ER-PM junction targeting have also been analysed during this PhD thesis, unravelling a complex and dynamic proteomic context. In addition, we have described for the first time the expression of Kv1.3, Kv1.5, Kv7.1 and Kv7.5 in endothelial cells of human veins and arteries. A remodelling of this composition is observed in different vascular cancers, related with the malignancy of the tumor in some of the cases., [cat] ls canals de potassi dependents (Kv) regulen processos fisiològics molt importants, com la proliferació, la migració o el volum cel·lular. La seva rellevància es posa de manifest amb les diferents patologies associades a alteracions en la expressió dels canals, incloent malalties cardiovasculars, cerebrals, autoimmunes o càncer. Es tracta de proteïnes transmembrana formades per l’associació de 4 subunitats α que s’uneixen per formar el por. La gran varietat de diversitat funcional és deguda a la capacitat de heterotetramerització dels canals, variants d’splicing, modificacions post-traduccionals o la associació a subunitats reguladores KCNE, entre d’altres. En cardiomiòcits, Kv7.1 s’associa a KCNE1 per generar les corrents IKs, encarregades de la repolarització del potencial cardíac. La seva associació i tràfic són tema de debat des de fa anys, amb dues escoles defensant idees oposades. La primera, que les dues proteïnes s’associen en les fases inicials de la biogènesi; la segona, que trafiquen independent cap a la membrana, on difondran per trobar-se. Els Kv també s’han detectat a musculatura vascular llisa, on mantenen el potencial de repòs i controlen així el to vascular. Kv7.1, Kv7.4 i Kv7.5 es troben en diferents venes i arteries, on una expressió aberrant provoca alteracions fisiològiques. Tot i així, el seu paper concret encara es desconeix. En la present tesi doctoral hem comprovat que Kv7.1 i KCNE1 utilitzen vies diferents per arribar a la membrana plasmàtica. KCNE1 viatja per la via convencional, mentre que Kv7.1 utilitza una ruta no convencional que escapa del Golgi. Quan co-expressats, Kv7.1 redirigeix KCNE1 cap aquesta via alternativa. Hem demostrat que aquesta via són les ER-PM junctions, que també són el compartiment on la seva associació té lloc. Els interactors moleculars del canal durant el seu tràfic cap a ER-PM junctions també s’ha estudiat durant aquest treball. A més a més, hem descrit per primer cop l’expressió de Kv1.3, Kv1.5, Kv7.1 i Kv7.5 en l’endoteli de venes i artèries humanes. Hem vist un remodelatge en aquesta expressió en diferents càncers vasculars, en alguns casos relacionat amb la malignitat del tumor.

[eng] Kv1.3 is a voltage-gated potassium channel mainly expressed at the plasma membrane and at the inner mitochondrial membrane of different cell types. A dual role for the channel has been suggested: Plasma membrane channel is linked to proliferation and activation while mitochondrial channel controls apoptosis. In this thesis, we analyse in depth the duality of Kv1.3 function. We show that Kv1.3 orchestrates cellular physiology in a way even more complex than expected. The thesis is structured in two parts: Plasma membrane Kv1.3 and Mitochondrial Kv1.3. Each part contains three contributions addressed to the understanding of Kv1.3 traffic and physiological implications of its localization. In the first part (Plasma membrane Kv1.3), we describe the caveolin-binding domain (CBD) of Kv1.3 as the determinant for the lipid raft targeting of the channel in cells expressing caveolin (Contribution I). The caveolar localization of the channel is required for proper insulin signalling in adipocytes. In these cells, Kv1.3 controls glucose uptake and insulin resistance. Kv1.3 is a target of Insulin Receptor activation via its localization at caveolae (Contribution II). Finally, we focus on the mechanisms of membrane arrangement of Kv1.3 in systems with limited Caveolin expression, such as lymphocytes, where the expression of Kv1.3 at the plasma membrane is also crucial. In this context, Kv1.3 palmitoylation resulted to be important for the Kv1.3 organization at the immunological synapse. This is the first time that Kv1.3 lipidation is reported and that Kv1.3 is visualized at the immunological synapse platform. Thus, our work expands our knowledge to define better therapeutic strategies in autoimmune diseases, where Kv1.3 is aberrantly expressed at the immunological synapse (Contribution III). In the second part (Mitochondrial Kv1.3), we first identify caveolin interaction as regulator of Kv1.3 pro-apoptotic activity. Kv1.3/Caveolin axis is an important regulator of cell apoptosis, with important consequences for chemotherapy resistance (Contribution IV). We also describe, for the first time, the mechanisms of Kv1.3 mitochondrial targeting, which involve an unconventional TIM/TOM pathway. We identify cytosolic chaperones as key regulators of Kv1.3 mitochondrial import and show how transmembrane domains cooperate to mediate such process (Contribution V). Finally, we show, for the first time, that mitochondrial Kv1.3 can also regulate proliferation through the control of mitochondrial fusion/fission equilibrium. This is a novel finding because reveals a novel function for mitochondrial Kv1.3 beyond apoptosis. At the same time, puzzle out Kv1.3 as an important component of the mitochondrial fusion/fission machinery (ContributionVI). In conclusion, this thesis includes novel and relevant findings about Kv1.3 function linked to its localization in the cell., [cat] Kv1.3 és un canal de potassi depenent de voltatge expressat, principalment, a la membrana plasmàtica i a la membrana mitocondrial interna. La localització dual del canal a membrana i a mitocondri s’ha associat amb la seva funció antagònica, on el canal regula proliferació i apoptosi, respectivament. En el context d’aquesta tesi hem estudiat els mecanismes que regulen l’arribada del canal al seu destí, i les implicacions funcionals de la seva presència en aquests compartiments subcel·lulars. La tesi conté 6 contribucions que inclouen els següents resultats: En primer lloc, es descriu la interacció del Kv1.3 amb la Caveolina com a mecanisme principal de tràfic a estructures de membrana lípid raft, on el canal exerceix la seva funció. En segon lloc, es demostra que la localització del canal a les caveoles és necessària per a la seva participació en la cascada de senyalització per insulina en adipòcits, gràcies a la qual el canal regula la fisiologia adipocitària i la captació de glucosa dependent d’insulina. En la tercera contribució, descrivim la palmitoilació de Kv1.3 com a mecanisme de regulació de la distribució del canal a la sinapsi immunològica en limfòcits T. Durant aquest fenomen, Kv1.3 té un paper clau per a l’activació del limfòcits i aberracions en la seva localització i activitat s’han relacionat en el desenvolupament de malalties autoimmunitàries. A la contribució quarta, descrivim la modulació de la caveolina sobre l’activitat pro-apoptòtica del canal a mitocondris. A la cinquena contribució demostrem que el canal segueix un mecanisme no convencional d’import a mitocondris i que els dominis transmembrana defineix motius estructurals per tal de promoure aquest procés. Finalment, amb la última contribució, demostrem que el canal està implicat en la regulació de l’equilibri de fusió/fissió mitocondrial, amb importants conseqüències per al cicle cel·lular.

