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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.

[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.

[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.

[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.