Heteroligomeric interactions of the Kv1.3 channelosome

Abstract

Voltage-gated potassium channels are proteins that allow the flux of potassium ions across the plasma membrane in response to a voltage stimulus. Those proteins were initially described in nervous system as the repolarization entities posterior to a depolarization. However, several different roles have been discovered to be enhanced, mediated or influenced by those entities. Cell cycle progression, homeostasis, proliferation or activation and apoptosis program are some of those functions. Kv1.3 is the third member of the first family of voltage-gated potassium channels in humans. This specific entity is mainly expressed in nervous and immune system. It has been associated with the repolarization of neurons, the activation and proliferation of leukocytes and apoptosis. Moreover, its dysfunctionality has been related to some autoimmune disease. The fine-tunning of the channel is highly relevant to control the final cell decision. The subunits that accompany the channel were classically named as β- subunits. Several different families have been described to modulate some channel features. Kvβ family are cytoplasmic proteins that can enhance the traffic to the plasma membrane and promote a switch towards negative voltages of the channel activation. However, few are known about alternative locations and Kv1.3 modulation. KCNE family are single spanning proteins highly promiscuous that modulate several different Kvα-subunits. Depending on the KCNE subtype, the effect on the channels can present different natures. This dissertation is focused on KCNE4 member, a peptide which generally negatively regulates Kv channels. This thesis described the positioning of Kvβ2.1, but not Kvβ1.1, in specific regions of the plasma membrane: lipid raft microdomains. Those are considered as signalling platforms at the plasma membrane highly relevant for several cellular processes. The possible mechanism that drives Kvβ2.1 is the palmitoylation of its amino acidic sequence; even other causes are not discarded. Proliferation signals are enhancing this localization while PMA treatment generates the opposite effect. This protein, as well as its partner Kvβ1.1, can form homo and heteroligomers. Their affinity and stoichiometry was addressed. Furthermore, multiprotein complexes were detected at membrane associated environments. Traffic and electrophysiological consequences on the channel were analysed upon coexpression with those subunits. Kv1.3 was removed from lipid raft microdomains and Kvβs prevented partially its PMA-dependent internalization. The molecular determinants involved in the Kv1.3 traffic to the plasma membrane were localised at the C-terminal domain. Previous results from the laboratory determined that KCNE4 is impairing the traffic of the channel. This thesis deciphered the molecular mechanisms involved in this effect concluding a bipartite system: (i) the masking of Kv1.3 export signal and (ii) the transference of a retention signal to the channelosome. Moreover, the specific domains of Kv1.3 and KCNE4 implicated in their interaction were mapped and pointed out to the C-terminal regions of both peptides. KCNE4 was also found to form oligomers and present several signals for its retention at the endoplasmic reticulum. Finally, the combination of both subunits (Kvβ2.1 and KCNE4) on the channel showed a dominance of KCNE4 effects, but an electrophysiological function of Kvβ2.1 on Kv1.3 kept preserved. Thus, the present thesis brought light to the comprehension of Kv1.3 channelosome.