[eng] Voltage-gated potassium channels are proteins that allow the flux of potassium ions across the plasma membrane in response to a voltage stimulus. Those proteins were initially described in nervous system as the repolarization entities posterior to a depolarization. However, several different roles have been discovered to be enhanced, mediated or influenced by those entities. Cell cycle progression, homeostasis, proliferation or activation and apoptosis program are some of those functions. Kv1.3 is the third member of the first family of voltage-gated potassium channels in humans. This specific entity is mainly expressed in nervous and immune system. It has been associated with the repolarization of neurons, the activation and proliferation of leukocytes and apoptosis. Moreover, its dysfunctionality has been related to some autoimmune disease. The fine-tunning of the channel is highly relevant to control the final cell decision. The subunits that accompany the channel were classically named as β- subunits. Several different families have been described to modulate some channel features. Kvβ family are cytoplasmic proteins that can enhance the traffic to the plasma membrane and promote a switch towards negative voltages of the channel activation. However, few are known about alternative locations and Kv1.3 modulation. KCNE family are single spanning proteins highly promiscuous that modulate several different Kvα-subunits. Depending on the KCNE subtype, the effect on the channels can present different natures. This dissertation is focused on KCNE4 member, a peptide which generally negatively regulates Kv channels. This thesis described the positioning of Kvβ2.1, but not Kvβ1.1, in specific regions of the plasma membrane: lipid raft microdomains. Those are considered as signalling platforms at the plasma membrane highly relevant for several cellular processes. The possible mechanism that drives Kvβ2.1 is the palmitoylation of its amino acidic sequence; even other causes are not discarded. Proliferation signals are enhancing this localization while PMA treatment generates the opposite effect. This protein, as well as its partner Kvβ1.1, can form homo and heteroligomers. Their affinity and stoichiometry was addressed. Furthermore, multiprotein complexes were detected at membrane associated environments. Traffic and electrophysiological consequences on the channel were analysed upon coexpression with those subunits. Kv1.3 was removed from lipid raft microdomains and Kvβs prevented partially its PMA-dependent internalization. The molecular determinants involved in the Kv1.3 traffic to the plasma membrane were localised at the C-terminal domain. Previous results from the laboratory determined that KCNE4 is impairing the traffic of the channel. This thesis deciphered the molecular mechanisms involved in this effect concluding a bipartite system: (i) the masking of Kv1.3 export signal and (ii) the transference of a retention signal to the channelosome. Moreover, the specific domains of Kv1.3 and KCNE4 implicated in their interaction were mapped and pointed out to the C-terminal regions of both peptides. KCNE4 was also found to form oligomers and present several signals for its retention at the endoplasmic reticulum. Finally, the combination of both subunits (Kvβ2.1 and KCNE4) on the channel showed a dominance of KCNE4 effects, but an electrophysiological function of Kvβ2.1 on Kv1.3 kept preserved. Thus, the present thesis brought light to the comprehension of Kv1.3 channelosome.

Voltage-gated K channels (Kv) is large family of channels that are expressed in both excitable and non-excitable cells. In excitable cells they contribute to the control of resting membrane potential and action potentials frequency and duration. In non-excitable tissues they are involved in many processes such as secretion to cell proliferation. Kv1.3 channel plays a key role in a wide range of physiological phenomenon. Regulation of this transmembrane protein is therefore essential for a correct function of the living cell. The balance between synthesis and degradation is highly important and must be tightly regulated. The present dissertation is focused in investigating endocytosis mechanisms of Kv1.3, as a process controlling number of the channel on the cell surface and the possible implication in cell destiny. We deciphered major endocytosis mechanisms triggered by EGF and Adenosine (ADO) in HeLa and HEK 293 heterologous cell systems as well as in native cell lines (macrophages, dendritic or neuronal precursor). These studies pointed out the impact of endocytosis in turnover and homeostasis of Kv1.3 and possible physiological relevance of these finding. Our experiments showed two different ways to control abundance of the Kv1.3 channel by EGF: via tyrosine phosphorylation and unconventional ERK1/2-dependent mechanisms. EGF triggered clathrin-dependent lysosomal degradation of Kv1.3. Moreover, this study show a high physiological relevance, pointing to EGF as a Kv1.3 inhibitor that might therefore reduce radiation-induced brain injury by targeting the key cells involved in the inflammatory process. As next, study was to investigating PMA-induced PKC-dependent endocytosis and ubiquitination. We revealed that PMA triggered PKC-dependent ubiquitin-mediated lysosomal degradation of Kv1.3. Next, we show that adenosine (ADO), which is a potent endogenous modulator, similar to PMA, stimulates PKC, thereby causing immunosuppression. PKC activation triggers down-regulation of Kv1.3 by inducing a clathrin-mediated endocytosis which targets the channel to lysosomal-degradative compartments. Therefore, the abundance of Kv1.3 at the cell surface decreases, which is clearly compatible with an effective anti-inflammatory response. This mechanism requires ubiquitination of Kv1.3, catalyzed by the E3 ubiquitin-ligase Nedd4-2. However, we discover that ADO activates both PKC and PKA signaling pathways. To further investigate the molecular mechanisms of the Kv1.3 internalization in response to ADO, we have examined the effects of PKA antagonists. Our results, for the first time, provided evidence on the effect of PKA activation on the Kv1.3 trafficking. Our findings indicated that PKA adenosine activation triggered Kv1.3 endocytosis redundantly to PKC. In addition, we put a hypothesis that PKA downregulated Kv1.3 in an ubiquitin-independent manner. In the last part of this dissertation we concentrated at molecular determinants involved in Kv1.3 ubiquitination. We found that complementary and redundant lysines participate in the ubiquitin-dependent PKC and PKA regulation of Kv1.3.

