Pablo Ávalos Prado

Arnaud Landra-Willm

Clément Verkest

Anne-Amandine Chassot

Anne Baron

Guillaume Sandoz
email: sandoz@unice.fr

TREK channel activation suppresses migraine pain phenotype

Keywords: Allodynia, Pain, K2P channels, Migraine, Neuronal excitability, Trigeminal neurons

Activation and sensitization of trigeminal (TG) ganglia sensory neurons, leading to the release of pro-inflammatory peptides such as calcitonin gene-related peptide (CGRP), are likely a key component in migraine-related headache induction. Reducing TG neuron excitability represents therefore an attractive alternative strategy to relieve migraine pain. Here by using pharmacology and genetic invalidation ex vivo and in vivo, we demonstrate that activating TREK1-and TREK2-Two-Pore-Domain potassium (K2P) channels inhibits TG neuronal firing sufficiently to fully reverse the migraine-like phenotype induced by NO-donors in rodents. Finally, targeting TREK is as efficient as treatment with CGRP antagonists, which represents one of the most effective migraine therapies. Altogether, our results demonstrate that inhibiting TG excitability by pharmacological activation of TREK channels should be considered as an alternative to the current migraine treatment.

INTRODUCTION

Migraine is considered one of the most disabling conditions worldwide, affecting around 15% of the global population [START_REF] Burch | The prevalence and burden of migraine and severe headache in the United States: updated statistics from government health surveillance studies[END_REF]. It manifests as a unilateral throbbing headache and is accompanied by multiple symptoms such as nausea, vomiting, photophobia, phonophobia or cutaneous allodynia. It is generally assumed that migraine attacks start with the activation of sensory neurons in the trigeminal ganglia (TG) [START_REF] Strassman | Sensitization of meningeal sensory neurons and the origin of headaches[END_REF].

Hyperexcitability of TG fibers innervating meninges induces the release of neuropeptides (e.g. substance P and the calcitonin gene-related peptide CGRP) that triggers vasodilatation of surrounding blood vessels and local neurogenic inflammation, leading to migraine [START_REF] Frederiksen | Perivascular neurotransmitters: Regulation of cerebral blood flow and role in primary headaches[END_REF][START_REF] Goadsby | Pathophysiology of Migraine: A Disorder of Sensory Processing[END_REF][START_REF] Khan | Meningeal contribution to migraine pain: a magnetic resonance angiography study[END_REF]. Targeting the pro-inflammatory peptides secreted by TG neurons such as CGRP constitutes one of the most effective current strategy to treat migraine [START_REF] Edvinsson | CGRP as the target of new migraine therapies -successful translation from bench to clinic[END_REF]. However, about 50% of the patients do not respond to this treatment and suffer from secondary effects (joint pain, dizziness, constipation, flu-like symptoms) and alternative patient care needs to be developed.

Expression of several K2P channel subunits has been detected in nociceptive dorsal root ganglions and trigeminal neurons [START_REF] Alloui | TREK-1, a K+ channel involved in polymodal pain perception[END_REF][START_REF] Yamamoto | Immunohistochemical colocalization of TREK-1, TREK-2 and TRAAK with TRP channels in the trigeminal ganglion cells[END_REF]. We recently found that the human mutation of K2P-TRESK channel, "TRESK-MT", related to migraine, inhibits K2P-TREK1 and TREK2 channels and sufficiently increases sensory TG neuronal excitability to generate a migraine-like phenotype [START_REF] Royal | Migraine-Associated TRESK Mutations Increase Neuronal Excitability through Alternative Translation Initiation and Inhibition of TREK[END_REF]. These results demonstrate the importance of TREK1 and TREK2 channels in the regulation of TG excitability.

As the current most effective drugs against migraine target the neuropeptide CGRP released by TG neurons or its receptor [START_REF] Edvinsson | CGRP as the target of new migraine therapies -successful translation from bench to clinic[END_REF], it is attractive to act on TG excitability to prevent CGRP release and, consequently, migraine. We therefore made the hypothesis that enhancing TREK1 and TREK2 activity would reduce TG excitability, prevent the release of pro-inflammatory peptides and therefore suppress migraine pain. We show that, at a cellular level, ML67-33, a TREK1/2 agonist [START_REF] Bagriantsev | A high-throughput functional screen identifies small molecule regulators of temperature-and mechanosensitive K2P channels[END_REF], reduces TG sensory neuronal excitability, explaining its "anti-migraine" properties. Using behavioral tests in wildtype and double knock-out mice for Trek1/Trek2 (Trek1 -/--Trek2 -/-mice), we demonstrate that ML67-33 reverses the NO donor-induced migraine-like phenotype. This reversion exhibits a similar potency to BIBN4096, a selective CGRP antagonist used against migraine [START_REF] Olesen | Calcitonin gene-related peptide receptor antagonist BIBN 4096 BS for the acute treatment of migraine[END_REF]. Finally, we demonstrate that ML67-33 completely reversed the NO donor-induced Electronic copy available at: https://ssrn.com/abstract=3850367 facial allodynia in rats, which is a direct index of TG excitability. Therefore, targeting TREK channels to inhibit TG firing and prevent CGRP release underlying migraine should therefore be considered when developing new molecules to treat migraine.

