Dimerization of the voltage-sensing phosphatase controls its voltage-sensing and catalytic activity.

Multimerization is a key characteristic of most voltage-sensing proteins. The main exception was thought to be the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP). In this study, we show that multimerization is also critical for Ci-VSP function. Using coimmunoprecipitation and single-molecule pull-down, we find that Ci-VSP stoichiometry is flexible. It exists as both monomers and dimers, with dimers favored at higher concentrations. We show strong dimerization via the voltage-sensing domain (VSD) and weak dimerization via the phosphatase domain. Using voltage-clamp fluorometry, we also find that VSDs cooperate to lower the voltage dependence of activation, thus favoring the activation of Ci-VSP. Finally, using activity assays, we find that dimerization alters Ci-VSP substrate specificity such that only dimeric Ci-VSP is able to dephosphorylate the 3-phosphate from PI(3,4,5)P3 or PI(3,4)P2 Our results indicate that dimerization plays a significant role in Ci-VSP function

Migraine-Associated TRESK Mutations Increase Neuronal Excitability through Alternative Translation Initiation and Inhibition of TREK

Mutations in ion channels contribute to neurological disorders, but determining the basis of their role in pathophysiology is often unclear. In humans, 2 mutations have been found to produce a dominant negative for TRESK, a two-pore-domain K+ channel implicated in migraine: TRESK-MT, a 2 bp frameshift mutation (F139WfsX24) and TRESK-C110R, a missense mutation. Despite the fact that both mutants strongly inhibit TRESK, only TRESK-MT leads to an increase in sensory neuron excitability and is associated with a migraine phenotype. Here, we identify a new mechanism, termed frameshift mutation induced Alternative Translation Initiation (fsATI) that may explain why TRESK-MT but not TRESK-C110R is associated with migraine disorder. fsATI leads, from the same TRESK-MT mRNA, to two proteins: TRESK-MT1 and TRESK-MT2. We show that by co-assembling with and inhibiting TREK1 and TREK2, another subfamily of K2P channels, overexpression of TRESK-MT2 increases trigeminal sensory neuron excitability, a key component of migraine induction, leading to a migraine-like phenotype. This finding identifies TREK as a potential molecular target in migraine pathophysiology and resolves the contradictory lack of effect of TRESK-C110R which targets only TRESK and not TREK. Finally, taking into account the potential for fsATI allowed us to identify a new migraine-related TRESK mutant, Y121LfsX44, which also leads to the production of two TRESK fragments, indicating that this mechanism may be widespread. Together, our results suggest that genetic analysis of disease-related mutations should consider fsATI as a distinct class of mutations

La diversité combinatoire des canaux potassiques à deux domaines pore et son implication dans la migraine