El Síndrome de Dravet es una de las 6.500‐8.000 enfermedades raras reconocidas por la Organización Mundial de la Salud (ORPHA:33069). Es una encefalopatía grave, caracterizada por el comienzo, antes del primer año de vida, de convulsiones recurrentes, febriles y/o afebriles, hemiclónicas o generalizadas, (status epiléptico), en niños anteriormente sanos. Este cuadro inicial es seguido de múltiples convulsiones, generalmente resistentes a fármacos antiepilépticos, con retraso o parada del desarrollo cognitivo y psicomotor. Está catalogada como canalopatía ya que más del 80% de los pacientes presentan mutaciones con pérdida de función en un alelo del gen SCN1A, que codifica para la subunidad alfa del canal de sodio dependiente de voltaje Nav1.1. Una función disminuida altera severamente la corriente de sodio y por tanto el disparo del potencial de acción. Debido a que Nav1.1 está expresado en interneuronas inhibitorias GABAérgicas, mayoritariamente del hipocampo y cerebelo, la caída de la actividad, provoca hiperexcitabilidad por defecto de inhibición. Además, parece que, como mecanismo compensatorio, se aumenta la expresión y/o la actividad de Nav1.6, canal de las neuronas excitatorias de Purkinje del cerebelo, lo que contribuye a la ataxia. El descubrimiento de la etiología genética del Síndrome de Dravet en 2001, sentó las bases para la búsqueda de tratamientos efectivos, principalmente paliativos, debido a la propia complejidad de los canales de sodio y las técnicas de electrofisiología. En esta tesis doctoral se ha realizado la puesta a punto de una técnica de medida que permite realizar in vitro, cribados de alto rendimiento de librerías de compuestos en una plataforma de electrofisiología automatizada. Cómo primera aproximación, se consideró que la búsqueda de activadores de la copia no mutada del Nav1.1 y bloqueadores de Nav1.6, mediante el reposicionamiento de fármacos podía ser el inicio para futuros ensayos de drug discovery para el Síndrome de Dravet. En primer lugar, se han caracterizado biofísica y farmacológicamente dos líneas celulares que expresan de forma estable los canales implicados. Los compuestos probados pertenecían a una librería de moléculas bioactivas (NIHCC) que, o son fármacos genéricos o han superado las fases I‐III de ensayos clínicos, disminuyendo así el tiempo potencial de llegada a los pacientes en el caso de la obtención de resultados positivos. El volumen de información obtenida obligó a centrarnos en los posibles activadores del Nav1.1, que no afectaran o bloquearan a Nav1.6. La dificultad de la búsqueda era esperable, ya que actualmente solo se conocen neurotoxinas que aumentan la actividad de los canales Nav. Se seleccionaron 4 posibles candidatos que parecían generar los efectos deseados, pero su validación en dosis‐respuesta no mostró diferencias significativas en la actividad de los canales. Nuestros estudios justifican e indican que 3 de estos candidatos necesitarían ser estudiados en profundidad mediante técnicas manuales y así profundizar en la información sobre los posibles bloqueadores específicos de Nav1.6 cómo dianas farmacológicas. En resumen, la técnica de patch‐clamp automático, que hemos puesto a punto, ofrece la posibilidad del cribado masivo de colecciones de compuestos para la búsqueda de un fármaco para el Síndrome de Dravet y por extensión de otras canalopatías.

Voltage dependent potassium channels are a group of plasma membrane ion channels with a key role in the immune system as the predominant ion channels controlling the resting membrane potential and tuning intracellular Ca2+ signaling in lymphocytes, monocytes, macrophages, and dendritic cells. Leukocytes present a limited Kv repertoire, including Kv1.3 and Kv1.5 channel isoforms. Kv1.3 is expressed in the immune system, and the blockade of this channel is associated with selective inhibition of T cell activation and proliferation. A functional Kv channel is an oligomeric complex composed of pore-forming and ancillary subunits. The KCNE gene family (KCNE1-5) is a novel group of modulatory Kv channel elements expressed in several tissues including leukocytes. KCNE peptides are small single spanning membrane proteins known to modulate Kv channels trafficking and biophysical properties. The hypothesis of the present PhD thesis entitled “Kv1.3 and Kv1.5 channels in leukocytes” was that changes in the channelosome composition by modulating the heterooligomeric combinations of the Kv1.3 channelosome control physiological and neoplastic cell growth as well as leukocyte responses. Evidence suggests that Kv channels are involved in cell differentiation and cell cycle control (because non-specific drugs, such as 4-AP and TEA, impaire proliferation), and they are also known to be remodeled during carcinogenesis. Thus, we elucidated the role of Kv1.3 and Kv1.5 channels in cell growth and their relationship with cancer, in models such as B lymphocytes and lymphomas (non-Hodgkin lymphomas), pancreatic ductal adenocarcinoma (PDAC) and glioblastomas. In spite of its significance, the mechanisms that regulate Kv1.3 and its role in the T cell activation are not well known. To that end, we analyzed the expression of KCNEs ancillary subunits upon different states of activation and proliferation of leukocytes (macrophages, T and B lymphocytes). In addition, recent data from our laboratory demonstrate that KCNE4, acting as a dominant negative ancillary subunit, physically interacts with Kv1.3 inhibiting K+ currents and retaining the channel intracellularly. Therefore, we studied the Kv1.3 modulation by the auxiliary subunit KCNE4 in leukocytes.

