Protein kinase-dependent Kv1.3 biology

Abstract

[eng] The voltage gated potassium channel Kv1.3 is a transmembrane protein that selectively drives potassium ions participating in the electrochemical gradient of cell membranes. This channel presents a wide distribution within the body, thereby playing an important role in several physiological processes, such as regulating the cell volume, proliferation and apoptosis, and leukocyte activation. Of particular interest in this dissertation is the role of Kv1.3 in the immune system, where its activity is crucial to initiate the immune response. Moreover, an increased and/or delocalized expression of the channel is observed at the onset of autoimmune diseases pointing Kv1.3 as a potential therapeutic target. In this contest, the study of the mechanisms involved in the modulation of the amount of Kv1.3 at the plasma membrane deserves considerable attention.

Kv1.3 activity mostly relies in its abundance and proper plasma membrane location, which is tightly regulated by a balance between the forward trafficking and the endocytic machinery. Thus, the control of channel internalization and degradation influences the inflammatory response. The endocytosis of Kv1.3 has been extensively studied in our laboratory and we claim that ubiquitination mediates the internalization and further lysosomal degradation of Kv1.3 via both PKC and EGFR activation. However, the specific residues, among all Kv1.3 intracellular lysines, which play a major role in channel turnover are still unknown. Moreover, although the ubiquitin ligase Nedd4-2 has been proposed to downregulate Kv1.3 activity, there is no clue about how the interaction takes place. In this context, adenosine (ADO), an endogenous key mediator in the immune response, triggers Kv1.3 endocytosis via PKC activation. However, via its A1 and A2A receptors, ADO not only activates PKC but also PKA. The ADO-dependent Kv1.3 modulation via such differential mechanisms had not been explored yet in immune cells. For that reason, in the present work we aimed to shed light to the mechanism involved in Kv1.3 turnover and thus, provide new knowledge of the molecular physiology of the immune system.

We have deciphered the molecular determinants involved in Ser/Thr kinase (PKC)- and Tyrosine kinase (EGF)-mediated Kv1.3 turnover. Activation of either pathway internalized the channel by the specific ubiquitination of the lysines K70, 84, 476, 498 and 519 which form two clusters at the amino and carboxy terminal domain of the channel. Moreover, our results suggested that these two clusters are also involved in the association between Kv1.3 and Nedd4-2. We confirmed that the Kv1.3-Nedd4-2 interaction is not direct but, a physical proximity (< 40 nm) between the proteins suggested the participation of adaptor proteins. We have also characterized the PKA-mediated Kv1.3 downregulation which, unlike PKC, did not triggered the endocytosis of the channel targeting Kv1.3 to proteasomal degradation. Moreover, the activation of PKC and PKA pathways using specific ADO receptors agonists efficiently modulate Kv1.3-mediated leukocyte physiology. Thus, ADO exerts an efficient anti-inflammatory response by the activation of two complementary and synergic signalling pathways.

In conclusion, this thesis further expands the knowledge of the molecular mechanisms involved in Kv1.3 turnover. We provide an insight from the molecular determinants to the functional consequences of Kv1.3 downregulation controlling the immune response and the leukocyte physiology. Our results are of considerably physiological interest due to the combination of the multitherapeutic potential of Kv1.3 and ADO.