Maintenance of a negative resting membrane potential underlies the basis of neuronal excitability. This negative potential is generated by a potassium leak current mediated by two-pore-domain potassium channels (K2P). Over the years, they have been shown to be involved in many physiological and pathophysiological mechanisms such as depression, neuroprotection, anesthesia, migraine and pain perception. Heteromultimerization is a mechanism commonly used to increase the functional diversity of protein complexes. For example, with 15 genes classified in 6 subfamilies, the K2P channel family can potentially generates 120 combinations and, in theory, each of them would show different functional properties. Here, we first investigated the ability of the members from the same K2P subfamily (TREK subfamily) to assemble and form functional heteromeric channels with novel properties. Using single molecule pulldown (SiMPull) from HEK cell lysates, subunit counting in the plasma membrane of living cells and opto-pharmacology, we show that the TREK channel members TREK1, TREK2, and TRAAK readily co-assemble. We functionally characterized the heterodimers and found that all combinations form outwardly rectifying potassium-selective channels but with variable voltage sensitivity and pH regulation. Having found that heteromerization is possible within the same subfamily we wonder if it can happen between members from different subfamilies with lower sequence homology and what could be the pathophysiological consequences. We found that TREK1 and TREK2 are able to heterodimerize with the distantly-related TRESK, a two-pore-domain K+ channel implicated in migraine. Notably, in humans, TRESK-MT, a 2 bp frameshift mutation (F139WfsX24), which induced the formation of TRESK-MT1 a dominant negative for TRESK, was found to perfectly segregate with typical migraine in a large pedigree. Strikingly, we found that the 2 bp frameshift mutation induced an alternative translation initiation (fsATI) which leads to the translation of a second TRESK fragment, termed TRESK-MT2. We show that by co-assembling with and inhibiting TREK1 and TREK2, TRESK-MT2 increases trigeminal sensory neuron excitability, a key component of migraine induction, leading to a migraine-like phenotype. Together these findings demonstrate that K2P heteromerization is not rare and needs to be considered to understand their pathophysiological functions and that genetic analysis of disease-related mutations should consider fsATI as a distinct class of mutations.Le maintien d'un potentiel de membrane de repos négatif est à la base de l'excitabilité neuronale. Ce potentiel négatif est généré par un courant de fuite de potassium induit par les canaux potassiques à deux domaines pore (K2P). Ils se sont révélés impliqués dans de nombreux mécanismes physiologiques et physiopathologiques tels que la dépression, la neuroprotection contre les ischémies, l'anesthésie, la migraine et la perception de la douleur. L'hétéromultimérisation est un mécanisme couramment utilisé dans la nature pour augmenter la diversité fonctionnelle des complexes protéiques. Par exemple, avec 15 gènes classés en 6 sous-familles, les canaux K2P pourraient générer 120 combinaisons et, en théorie, chacune d’elles possèderait des caractéristiques bien distinctes. Ici, nous avons d’abord étudié la capacité des membres de la même sous-famille K2P (sous-famille TREK) à s’assembler pour former des hétéromères fonctionnels dotés de nouvelles propriétés. En alliant l’optopharmacologie, une technique de précipitation de molécules uniques (SiMPull) et une technique de co-localisation à l’échelle de la molécule unique à la membrane plasmique, nous avons déterminé l’existence ainsi que la stœchiométrie des complexes créés entre TREK1, TREK2 et TRAAK. Nous avons caractérisé fonctionnellement les hétérodimères et avons constaté qu'ils formaient tous des canaux sélectifs au potassium rectifiant vers l'extérieur avec une sensibilité à la tension et aux pH variables. Ayant constaté que l’hétéromérisation est possible dans la même sous-famille, nous nous demandons si cela peut être fait entre membres de familles différentes et quelles pourraient en être les conséquences pathophysiologiques. Nous avons trouvé que TREK1 et TREK2 sont capable d’hétéromériser avec le canal plus distant TRESK, un canal K2P impliqué dans la migraine. Chez l'homme, la mutation TRESK-MT, une délétion de 2 paires de base (F139WfsX24) qui induit la formation de TRESK-MT1, un dominant négatif de TRESK, a été corrélé à la migraine. De manière surprenante, nous avons découvert que cette délétion induit un site alternatif de traduction (fsATI), menant à la formation d’un second fragment de TRESK, TRESK-MT2 qui s’assemble spécifiquement avec TREK1 et TREK2. Cet assemblage induit l’extinction des courants TREK, ce qui va augmenter l’excitabilité des neurones trijumeaux, une composante clé dans l’induction de la migraine, à l’origine du phénotype migraineux observé. Ensemble, ces résultats démontrent que l’hétéromérisation des canaux K2P n’est pas rare et doit être considérée pour comprendre leurs fonctions pathophysiologiques. Enfin, les analyses génétiques des mutations liées à des pathologies devraient désormais prendre en compte les fsATI

Combinatorial diversity of two-pore-domain k+ channels and its involvement in migraine