La cromatina és una estructura nucleoproteica, dinàmica i jeràrquicament organitzada dins del nucli cel•Iular. La seva estructura, organització i funció està controlada al llarg del cicle cel.lular, per processos epigenètics que produeixen canvis heretables que no afecten la seqüència de DNA. Entre aquests canvis es troben la metilació del DNA, les modificacions postraduccionals de les histones i desplegament de la cromatina orquestrat pels complexes remodeladors i proteïnes estructurals. Les modificacions postraduccionals de les histones es donen principalment de forma dinàmica i regulada a l'extrem N-terminal, canviant la seva càrrega i funció. Entre les diferents modificacions descrites, l'acetilació i la metilació de les histones H3 i H4, semblen tenir una importància especial sobre l'estructura i estat de la cromatina. En particular, l'acetilació de la lisina 16 de la histona H4 (H4K16Ac) per se impedeix la formació d'estructures compactes de cromatina. D'aquesta manera, s'associa l'estat desacetilat d'aquest residu a zones d'heterocromatina i transcripcionalment inactives, mentre que la hiperacetilació d'H4K16 està estretament vinculada a zones eucromàtiques i transcripcionalmentactives. La dinàmica d'acetilació-desacetiIació d' H 4K 16 està controlada principalment per l'equilibri de les activitats acetiltransferasa de MOF i desacetilasa de Si rT1 i Si rT2. Per tant, aquests dos grups d'enzims són essencials per el control de l'expressió gènica i de l'arquitectura de la cromatina dins del nucli, regulant la transició entre l'estat transcripcionalment actiu i inactiu de la cromatina. MOF és crucial per el desenvolupament embrionari , I a reparació del DNA i la progressió del cicle cel .lular, i la seva pèrdua està associada a l'augment de les aberracions cromosòmiques, a l'aturada del cicle cel.lular en G2/M i a l'augment de la inestabilitat genòmica. SirT1 i Si rT2 pertanyen a la classe III de desacetilases d'histones (HDACs), conegudes com sirtuïnes, i són crucials per el manteniment de la integritat genòmica, l'adaptació a l'entorn i l'envelliment, entre altres funcions. No obstant, dels set membres de sirtuïnes presents a mamífers, només Si rT2 i en menor mesura SirT1, han estat vinculades amb el control del cicle cel.lular. Concretament, Si rT2, tot i ser principalment citoplasmàtica, és transportada al nucli durant I a transició G2/M , moment en el que a través de la desacetilació d'H4K16 permet la monometilació d'H4K20 per PR-SET7 (la monometiI transferasa d'H4K20), determinant els nivells d'H4K2Ome2,3 en la resta del cicle cel.lular. Com a conseqüència, SirT2 està implicat en processos estrictament associat a aquestes modificacions, com la compactació dels cromosomes metafàsics, Ia progressió a través de mitosi, la replicació i reparació del DNA o la formació de l'heterocromatina. Fins al moment el paper de Si rT2 en el control de I a progressió a través de la mitosi ha estat atribuït a la regulació de diferents substrats. En particular, el control d'H4K2Ome1 dependent de SirT2 ha estat relacionat amb l'activació del checkpoint de G2/M en resposta a estrès, tot i que els mecanismes involucrats en l'aturada del cicle cel.lular eren del tot desconeguts. En aquest treball hem volgut indagar sobre com Si rT2 regula la transició G2/M i la seva coordinació amb la maquinària regulatória del checkpoint. Les nostres dades semblen indicar que SirT2 condueix a l'activació del checkpoint de G2/M a través de la regulació d' H4K 1 6Ac, H4K2Ome1 i el control de I a transcripció de gens relacionats amb el cicle cel.lular. Es descriu per primera vegada, com SirT2 controla H4K16Ac a través de la regulació de l'activitat i de l'estabilitat proteica de MOF durant G2/M. Concretament, SirT2 desacetila MOF durant G2/M i en promou la sortida de la cromatina, la inactivació i degradació, afavorint l'estat desacetilat d'H4K16 i la monometilació d'H4K20. En concordança, hem vist que MOF controla negativament la presència de PR-SET7 a la cromatina, mantenint així uns nivells adequats d'H4K2Omel abans d'entrar a mitosi i evitant la condensació prematura dels cromosomes. El nostre estudi suggereix que la interconnexió entre SirT2 i MOF està directament implicada en el control epigenètic del cicle cel.lular, contribuint al manteniment de l'estabilitat genòmica., Chromatin is a dynamical structure hierarchically organized to fit inside the nucleus. The structure, organization and function of chromatin are tightly controlled throughout the cell cycle by different epigenetic mechanisms, including DNA methylation and histone modifications. The histone post-translational modifications occur mainly in their N-terminal tail, and give rise to changes in the charge and function of the protein. Among the different histone modifications, lysine acetylation (K) is one of the best characterized. Acetylation of lysine 16 of histone H4 is the most frequently acetylated residue in eukaryotes and is a key regulator of high orders of chromatin structure. Thus, the deacetylated state of this residue is associated with heterochromatic and transcriptional inactive regions, whereas the acetylated form is found in euchromatic and transcriptional active regions. The dynamics of this histone mark is mainly governed by the acetyltransferase MOF and the NAD±-dependent deacetylases SirT1 and SirT2, which makes both groups of enzymes essential for the regulation of the gene expression and the control of chromatin organization. MOF is crucial in embryogenesis, DNA repair and the cell cycle progression. In fact, loss of MOF has been shown to induce cell cycle arrest during G2/M transition, increased chromosomal aberrations and genome instability. SirT 1 and SirT2 belong to Class III of histone deacetylases (HDACs), commonly referred as sirtuins. They play a key role in stress response, and in particular in protecting genome integrity. Among the seven mammalian sirtuins (SirT1-7), only SirT2 and to a lesser extend SirT1, have been linked with cell cycle regulation. In particular, SirT2, which mainly localizes to the cytoplasm during most of the cell cycle, shuttles to the nucleus in G2/M transition, where deacetylates H4K16Ac driving, among other things, H4K2Omel deposition by the histone methyltransferase PR-SETT. The control of H4K2Omel deposition determines the levels of H4K2Ome2,3 in the next cell cycle, which links SirT2 to the regulation of DNA replication and repair, as well as heterochromatin formation. Work from our group and others have shown that SirT2 plays a role in the control of mitosis progression. In particular, SirT2 is required for the cell cycle arrest in the G2/M checkpoint during stress response, process that has been related to SirT2-dependent regulation of H4K2Omel. However, the mechanisms behind the cell cycle arrest are still undefined. In the present work we aimed to elucidate the function of SirT2 in G2/M transition and its coordination with the checkpoint regulatory machinery. Our results seem indicate that SirT2 drives the G2/M checkpoint activation through the regulation of H4K16Ac, H4K2Omel and the control of the expression of cell cycle related genes. We also describe for the first time, a complementary mechanism whereby SirT2 regulates the levels of H4K16Ac during mitosis. We observe that SirT2 not only deacetylates MOF during G2/M, suppressing its acetyltransferase activity, but also induces both chromatin eviction and degradation of MOF. This in turn, results in H4K16 hypoacetylation and subsequent monomethylation of H4K20. Additionally, we show that MOF inhibits PR-SETT chromatin localization, maintaining the appropriate levels of H4K2Omel before entering mitosis and avoiding premature chromosome condensation. Our study suggests that the crosstalk between MOF and SirT2 is directly involved in the epigenetic control of the cell cycle, contributing to the maintenance of genome stability.

