Regulatory subunits controlling the Kv1.3 channelosome

Abstract

[eng] The Kv1.3 voltage-dependent potassium channel plays a crucial role in the immune response participating in various cellular functions like proliferation, activation, and apoptosis. The aberrant expression of this channel is associated with autoimmune diseases, highlighting the need for precise regulation in leukocyte physiology. Kvβ proteins, the first identified modulators of Kv channels, have been extensively studied in their regulation of α-subunit kinetics and traffic. However, limited information is available regarding their own biology. Despite their cytosolic distribution, Kvβ subunits show spatial localization near plasma membrane-Kv channels for an effective immune response. Our study focused on the structural elements influencing Kvβ distribution. We discovered that Kvβ peptides could target the cell surface independently of Kv channels. Additionally, Kvβ2.1, but not Kvβ1.1, targeted lipid raft microdomains via S-acylation of two C-terminal cysteines (C301/C311), concomitantly with the peptide localization at the immunological synapse. Moreover, growth factor-dependent proliferation increased the Kvβ2.1 surface targeting, whereas PKC activation disrupted lipid raft localization, but PSD95 counteracted this action. These findings elucidate the mechanisms by which Kvβ2 clusters within immunological synapses during leukocyte activation. Kvβ peptides, interacting with Kv channels, exhibited a suggested α4/β4 conformation. While Kvβ2 and Kvβ1 can form homo- and heterotetramers with similar affinities, only Kvβ2.1 forms tetramers independently of α subunits. Thus, Kvβ oligomers stoichiometry fine-tunes hetero-oligomeric Kv channel complexes. Similar to Kvβ1.1, Kvβ1.1/Kvβ2.1 heteromers did not target lipid rafts. Therefore, because Kvβ2 is an active partner of the Kv1.3-TCR complex at the immunological synapse, an association with Kvβ1 would alter its location, impacting on immune responses. Differential regulation of Kvβs influences the traffic and architecture of Kvβ heterotetramers, modulating Kvβ-dependent physiological responses. Regulatory KCNE subunits are expressed in the immune system and KCNE4 tightly regulates Kv1.3. KCNE4 modifies Kv1.3 currents altering kinetics and retaining the channel at the endoplasmic reticulum (ER). This function affects in turn membrane localization of the channel. Our research showed that KCNE4 can dimerize via the juxtamembrane tetraleucine carboxyl-terminal domain of KCNE4. This cluster serves as a competitive structural platform for Kv1.3, Ca2+/calmodulin (CaM) and KCNE4 dyads. While KCNE4 is typically retained in the ER, the association with CaM leads to COP-II-dependent forward trafficking. Consequently, CaM plays a vital role in controlling the dimerization and membrane targeting of KCNE4, affecting the regulation of Kv1.3 and, subsequently, leukocyte physiology. Kv1.3, localized in membrane lipid rafts, accumulates at immunological synapses during cell activation, influencing membrane potential and downstream calcium-signalling pathways. KCNE4 acts as a dominant negative regulatory subunit on Kv1.3, causing intracellular retention. Palmitoylation, a reversible post- translational modification, enhances protein hydrophobicity, facilitating membrane association, protein interactions, and subcellular trafficking. Our data demonstrated the S-acylation of KCNE4, resulting in spatial rearrangements that reduce ER distribution, which in turn affects Kv1.3 regulation. KCNE4 partially traffics to the cell surface with Kv1.3 in activated dendritic cells but alters immunological synapse targeting. This highlights the significance of KCNE4 palmitoylation in regulating protein subcellular localization and oligomeric state, subsequently affecting channel membrane expression. Given the role of Kv1.3 as an immunomodulatory target, these findings offer insights for future clinical and pharmacological studies.