Cardiovascular Kv7 channels in health and in disease

Abstract

Voltage-gated K+ channels, Kv channels, are the most diverse group of K+ channels. They have several key functions in cardiovascular and nervous system; setting the resting membrane potential and controlling the repolarization of the action potential. In non-excitable cells they also play crucial role in water and ion transport across the epithelia or cell volume regulation. Such extraordinary heterogeneity in functions is closely related with a remarkable genetic and structural heterogeneity of Kv channels.

Among Kv channels, the Kv7 family comprises 5 members, Kv7.1-Kv7.5. Like other Kvs, Kv7 channels are tetramers of 4 α subunits. The topological arrangement of every α subunit consists in 6 transmembrane segments: S1-S4 form the voltage sensing domain and the S5-S6 region the ion-selective pore. Kv7 channels use to assemble as homotetramers, but most of the members of the family are also able to form heterotetrameric channels. Heterotetramerization is one of most important mechanisms to achieve the vast functional diversity that they display. Moreover, Kv7 channel function is also influenced by regulatory subunits, the KCNE family. They regulate various gating parameters, current density, channel assembly, ER retention, sorting into different traffic pathways, subcellular localization, regulation of the post-translational modifications or sensibility to pharmacological agents.

In the cardiovascular system, both Kv7.1 and Kv7.5 play a pivotal role. In cardiac myocytes, Kv7.1 homotetramers combine with KCNE1 β subunit to form functional channels. They are the molecular correlate of the IKs current, a slow delayed-rectifier K+ current involved in the repolarization of the cardiac action potential and an important determinant of the duration of the QT interval of the electrocardiogram. Mutation in either, Kv7.1 and KCNE1 result in long QT or short QT syndromes, which can lead to fatal severe arrhythmias. Kv7 have been also recognized to be key regulators of the vascular physiology, setting the resting membrane potential in vascular smooth muscle myocytes and therefore controlling the vascular smooth muscle tone. In particular, Kv7.1, Kv7.4 and Kv7.5 had been detected in numbers of veins and arteries, but the specific contribution of every subunit to physiological relevant K+ currents is still under debate.

Within this scenario, the proposed objectives for the currently discussed PhD dissertation were focused on further study the heteroligomerization process of the Kv7 channel family, either by heterotetramerization or by interaction with β regulatory subunits. In the one hand corroborate the functional assembly of Kv7.1-Kv7.5 heterotetramers in vascular smooth muscle. On the other hand, we were also committed to explore the molecular pathological mechanisms of Kv7.1 mutations linked to cardiac channelopathies as well as dissect the traffic and assembly process of the Kv7.1-KCNE1 complex.

We had been able to prove that Kv7.1-Kv7.5 form heterotetrameric channels in vascular smooth muscle cells. Moreover, upon heterotetramer assembly the special distribution of those channels within plasma membrane microdomains is rearrange and Kv7.1 shifted out of lipid raft structures. In addition, we had studied 2 different mutations in Kv7.1, F279I and D242N, responsible for short QT and long QT syndromes, respectively. Those mutations uncover 2 Kv7.1 residues important for KCNE1 interaction: F279 physically interacts with KCNE1 and D242 is involved in the KCNE1-dependent modulation of the voltage-dependent gating mechanism of the Kv7.1 channel. Furthermore, our results revealed that Kv7.1 and KCNE1 are not assembled in the very first stages of the biosynthetic pathway. On its own, KCNE1 traffics through the classical secretion pathway to the cell surface, in contrast, Kv7.1 takes an unconventional pathway. Upon co-assembly, Kv7.1 redirects KCNE1 to the unconventional pathway that lead to the concentration of both subunits in the endoplasmatic reticulum-plasma membrane junctions, where complexes are assembled.

Els canals de potassi dependents de voltatge Kv7.1 i Kv7.5 juguen un paper molt important en la fisiologia del sistema cardiovascular. Aquest canals iònics estan formats per la tetramerització de 4 subunitats α. La heterotetramerizació és un dels mecanisme principals que dona lloc a la immensa diversitat funcional que mostra aquesta família. A més a més, la seva funció també es veu influenciada per la interacció amb subunitats β reguladores de la família KCNE. Tot i ser petits pèptids, els KCNEs poden modular un gran ventall de característiques de la biologia i electrofisiologia de les corrents generades per la família Kv7. En els cardiomiòcits, Kv7.1 interacciona amb KCNE1 per donar lloc el corrent IKs, una important corrent de K+ determinant per la repolarització del potencial d’acció cardíac i per la durada del interval QT de l’electrocardiograma. Mutacions tant en Kv7.1 com en KCNE1 donen lloc a les síndromes de QT llarg i QT curt, totes dues relacionades amb perilloses arítmies i mort sobtada. Kv7.1 juntament amb Kv7.4 i Kv7.5 també participen en el control del to de la musculatura llisa vascular, tot i que el paper específic de cada subunitat en les corrents detectades en aquest teixit encara genera una gran controvèrsia. En la present tesi doctoral demostrem l’existència d’heterotetramers Kv7.1-Kv7.5 en els miòcits de la musculatura llisa vascular. Aquesta nova interacció provoca el desplaçament de Kv7.1 fora dels microdominis de membrana tipus lipid raft. També hem estudiat els mecanismes patològics moleculars de dos mutacions en Kv7.1, F279I i D242N, responsables de les síndromes del QT curt i el QT llarg, respectivament. Aquestes dues mutacions han posat de manifest 2 residus en Kv7.1 importants per la interacció amb KCNE1: F279 participa en la interacció física amb KCNE1 i D242 en la regulació per KCNE1 del mecanisme, depenent de voltatge, d’obertura del canal. Per últim, hem comprovat que Kv7.1 i KCNE1 trafiquen per diferents vies secretores i que la formació dels complexes Kv7.1-KCNE1 té lloc a les unions reticle endoplasmatic-membrana plasmàtica.