Resumen de Role of kcne4 on the voltage gated potassium channel kv1.3
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.