[cat] El canal de potassi dependent de voltatge Kv1.3 està involucrat en múltiples funcions cel·lulars. Així, el Kv1.3 participa en la fisiologia cel·lular de leucòcits, neurones sensorials, el to de la musculatura llisa vascular, resistència a insulina i obesitat. Part del Kv1.3, que es localitza majoritàriament a la membrana plasmàtica, es distribueix en microdominis lípid raft. Aquests lípid rafts actuen com a plataformes de senyalització on múltiples molècules de senyalització convergeixen per coordinar la resposta cel·lular. Un tipus especialitzat d’aquests microdominis són les caveoles. Estructures amb forma d’omega gràcies a la presència de la proteïna estructural caveolina. Són presents en diversos tipus cel·lulars i especialment abundants en adipòcits on poden suposar fins un 30% de la superfície cel·lular. En aquesta tesi hem estudiat la interacció entre el Kv1.3 i la caveolina en el sistema d’expressió heteròleg HEK 293. Així, ambdues proteïnes interaccionen a través d’un motiu CBD present al Nt del Kv1.3. La disrupció d’aquest domini aboleix la interacció entre ambdues proteïnes i, més enllà, altera el trànsit del canal a través de la ruta secretora. Així, el Kv1.3mutCBD tot i mantenir la capacitat de tetrameritzar queda retingut en estructures intracel3lulars tipus Golgi presumiblement degut a un defecte de N-glicosilació. La interacció entre el Kv1.3 i caveolina va poder ser confirmada en adipòcits. més encara, la depleció de caveolina en adipòcits 3T3-L1 deslocalitza el canal de les caveoles. Més enllà d’intervenir en la localització del canal, la caveolina també canvia l’activitat electrofisiològica del canal. Així, en absència de caveolina el canal presenta una menor densitat de corrent i la seva presència canvia lleugerament les propietats biofísiques del canal, fent més ràpides l’activació i inactivació., [eng] The voltage-dependent K+ channel Kv1.3 participates in many physiological events like proliferation and activation of immunitary cells, membrane repolarization in sensory neurons, vascular smooth muscle tone, insulin resistance and obesity. In most cases, Kv1.3 concentrates in specific membrane microdomains called lipid rafts. These domains act as platforms where signaling pathways converge. Caveolae are a specialized form of lipid rafts with an omega shaped structure build up by the structural protein caveolin. These omega shaped structures are present in multiple cell lines and highly abundant in adipocytes where they account for up to 30 % of the plasma membrane surface, where they participate in lipid transport, lipotoxicity protection, mechanic stress protection and cell signaling. Evidence indicates that Kv1.3 functionality relays not only on expression but also on localization at the plasma membrane. The importance of this spatial regulation is manifest when an altered Kv1.3 distribution is related with the appearance of disease. Therefore, the mechanisms that govern Kv1.3 function are of considerable interest. In this study we characterized the relationship between Kv1.3 and caveolins. Thus Kv1.3 interacted mainly with caveolin 1. In addition, we analyzed the presence of putative caveolin-binding domain in Kv1.3 that could explain their interaction. To that end, we performed structure-function studies by using a repertoire of Kv1.3 mutants and stable HEK-293 cell lines with or without caveolins, characterized putative interactions and analyzed membrane dynamics by single molecule tracking. Thus, a CBD appeared in the Nt of the Kv1.3 (close to the T1 domain). This motive was not only involved in Kv1.3-caveolin interaction but also in Kv1.3 forward trafficking. Thus the Kv1.3mutCBD remained trapped in intracellular Golgi-like structures. Even so, the channel remained functional despite some biophysical properties alterations. Moreover, the channel suffered palmitoylation like Kv1.5 but, unlike Kv1.5 it was not the mail lipid raft targeting signal as it was caveolin. 2 palmitoilation sites would be involved in Kv1.3 palmitoylation. C51/52, C267 and C515. The role of Kv1.3 in adipocytes raises an important debate as it has been proposed that Kv1.3 could be a pharmacological target in obesity. What is more, Kv1.3 (-/-) mice presented a lean phenotype that could suggest the importance of the Kv1.3 in preadipocytes proliferation and differentiation. We have characterized the presence of Kv1.3 in rat, mice and human adipocytes and before and after adipogenesis induction of 3T3-L1 cell line. the adipogenesis induced caveolin 1 expression and a parallel Kv1.3 increase. Since adipogenesis induced both Kv1.3 and caveolin 1 expression and so the appearance of caveolae, we further analyzed the specific localization of Kv1.3. Our results indicate that adipogenesis triggers a relocalization of Kv1.3 in newly synthesized caveolae. Because most of insulin-dependent adipocyte signaling is located in these structures the presence of the channel in caveolae seemed to be important for adipocytes physiology. What is more, the caveolin expression disruption relocated Kv1.3 out of lipid raft caveolae structures. As Kv1.3 is considered an important pharmacological target for different autoimmunitary diseases and obesity, it is important to bring light to the Kv1.3 interactions with other partners that can regulate channel surface distribution and function.

