Deciphering the interaction between Kvl.3 and Caveolin

Abstract

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.

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.