Le maintien d'un potentiel de membrane de repos négatif est à la base de l'excitabilité neuronale. Ce potentiel négatif est généré par un courant de fuite de potassium induit par les canaux potassiques à deux domaines pore (K2P). Ils se sont révélés impliqués dans de nombreux mécanismes physiologiques et physiopathologiques tels que la dépression, la neuroprotection contre les ischémies, l'anesthésie, la migraine et la perception de la douleur. L'hétéromultimérisation est un mécanisme couramment utilisé dans la nature pour augmenter la diversité fonctionnelle des complexes protéiques. Par exemple, avec 15 gènes classés en 6 sous-familles, les canaux K2P pourraient générer 120 combinaisons et, en théorie, chacune d’elles possèderait des caractéristiques bien distinctes. Ici, nous avons d’abord étudié la capacité des membres de la même sous-famille K2P (sous-famille TREK) à s’assembler pour former des hétéromères fonctionnels dotés de nouvelles propriétés. En alliant l’optopharmacologie, une technique de précipitation de molécules uniques (SiMPull) et une technique de co-localisation à l’échelle de la molécule unique à la membrane plasmique, nous avons déterminé l’existence ainsi que la stœchiométrie des complexes créés entre TREK1, TREK2 et TRAAK. Nous avons caractérisé fonctionnellement les hétérodimères et avons constaté qu'ils formaient tous des canaux sélectifs au potassium rectifiant vers l'extérieur avec une sensibilité à la tension et aux pH variables. Ayant constaté que l’hétéromérisation est possible dans la même sous-famille, nous nous demandons si cela peut être fait entre membres de familles différentes et quelles pourraient en être les conséquences pathophysiologiques. Nous avons trouvé que TREK1 et TREK2 sont capable d’hétéromériser avec le canal plus distant TRESK, un canal K2P impliqué dans la migraine. Chez l'homme, la mutation TRESK-MT, une délétion de 2 paires de base (F139WfsX24) qui induit la formation de TRESK-MT1, un dominant négatif de TRESK, a été corrélé à la migraine. De manière surprenante, nous avons découvert que cette délétion induit un site alternatif de traduction (fsATI), menant à la formation d’un second fragment de TRESK, TRESK-MT2 qui s’assemble spécifiquement avec TREK1 et TREK2. Cet assemblage induit l’extinction des courants TREK, ce qui va augmenter l’excitabilité des neurones trijumeaux, une composante clé dans l’induction de la migraine, à l’origine du phénotype migraineux observé. Ensemble, ces résultats démontrent que l’hétéromérisation des canaux K2P n’est pas rare et doit être considérée pour comprendre leurs fonctions pathophysiologiques. Enfin, les analyses génétiques des mutations liées à des pathologies devraient désormais prendre en compte les fsATI.Maintenance of a negative resting membrane potential underlies the basis of neuronal excitability. This negative potential is generated by a potassium leak current mediated by two-pore-domain potassium channels (K2P). Over the years, they have been shown to be involved in many physiological and pathophysiological mechanisms such as depression, neuroprotection, anesthesia, migraine and pain perception. Heteromultimerization is a mechanism commonly used to increase the functional diversity of protein complexes. For example, with 15 genes classified in 6 subfamilies, the K2P channel family can potentially generates 120 combinations and, in theory, each of them would show different functional properties. Here, we first investigated the ability of the members from the same K2P subfamily (TREK subfamily) to assemble and form functional heteromeric channels with novel properties. Using single molecule pulldown (SiMPull) from HEK cell lysates, subunit counting in the plasma membrane of living cells and opto-pharmacology, we show that the TREK channel members TREK1, TREK2, and TRAAK readily co-assemble. We functionally characterized the heterodimers and found that all combinations form outwardly rectifying potassium-selective channels but with variable voltage sensitivity and pH regulation. Having found that heteromerization is possible within the same subfamily we wonder if it can happen between members from different subfamilies with lower sequence homology and what could be the pathophysiological consequences. We found that TREK1 and TREK2 are able to heterodimerize with the distantly-related TRESK, a two-pore-domain K+ channel implicated in migraine. Notably, in humans, TRESK-MT, a 2 bp frameshift mutation (F139WfsX24), which induced the formation of TRESK-MT1 a dominant negative for TRESK, was found to perfectly segregate with typical migraine in a large pedigree. Strikingly, we found that the 2 bp frameshift mutation induced an alternative translation initiation (fsATI) which leads to the translation of a second TRESK fragment, termed TRESK-MT2. We show that by co-assembling with and inhibiting TREK1 and TREK2, TRESK-MT2 increases trigeminal sensory neuron excitability, a key component of migraine induction, leading to a migraine-like phenotype. Together these findings demonstrate that K2P heteromerization is not rare and needs to be considered to understand their pathophysiological functions and that genetic analysis of disease-related mutations should consider fsATI as a distinct class of mutations

Canaux ioniques et mécanismes de transmission de maladies héréditaires à l’origine de la migraine

International audienc

Canaux potassiques à deux domaines P (K2P) et migraine

International audienceMigraine is a common, disabling neurological disorder with genetic, environmental and hormonal components and a prevalence estimated at ∼15%. Migraine episodes are notably related, among several factors, to electric hyperexcitability in sensory neurons. Their electrical activity is controlled by ion channels that generate current, specifically by the two-pore-domain potassium, K2P, channels, which inhibit electrical activity. Mutation in the gene encoding TRESK, a K2P channel, causes the formation of TRESK-MT1, the expected non-functional C-terminal truncated TRESK channel, and an additional unexpected protein, TRESK-MT2, which corresponds to a non-functional N-terminal truncated TRESK channel, through a mechanism called frameshift mutation-induced Alternative Translation Initiation (fsATI). TRESK-MT1 is inactive but TRESK-M2 targets two other ion channels, TREK1 and TREK2, inducing a great stimulation of the neuronal electrical activity that may cause migraines. These findings identify TREK1 and TREK2 as potential molecular targets for migraine treatment and suggest that fsATI should be considered as a distinct class of mutations