Voltage-gated K+ channels, Kv channels, are the most diverse group of K+ channels. They have several key functions in cardiovascular and nervous system; setting the resting membrane potential and controlling the repolarization of the action potential. In non-excitable cells they also play crucial role in water and ion transport across the epithelia or cell volume regulation. Such extraordinary heterogeneity in functions is closely related with a remarkable genetic and structural heterogeneity of Kv channels. Among Kv channels, the Kv7 family comprises 5 members, Kv7.1-Kv7.5. Like other Kvs, Kv7 channels are tetramers of 4 α subunits. The topological arrangement of every α subunit consists in 6 transmembrane segments: S1-S4 form the voltage sensing domain and the S5-S6 region the ion-selective pore. Kv7 channels use to assemble as homotetramers, but most of the members of the family are also able to form heterotetrameric channels. Heterotetramerization is one of most important mechanisms to achieve the vast functional diversity that they display. Moreover, Kv7 channel function is also influenced by regulatory subunits, the KCNE family. They regulate various gating parameters, current density, channel assembly, ER retention, sorting into different traffic pathways, subcellular localization, regulation of the post-translational modifications or sensibility to pharmacological agents. In the cardiovascular system, both Kv7.1 and Kv7.5 play a pivotal role. In cardiac myocytes, Kv7.1 homotetramers combine with KCNE1 β subunit to form functional channels. They are the molecular correlate of the IKs current, a slow delayed-rectifier K+ current involved in the repolarization of the cardiac action potential and an important determinant of the duration of the QT interval of the electrocardiogram. Mutation in either, Kv7.1 and KCNE1 result in long QT or short QT syndromes, which can lead to fatal severe arrhythmias. Kv7 have been also recognized to be key regulators of the vascular physiology, setting the resting membrane potential in vascular smooth muscle myocytes and therefore controlling the vascular smooth muscle tone. In particular, Kv7.1, Kv7.4 and Kv7.5 had been detected in numbers of veins and arteries, but the specific contribution of every subunit to physiological relevant K+ currents is still under debate. Within this scenario, the proposed objectives for the currently discussed PhD dissertation were focused on further study the heteroligomerization process of the Kv7 channel family, either by heterotetramerization or by interaction with β regulatory subunits. In the one hand corroborate the functional assembly of Kv7.1-Kv7.5 heterotetramers in vascular smooth muscle. On the other hand, we were also committed to explore the molecular pathological mechanisms of Kv7.1 mutations linked to cardiac channelopathies as well as dissect the traffic and assembly process of the Kv7.1-KCNE1 complex. We had been able to prove that Kv7.1-Kv7.5 form heterotetrameric channels in vascular smooth muscle cells. Moreover, upon heterotetramer assembly the special distribution of those channels within plasma membrane microdomains is rearrange and Kv7.1 shifted out of lipid raft structures. In addition, we had studied 2 different mutations in Kv7.1, F279I and D242N, responsible for short QT and long QT syndromes, respectively. Those mutations uncover 2 Kv7.1 residues important for KCNE1 interaction: F279 physically interacts with KCNE1 and D242 is involved in the KCNE1-dependent modulation of the voltage-dependent gating mechanism of the Kv7.1 channel. Furthermore, our results revealed that Kv7.1 and KCNE1 are not assembled in the very first stages of the biosynthetic pathway. On its own, KCNE1 traffics through the classical secretion pathway to the cell surface, in contrast, Kv7.1 takes an unconventional pathway. Upon co-assembly, Kv7.1 redirects KCNE1 to the unconventional pathway that lead to the concentration of both subunits in the endoplasmatic reticulum-plasma membrane junctions, where complexes are assembled., Els canals de potassi dependents de voltatge Kv7.1 i Kv7.5 juguen un paper molt important en la fisiologia del sistema cardiovascular. Aquest canals iònics estan formats per la tetramerització de 4 subunitats α. La heterotetramerizació és un dels mecanisme principals que dona lloc a la immensa diversitat funcional que mostra aquesta família. A més a més, la seva funció també es veu influenciada per la interacció amb subunitats β reguladores de la família KCNE. Tot i ser petits pèptids, els KCNEs poden modular un gran ventall de característiques de la biologia i electrofisiologia de les corrents generades per la família Kv7. En els cardiomiòcits, Kv7.1 interacciona amb KCNE1 per donar lloc el corrent IKs, una important corrent de K+ determinant per la repolarització del potencial d’acció cardíac i per la durada del interval QT de l’electrocardiograma. Mutacions tant en Kv7.1 com en KCNE1 donen lloc a les síndromes de QT llarg i QT curt, totes dues relacionades amb perilloses arítmies i mort sobtada. Kv7.1 juntament amb Kv7.4 i Kv7.5 també participen en el control del to de la musculatura llisa vascular, tot i que el paper específic de cada subunitat en les corrents detectades en aquest teixit encara genera una gran controvèrsia. En la present tesi doctoral demostrem l’existència d’heterotetramers Kv7.1-Kv7.5 en els miòcits de la musculatura llisa vascular. Aquesta nova interacció provoca el desplaçament de Kv7.1 fora dels microdominis de membrana tipus lipid raft. També hem estudiat els mecanismes patològics moleculars de dos mutacions en Kv7.1, F279I i D242N, responsables de les síndromes del QT curt i el QT llarg, respectivament. Aquestes dues mutacions han posat de manifest 2 residus en Kv7.1 importants per la interacció amb KCNE1: F279 participa en la interacció física amb KCNE1 i D242 en la regulació per KCNE1 del mecanisme, depenent de voltatge, d’obertura del canal. Per últim, hem comprovat que Kv7.1 i KCNE1 trafiquen per diferents vies secretores i que la formació dels complexes Kv7.1-KCNE1 té lloc a les unions reticle endoplasmatic-membrana plasmàtica.

