Heteromeric composition of the Kv 1.3 channelosome = Composició heteromèrica del canalosoma Kv1.3

Abstract

Ion channels are transmembrane proteins containing aqueous pores which, once open, permit the pass of ions through the plasma membrane. This ion flux takes places following the electrochemical gradient for the specific ion. Kv1.3 is a voltage-gated potassium channel which is member of the Shaker superfamily. Its basic structure consists in a protein with six transmembrane domains, while the functional channel is formed by 4 copies of this protein. Kv1.3 participates in a great amount of physiological functions: nervous system, immune system, insulin signaling or cell proliferation. In the immune system, Kv1.3 is highly expressed both in lymphocytes as well as in mononuclear phagocytes. In both cell types, Kv1.3 regulates the immune activation and cell proliferation. Moreover, Kv1.3 is coexpressed with other ion channel proteins like Kv1.5 or KCNE4 in the immune cells. Kv1.5 is able to heteromerize with Kv1.3, generating heterotetramers with variable stoichiometries. Those heterotetramers produce intermediate phenotypes depending on the ratio of the subunits that generate them. On the other side, KCNE4 may interact with Kv1.3, but not with Kv1.5. Kv1.3 is greatly inhibited by the association with either of the two proteins. In the present thesis we focus in characterizing these interactions and the importance of stoichiometry in their effects. We demonstrate that the associations between Kv1.3 and Kv1.5; and between Kv1.3 and KCNE4 take place in immune cells. Moreover, by using a fusion protein we get to fix the stoichiometry of the Kv1.3-Kv1.5 complex to 1:1. With this stoichiometry, Kv1.5 acts as a dominant negative toward Kv1.3 in the complex. Further interactions are characterized by using several chimeric proteins. By using those chimaeras, it is revealed that the carboxyterminal domain is necessary for the correct function of the channel. On the other hand, we demonstrate that KCNE4 is able to interact with Kv1.3 regardless of Kv1.5 presence. Furthermore, the presence of Kv1.5 in the Kv1.3-KCNE4 interaction results in this association potentiating the function of the channel, instead of inhibiting it. These results are replicated both in heterologous systems as well as in native cells. This discovery presents a new paradigm by which the association with several modulatory proteins may result in the modification of the effect of each one of them. Taking into account the sheer number of different ion channel subunits, the number of different potential phenotypes is increased by a huge margin. KCNE1 is a regulatory subunit, as well as KCNE4. Unlike KCNE4, though, KCNE1 can interact with Kv1.5. In the present thesis we demonstrate for the first time that KCNE1 is not only able to associate with Kv1.5, but to potentiate its activity by a huge amount. This interaction also seems to affect the membrane microdomain targeting of Kv1.5 Finally, the 4 studied proteins are expressed in T lymphocytes, which are the main actors in the pathogenicity of autoimmune diseases. Therefore, we genotyped those genes in multiple sclerosis patients to identify different polymorphisms which could be linked to immune overactivity. After analyzing the different polymorphisms, we located some which could be of special relevance for the physiopathology of autoimmune diseases.

Los canales iónicos son proteínas transmembrana que contienen poros acuosos que permiten el paso de iones a través de la membrana plasmática a favor de gradiente electroquímico. Kv1.3 es un canal de potasio dependiente de voltaje de la superfamilia Shaker. La estructura básica consiste en una proteína con seis dominios transmembrana y el canal funcional está formado por cuatro copias de esta proteína. Kv1.3 participa en multitud de funciones del organismo: sistema nervioso, sistema inmunitario, señalización de la insulina o proliferación celular. En el sistema inmunitario está altamente expresado tanto en linfocitos como en fagocitos mononucleares. En ambos tipos celulares regula la activación inmunitaria y la proliferación celular. Además, se ve coexpresado con otras proteínas de relevancia como Kv1.5 o KCNE4. Kv1.5 puede heteromerizar con Kv1.3, dando lugar a heterotrámeros de estequiometrias variables. Por otro lado, KCNE4 puede interaccionar con Kv1.3, pero no con Kv1.5. Kv1.3 se ve potentemente inhibido por ambas asociaciones. En la presente tesis nos centramos en caracterizar estas interacciones y el peso de la estequiometría en sus efectos. Demostramos que ambas asociaciones tienen lugar en células del sistema inmunitario. Además, mediante una proteína de fusión logramos fijar la estequiometría del complejo Kv1.3-Kv1-5 en 1:1. Así, Kv1.5 demuestra ejercer como dominante negativo respecto a Kv1.3 en el complejo. Estas interacciones intramoleculares son estudiadas mediante el uso de diversas proteínas quiméricas para dilucidar el peso de los extremos carboxiterminales en la formación del canal y su función. Por otro lado, demostramos que KCNE4 afecta el canal de estequiometría 1:1 aumentado su actividad, en lugar de reducirla. Este descubrimiento presenta un nuevo paradigma en que la asociación con varias proteínas reguladoras puede resultar en la modificación del efecto de cada una de ellas. KCNE1 es una proteína reguladora al igual que KCNE4, pero que interactúa con Kv1.5. En la presente tesis demostramos como KCNE1 no solo interacciona con Kv1.5, sino que aumenta en gran medida su actividad. Finalmente, también genotipamos estos genes en pacientes de una enfermedad autoinmune como es la esclerosis múltiple, llegando a localizar diversos polimorfismos de posible interés fisiopatológico.