Molecular determinants of the Kv1.3/KCNE4 interaction

Abstract

Kv1.3 plays a crucial role in leukocytes, where it regulates activation and proliferation. In these cells, Kv1.3 is coexpressed with modulatory subunit KCNE4. This ancillary peptide acts as a dominant negative regulator of the channel with a dual function. On the one hand, KCNE4 triggers the intracellular retention of Kv1.3 reducing K+ currents. On the other, KCNE4 enhances their C- type inactivation kinetics. Thus, the aim of this thesis was to characterize the molecular mechanisms of Kv1.3/KCNE4 association and modulation in the context of the immune system physiology. To do so, the thesis is structured in three chapters. Chapter I (KCNE4 in the immune system). We confirmed the physiological role of KCNE4 in immune cells like T lymphocytes and dendritic cells (Contribution 1). By manipulating the expression of KCNE4 in these cells, we were the first to describe a relationship between KCNE4 and the leukocyte function. Our results highlight a possible therapeutic role for KCNE4. Chapter II (A protein interaction hub (L69-72) in the C-terminus of KCNE4). We analysed the relationship between Kv1.3, KCNE4 and calmodulin (CaM). We identified a tetraleucine motif in the C-terminus of KCNE4 (L69-72) that determines association to Kv1.3 (Contribution 2). In a nearby position, an ER retention motif (ERRM) triggers intracellular retention of the channel. These results were replicated in a peptide containing just 22 residues of KCNE4, including both clusters. This peptide partially replicated the inhibitory effects of KCNE4 and thus, a potential pharmacological interest. We are also the first to propose a structural model for the Kv1.3/KCNE4 association. On the other hand, L69-72 in KCNE4 also determines CaM binding. The role of Ca2+/CaM is relevant in the context of the immune system physiology, where Kv1.3 potentiates the calcium signalling. Furthermore, we are the first to report dimerization of KCNE4 (Contribution 3), which modulates the traffic of KCNE4. Dimerization was also mediated by the leucine cluster, acting as a protein interaction hub. We propose CaM binding as a mechanism to fine-tune interactions with Kv1.3 and dimerization. Chapter III (Structural relationships of the Kv1.3/KCNE4 complex). We focused on the structural Kv1.3/KCNE4 association. A series of chimaeras between KCNE2 and KCNE4 allowed us to decipher roles for each domain of KCNE4 (Contribution 4). Thus, we confirmed that the C- terminus of KCNE4 mediates association and intracellular retention of Kv1.3. Moreover, for the first time we characterized the molecular determinants of KCNE4-dependent kinetic modulation of Kv1.3. These results were confirmed with Gibbs free energy calculations and the Kv1.3/KCNE4 structural model. Using alanine-scanning mutagenesis, we mapped some residues implicated in this process. Moreover, we found that, similar to other Kv/KCNE associations, Kv1.3/KCNE4 associated in an open stoichiometry with up to four KCNE4 subunits per complex (Contribution 5). The number of KCNE4 peptides in the channelosome was proportional to the effects on activation kinetics and current amplitude. However, the accentuation of Kv1.3 inactivation was independent of stoichiometry. The similarities in stoichiometry and docking to Kv channels suggest a conserved mechanism among KCNE peptides. Finally, because KCNE4 caused structural Kv1.3 changes, we studied the effect of KCNE4 on Kv1.3 pharmacology (Contribution 6). We studied margatoxin and Psora-4, two well-characterized Kv1.3 inhibitors with different binding sites. Our results point out the relevance of considering auxiliary subunits in the development of therapeutic approaches. In conclusion, this thesis deepens the knowledge of the molecular mechanisms of Kv1.3 modulation by KCNE4. We provide a structural insight into Kv channel modulation while considering the physiological context. Our results are of considerably physiological interest due to the multitherapeutic potential of Kv1.3.