[cat] Els canals de potassi dependents de voltatge (Kv) juguen un paper molt important tant en cèl•lules excitables com no excitables. La possibilitat de formar hetero-oligomers i la d’associació amb subunitats reguladores són uns dels mecanismes que existeixen per tal de proveir de diferents mecanismes per a respondre de manera diferent enfront a canvis en el potencial de membrana. La composició del canalosoma modula tant la seva expressió a superfície com l’activitat d’aquests. Aquesta tesi es centra en l’estudi de l’efecte del la família de les subunitats reguladores KCNE sobre diferents Kv. Primerament s’estudià l’efecte que causaven en el tràfic del canal Kv7.1 (canal model per a l’estudi dels KCNEs) i posteriorment s’amplià l’estudi a un altre membre de la mateixa família, Kv7.5. A continuació s’estudià un canal d’elevada importància per a l’activació i proliferació leucocitària: Kv1.3, centrant-nos sobretot en l’efecte causat per un dels KCNEs: KCNE4. Aquesta subunitat no només inhibeix dràsticament el corrent del canal Kv1.3, sinó que a més a més, modifica el seu tràfic i localització. Aquests canvis són deguts a una interacció directa entre ambdues proteïnes. A continuació s’estudià en detall el complex Kv1.3-KCNE4. Mitjançant la combinació d’experiments d’electrofisiologia i monitorització de fluorescència de molècules individuals en la membrana, es va poder establir l’estequiometria d’aquest complex. Posteriorment, mitjançant l’anàlisi de diverses proteïnes quimèriques i mutants, tant del canal com de la subunitat reguladora, es van cercar els determinants moleculars implicats en l’associació entre ambdues proteïnes. S’han pogut determinar els motius claus en KCNE4 i Kv1.3 implicats en la formació del complex, però no en la modulació del canal. Finalment, degut a la importància de Kv1.3 en el sistema immunitari, s’han analitzat els nivells d’expressió dels KCNEs en diferents línies leucocitàries. S’ha observat que aquestes subunitats pateixen una regulació diferencial en funció de la manera d’activació i al llarg de la proliferació del leucòcits, suggerint un possible paper en la regulació precisa de la resposta immunològica., [eng] Voltage gated potassium channels (Kv) play important roles in different biological process such as generation and propagation of the nerve pulse and the cardiac action potential, promotion of insulin secretion, cell volume control, induction of cell proliferation, apoptosis, migration and initiation of many signaling pathways. Kv channels can homo- or hetero- tetramerize. The composition of the channel modulates their surface expression and serves as a mechanism for regulating channel activity. Kv channel interaction with accessory subunits provides mechanisms for channels to respond to stimuli beyond changes in membrane potential. The present dissertation is focused in the analysis of the effect of one regulatory subunits family (KCNEs) on different Kv channels. The first channel analyzed is Kv7.1, which is one of the most well-known channels to interact with all the KCNE family members. In fact KCNE1-Kv7.1 complex is focus of a huge number of studies, due to its important role in heart. Most of the studies though are focused to their electrophysiological properties and molecular determinants involved in the interaction. We performed traffic analysis experiments of Kv7.1 in the present of KCNE1-5 and demonstrated that Kv7.1 membrane surface localization is modified by some of them. Next, analysis was expanded to another channel from the same family, less characterized: Kv7.5. We demonstrated that from the five KCNEs members, only KCNE1 and KCNE3 modulate Kv7.5 activity. Furthermore, we demonstrated that Kv7.5 association to KCNE3 modifies the targeting of the regulatory subunit. Next, we moved to a non-related Kv channel such as Kv1.3, which plays a crucial role in the immune system. We first focused into characterizing the modulation of Kv1.3. We demonstrated that KCNE4, but not KCNE2, functions as an inhibitory Kv1.3 partner. Kv1.3 trafficking, targeting and activity are altered by the presence of KCNE4. Furthermore, by the combination of a plethora of approaches such as electrophysiological experiments from chimeric proteins and GFP single bleaching counting steps methodology we deciphered the stoichiometry of the Kv1.3-KCNE4 complex. Next, by immunoprecipitation experiments, traffic analysis and electrophysiological experiments, we analyzed the molecular determinants involved in the association between Kv1.3 and KCNE4. We have map a domain of Kv1.3 and a specific motif of KCNE4 involved in the formation of Kv1.3-KCNE4 complex, but not in the modulation of the channel. We also proposed a 3D docking model of Kv1.3 and KCNE4. Finally, due to the importance of Kv1.3 in the immune system, the expression of all the KCNE family has been analyzed in several cell lines of leukocytes. We have demonstrated that all KCNEs suffer a differential regulation among proliferation of leukocytes. Furthermore, a different regulation can be observed, depend on the mode of leukocytes’ activation. Our results further suggest a new and yet unidentified physiological role for KCNE subunits in the immune system. Putative associations of these ancillary proteins with Kv channels would yield a wide variety of biophysically and pharmacologically distinct channels that fine-tune the immunological response.

[spa] En esta tesis doctoral se presentan los estudios realizados sobre Kv1.3, uno de los canales de potasio existentes en macrófagos y su regulación ante distintos estímulos de proliferación y activación celular. En concreto, en la primera contribución que se adjunta se describe cómo los canales de potasio Kv1.3, Kv1.5 y Kir2.1 son los responsables de las corrientes de salida dependiente de voltaje y de rectificación de entrada que se detectan en estas células. En este trabajo se muestra cómo Kv1.3 y Kir2.1 están altamente regulados tanto por el factor de crecimiento MCSF, como por factores inductores de la activación de los macrófagos como son el lipopolisacárido y la citoquina TNF-alfa, existiendo cambios electrofisiológicos en la corriente Kv. Estos estudios muestran cómo, no sólo Kv1.3 se ve regulado en los procesos de proliferación y activación, sino que se requiere su participación en estos procesos. Como complemento a esta regulación se adjunta un trabajo donde se muestra que esta modulación es un mecanismo general que puede ser importante en diferentes patologías sistémicas en las que estos mediadores estén presentes como caquexia, sepsis o inflamación crónica. Por otro lado, un segundo apartado en esta tesis doctoral se centra en los cambios electrofisiológicos que presentan en la Kv en proliferación y activación. Se analiza la posibilidad de que sean debidos a cambios en la composición del complejo funcional. En este sentido se presenta una contribución donde se describe la presencia en macrófagos de todas las subunidades Kv-beta estudiadas menos la Kv-beta4. La proliferación inducida por MCSF incrementaría la expresión de todas las subunidades auxiliares mientras que distintos estímulos de activación, LPS y TNF-alfa, regulan la expresión génica de estas proteínas de distinta forma. Los parámetros cinéticos de la corriente de salida de potasio sobre los que estas subunidades actúan, como son las constantes de activación, inactivación y deactivación, han sido analizados en un intento de relacionar cambios moleculares con cambios electrofisiológicos en macrófagos proliferantes y activados. Otra aproximación en el análisis de los cambios electrofisiológicos presentes en macrófagos activados se presenta como una publicación en preparación en la que se describe cómo Kv1.5 es capaz de asociarse con Kv1.3 para formar un complejo funcional generador de corrientes de salida de potasio. En este trabajo se muestran estudios de asociación por microscopía confocal y electrónica junto con estudios de farmacología que confirman la formación del complejo heteromérico. Los cambios en los parámetros electrofisiológicos de las corrientes, dependiendo de la composición del complejo se han analizado en distintos modelos de expresión heteróloga. Por último se sugiere que, los cambios en el gating que se producen en esta corriente ante distintos estímulos, como el TNF-alfa, podrían deberse a cambios en la estequiometría de las subunidades que conforman el complejo generador de la corriente de salida de potasio en macrófagos. La localización subcelular y el entorno que rodea a las proteínas de membrana son determinantes en la función de éstas. En el tercer apartado de esta memoria se presentan los estudios realizados sobre la biología celular del complejo formado por Kv1.3, tanto su tráfico a membrana como su localización. Se estudia la influencia de las distintas subunidades presentes en el complejo sobre la biología celular de éste, teniendo en cuenta la influencia de la subunidad Kv1.5 que ya se había descrito en macrófagos, y se acompaña por último de los primeros estudios de internalización del canal a través de vesículas recubiertas de clatrina., [eng] Voltage-dependent potassium channels (Kv) play a crucial role in non excitable cells by determining membrane potential. In addition, they are also involved in the activation and proliferation of leukocytes. Bone marrow-derived macrophages are cells fully differentiated, and proliferate and activate separately. The present study identifies the voltage-gated potassium channels expressed in macrophages. We found Kv1.3 and Kir2.1 potassium channels are differentially regulated under proliferation and activation. Both isoforms are required for these processes. In addition, our results indicate that posttranslational events are involved in the differential Kv regulation in response to different stimuli. In this vein, we describe the expression of modulatory Kvβ subunits. This work shows that differential expression of these regulatory subunits modulate activation, inactivation and deactivation parameters of the Kv current. We have also explored whether Kv1.5 play a role in the major voltage-dependent K+ current generating functional heterotetrameric Kv1.3/Kv1.5 complexes in macrophages. Different approaches demonstrate that Kv1.5 co-associates to Kv1.3 generating functional heterotetrameric channels in these cells. Upon different physiological stimuli, changes in the oligomeric composition of functional Kv could be crucial in intracellular signals determining the specific macrophage response. We also report that Kv1.3 traffic, targeting to raft domains and activity are dramatically changed by the presence of Kv1.5. Finally this work includes the first evidences for Kv1.3 internalization by clathrin coated pits.

