The Kv1.3 channel: Functional consequences of cellular and spatial reorganization

Abstract

[eng] The voltage-gated potassium channel Kv1.3 is a transmembrane protein that facilitates the selective flow of K+ ions across cell membranes, playing a crucial role in regulating cellular excitability and maintaining the electrochemical gradient. This channel is ubiquitously expressed within the human body and participates in several physiological processes, including proliferation, apoptosis, and leukocyte activation. The functional role of Kv1.3 is influenced by its dual localisation at the cell surface and the inner mitochondrial membrane. Hence, while Kv1.3 at the plasma membrane promotes cell proliferation, its mitochondrial counterpart is associated with the regulation of apoptosis. In this context, the present thesis aims to further explore this channel dichotomy, from the early biogenesis to the functional implications. The interaction between caveolin and Kv1.3 via a caveolin binding domain (CBD) is crucial for channel localisation at the plasma membrane. Abrogation of the CBD redirects Kv1.3 to mitochondria, where its accumulation affects organelle physiology and compromises cell survival. Thus, our findings reveal an unexpected role of the mitochondrial caveolin-Kv1.3 axis in regulating cell survival and apoptosis, with significant implications for chemotherapy resistance. Moreover, we decipher a new role for mitochondrial Kv1.3 in regulating cell proliferation by modulating the balance between mitochondrial fusion and fission. This function, extending beyond apoptosis, adds further complexity to the role of the mitochondrial channel. Finally, we describe for the first time the mitochondrial route of Kv1.3. Interestingly, the channel uses a pre-sequence independent pathway where transmembrane domains function as redundant motifs mediating the import process. The interaction with cytosolic chaperones is essential for the subsequent translocation through unconventional TIM/TOM complexes. Kv1.3 plays a crucial role in T cell activation, and channel delocalisation or increased activity is linked to the onset of diverse autoimmune diseases. For this reason, we studied the plasma membrane functions of Kv1.3 in the context of the immunological synapse (IS). In a pioneering effort to increase the resolution of Kv1.3 distribution at the IS, we reveal, for the first time, its organisation into a peripheral ring. As the contact matures, Kv1.3 migrates to the centre of the synapse, where it becomes more static to facilitate endocytosis for degradation, recycling, or exocytosis into extracellular vesicles. The internalisation process is regulated by CaMKII- dependent palmitoylation of Kv1.3, which facilitates channel ubiquitination and subsequent endocytosis. CaMKII also mediates the impact of Ca2+ fluctuations on Kv1.3 regulation, influencing the channel localisation in lipid rafts and its role T cell activation, among others. Thus, our work deepens the knowledge of Kv1.3 biology at the IS, contributing to the refinement of therapeutic strategies for autoimmune diseases characterised by channel dysregulations. Finally, we investigate alternative roles of Kv1.3 cysteine residues during channel biogenesis and sorting. Absence of palmitoylation in intracellular cysteines reduces Kv1.3 interaction with caveolin, redirecting the channel to mitochondria where it promotes apoptosis. Moreover, we identify two transmembrane cysteines that participate in the oxidative folding of Kv1.3 mediated by protein disulphide isomerase. Overall, the present thesis offers a broader view of Kv1.3 functions regarding the spatiotemporal context. We introduce novel roles and regulatory mechanisms that could contribute to the development of new therapies for cancer, obesity, and autoimmune diseases.