Heterodimerization within the TREK channel subfamily produces a diverse family of highly regulated potassium channels

Twik-related K(+) channel 1 (TREK1), TREK2, and Twik-related arachidonic-acid stimulated K(+) channel (TRAAK) form the TREK subfamily of two-pore-domain K(+) (K2P) channels. Despite sharing up to 78% sequence homology and overlapping expression profiles in the nervous system, these channels show major differences in their regulation by physiological stimuli. For instance, TREK1 is inhibited by external acidification, whereas TREK2 is activated. Here, we investigated the ability of the members of the TREK subfamily to assemble to form functional heteromeric channels with novel properties. Using single-molecule pull-down (SiMPull) from HEK cell lysate and subunit counting in the plasma membrane of living cells, we show that TREK1, TREK2, and TRAAK readily coassemble. TREK1 and TREK2 can each heterodimerize with TRAAK, but do so less efficiently than with each other. We functionally characterized the heterodimers and found that all combinations form outwardly rectifying potassium-selective channels but with variable voltage sensitivity and pH regulation. TREK1-TREK2 heterodimers show low levels of activity at physiological external pH but, unlike their corresponding homodimers, are activated by both acidic and alkaline conditions. Modeling based on recent crystal structures, along with mutational analysis, suggests that each subunit within a TREK1-TREK2 channel is regulated independently via titratable His. Finally, TREK1/TRAAK heterodimers differ in function from TRAAK homodimers in two critical ways: they are activated by both intracellular acidification and alkalinization and are regulated by the enzyme phospholipase D2. Thus, heterodimerization provides a means for diversifying functionality through an expansion of the channel types within the K2P channels

Heterodimerization within the TREK channel subfamily produces a diverse family of highly regulated potassium channels

Twik-related K(+) channel 1 (TREK1), TREK2, and Twik-related arachidonic-acid stimulated K(+) channel (TRAAK) form the TREK subfamily of two-pore-domain K(+) (K(2P)) channels. Despite sharing up to 78% sequence homology and overlapping expression profiles in the nervous system, these channels show major differences in their regulation by physiological stimuli. For instance, TREK1 is inhibited by external acidification, whereas TREK2 is activated. Here, we investigated the ability of the members of the TREK subfamily to assemble to form functional heteromeric channels with novel properties. Using single-molecule pull-down (SiMPull) from HEK cell lysate and subunit counting in the plasma membrane of living cells, we show that TREK1, TREK2, and TRAAK readily coassemble. TREK1 and TREK2 can each heterodimerize with TRAAK, but do so less efficiently than with each other. We functionally characterized the heterodimers and found that all combinations form outwardly rectifying potassium-selective channels but with variable voltage sensitivity and pH regulation. TREK1-TREK2 heterodimers show low levels of activity at physiological external pH but, unlike their corresponding homodimers, are activated by both acidic and alkaline conditions. Modeling based on recent crystal structures, along with mutational analysis, suggests that each subunit within a TREK1-TREK2 channel is regulated independently via titratable His. Finally, TREK1/TRAAK heterodimers differ in function from TRAAK homodimers in two critical ways: they are activated by both intracellular acidification and alkalinization and are regulated by the enzyme phospholipase D2. Thus, heterodimerization provides a means for diversifying functionality through an expansion of the channel types within the K(2P) channels

Migraine-Associated TRESK Mutations Increase Neuronal Excitability through Alternative Translation Initiation and Inhibition of TREK

International audienceIt is often unclear why some genetic mutations to a given gene contribute to neurological disorders and others do not. For instance, two mutations have previously been found to produce a dominant negative for TRESK, a two-pore-domain K+ channel implicated in migraine: TRESK-MT, a 2-bp frameshift mutation, and TRESK-C110R. Both mutants inhibit TRESK, but only TRESK-MT increases sensory neuron excitability and is linked to migraine. Here, we identify a new mechanism, termed frameshift mutation-induced alternative translation initiation (fsATI), that may explain why only TRESK-MT is associated with migraine. fsATI leads to the production of a second protein fragment, TRESK-MT2, which co-assembles with and inhibits TREK1 and TREK2, two other two-pore-domain K+ channels, to increase trigeminal sensory neuron excitability, leading to a migraine-like phenotype in rodents. These findings identify TREK1 and TREK2 as potential molecular targets in migraine and suggest that fsATI should be considered as a distinct class of mutations