En esta tesis doctoral se presentan los estudios realizados sobre Kv1.3, uno de los canales de potasio existentes en macrófagos y su regulación ante distintos estímulos de proliferación y activación celular. En concreto, en la primera contribución que se adjunta se describe cómo los canales de potasio Kv1.3, Kv1.5 y Kir2.1 son los responsables de las corrientes de salida dependiente de voltaje y de rectificación de entrada que se detectan en estas células. En este trabajo se muestra cómo Kv1.3 y Kir2.1 están altamente regulados tanto por el factor de crecimiento MCSF, como por factores inductores de la activación de los macrófagos como son el lipopolisacárido y la citoquina TNF-alfa, existiendo cambios electrofisiológicos en la corriente Kv. Estos estudios muestran cómo, no sólo Kv1.3 se ve regulado en los procesos de proliferación y activación, sino que se requiere su participación en estos procesos. Como complemento a esta regulación se adjunta un trabajo donde se muestra que esta modulación es un mecanismo general que puede ser importante en diferentes patologías sistémicas en las que estos mediadores estén presentes como caquexia, sepsis o inflamación crónica.Por otro lado, un segundo apartado en esta tesis doctoral se centra en los cambios electrofisiológicos que presentan en la Kv en proliferación y activación. Se analiza la posibilidad de que sean debidos a cambios en la composición del complejo funcional.En este sentido se presenta una contribución donde se describe la presencia en macrófagos de todas las subunidades Kv-beta estudiadas menos la Kv-beta4. La proliferación inducida por MCSF incrementaría la expresión de todas las subunidades auxiliares mientras que distintos estímulos de activación, LPS y TNF-alfa, regulan la expresión génica de estas proteínas de distinta forma. Los parámetros cinéticos de la corriente de salida de potasio sobre los que estas subunidades actúan, como son las constantes de activación, inactivación y deactivación, han sido analizados en un intento de relacionar cambios moleculares con cambios electrofisiológicos en macrófagos proliferantes y activados.Otra aproximación en el análisis de los cambios electrofisiológicos presentes en macrófagos activados se presenta como una publicación en preparación en la que se describe cómo Kv1.5 es capaz de asociarse con Kv1.3 para formar un complejo funcional generador de corrientes de salida de potasio. En este trabajo se muestran estudios de asociación por microscopía confocal y electrónica junto con estudios de farmacología que confirman la formación del complejo heteromérico. Los cambios en los parámetros electrofisiológicos de las corrientes, dependiendo de la composición del complejo se han analizado en distintos modelos de expresión heteróloga. Por último se sugiere que, los cambios en el gating que se producen en esta corriente ante distintos estímulos, como el TNF-alfa, podrían deberse a cambios en la estequiometría de las subunidades que conforman el complejo generador de la corriente de salida de potasio en macrófagos.La localización subcelular y el entorno que rodea a las proteínas de membrana son determinantes en la función de éstas. En el tercer apartado de esta memoria se presentan los estudios realizados sobre la biología celular del complejo formado por Kv1.3, tanto su tráfico a membrana como su localización. Se estudia la influencia de las distintas subunidades presentes en el complejo sobre la biología celular de éste, teniendo en cuenta la influencia de la subunidad Kv1.5 que ya se había descrito en macrófagos, y se acompaña por último de los primeros estudios de internalización del canal a través de vesículas recubiertas de clatrina., Voltage-dependent potassium channels (Kv) play a crucial role in non excitable cells by determining membrane potential. In addition, they are also involved in the activation and proliferation of leukocytes. Bone marrow-derived macrophages are cells fully differentiated, and proliferate and activate separately. The present study identifies the voltage-gated potassium channels expressed in macrophages. We found Kv1.3 and Kir2.1 potassium channels are differentially regulated under proliferation and activation. Both isoforms are required for these processes. In addition, our results indicate that posttranslational events are involved in the differential Kv regulation in response to different stimuli. In this vein, we describe the expression of modulatory Kvβ subunits. This work shows that differential expression of these regulatory subunits modulate activation, inactivation and deactivation parameters of the Kv current. We have also explored whether Kv1.5 play a role in the major voltage-dependent K+ current generating functional heterotetrameric Kv1.3/Kv1.5 complexes in macrophages. Different approaches demonstrate that Kv1.5 co-associates to Kv1.3 generating functional heterotetrameric channels in these cells. Upon different physiological stimuli, changes in the oligomeric composition of functional Kv could be crucial in intracellular signals determining the specific macrophage response. We also report that Kv1.3 traffic, targeting to raft domains and activity are dramatically changed by the presence of Kv1.5. Finally this work includes the first evidences for Kv1.3 internalization by clathrin coated pits.