RESULTS

TREK1 and TREK2 activation by ML67-33 reverses the migraine-like cutaneous allodynia in mice

To investigate whether targeting TREK1 and TREK2 activity would treat migraine, we first tested whether the TREK agonist ML67-33 [START_REF] Bagriantsev | A high-throughput functional screen identifies small molecule regulators of temperature-and mechanosensitive K2P channels[END_REF] specifically activated TREK1 and TREK2 channels. We validated that ML67-33 selectively activated both TREK1 homodimer and TRESK-TREK1 heterodimer currents whereas TRESK alone was not activated by ML67-33 (Figure S1). This shows that ML67-33, by binding to only one channel moiety, can agonize the TREK channels, making it the molecule of choice for studying TREK1/2 homoand heterodimer channel function.

Once validated, we next monitored the effects of ML67-33 in a well-described model of chronic migraine in mice in which migraine is induced by nitric oxide (NO) donors [START_REF] Bates | Sumatriptan alleviates nitroglycerin-induced mechanical and thermal allodynia in mice[END_REF] such as isosorbide dinitrate (ISDN) [START_REF] Dallel | Recurrent administration of the nitric oxide donor, isosorbide dinitrate, induces a persistent cephalic cutaneous hypersensitivity: A model for migraine progression[END_REF][START_REF] Royal | Migraine-Associated TRESK Mutations Increase Neuronal Excitability through Alternative Translation Initiation and Inhibition of TREK[END_REF][START_REF] Verkest | Effects of systemic inhibitors of acid-sensing ion channels 1 (ASIC1) against acute and chronic mechanical allodynia in a rodent model of migraine[END_REF]. We induced and evaluated cutaneous allodynia, a quantifiable marker of migraine, after four consecutive days of ISDN injections in wild-type and Trek1 -/--Trek2 -/-mice by measuring paw withdrawal mechanical threshold using a dynamic von Frey aesthesiometer before each injection (Figure 1A). We first measured the consequences of ISDN injection on the mechanical threshold. In wild-type animals, we observed a significant decrease of this threshold when we compared the first and last measurements (4.04 ± 0.06 g vs 2.87 ± 0.04 g, P < 0.001). Trek1 -/--Trek2 -/-mice already exhibited a low mechanical threshold, as previously reported [START_REF] Royal | Migraine-Associated TRESK Mutations Increase Neuronal Excitability through Alternative Translation Initiation and Inhibition of TREK[END_REF] (4.04 ± 0.06 g vs 3.11 ± 0.08 g for wild-type and Trek1 -/--Trek2 -/- mice before ISDN i.p., respectively, P > 0.001) (Figure 1B).

We then compared the effect of ML67-33 with the effect of BIBN4096, a CGRP antagonist used for migraine treatment [START_REF] Olesen | Calcitonin gene-related peptide receptor antagonist BIBN 4096 BS for the acute treatment of migraine[END_REF], and challenged the capacity of ML67-33 to increase the lowered mechanical threshold induced by ISDN injections. For this purpose, wild-type or Trek1 -/--Trek2 -/-mice were classified into three groups (for each), following a series of two different injections spaced four hours in time (Figure 1D,F). The first Electronic copy available at: https://ssrn.com/abstract=3850367 group was first injected with a vehicle solution (saline + DMSO 0.1%) then with BIBN4096.

The second group was first injected with BIBN4096 then with the vehicle solution. The third group was first injected with ML67-33 and then BIBN4096.

The efficacy of BIBN4096 was confirmed in both wild-type and Trek1 -/--Trek2 -/-mice from the first group, since the second injection with BIBN4096 strongly increased their mechanical threshold compared to the first injection with the vehicle solution (2.92 ± 0.10 g vs 3.47 ± 0.11 g and 2.79 ± 0.13 g vs 3.28 ± 0.26 g, for wild-type and Trek1 -/--Trek2 -/-mice, respectively, P > 0.2 for both conditions) (Figures 1D andF, black graphs). In the second group, the mechanical threshold rapidly increased in both wild-type and Trek1 -/--Trek2 -/- animals after the first injection with BIBN4096 (2.89 ± 0.07 g vs 3.61 ± 0.11 g, P < 0.01; and 2.87 ± 0.18 g vs 3.87 ± 0.17 g, P < 0.001, for wild-type and Trek1 -/--Trek2 -/-mice, respectively) (Figures 1C andE, blue traces), and exhibited no additive effects following the second injection with the vehicle solution (3.61 ± 0.11 g vs 3.85 ± 0.17 g and 3.87 ± 0.17 g vs 3.82 ± 0.18 g, for wild-type and Trek1 -/--Trek2 -/-mice, respectively, P < 0.01 for both conditions) (Figures 1D andF, blue graphs). Nevertheless, wild-type and Trek1 -/--Trek2 -/-mice from the third group responded differently: whereas the first injection of ML67-33 increased the mechanical threshold of wild-type animals compared to the control (2.78 ± 0.06 g vs 3.92 ± 0.18, P < 0.001) (Figure 1C, red trace), it had no significant impact on Trek1 -/--Trek2 -/-mice (2.69 ± 0.11 g vs 3.00 ± 0.16, P > 0.1) (Figure 1E, red trace). Interestingly, the second injection of BIBN67-33 had no additive effect on the threshold in wild-type mice after the first injection (3.92 ± 0.18 g vs 3.65 ± 0.19, P > 0.1) (Figure 1D, red graph), while it increased the threshold in Trek1 -/--Trek2 -/-mice (3.00 ± 0.16 g vs 3.61 ± 0.18, P < 0.05) (Figure 1F, red graph).

Together, these results indicate that either antagonizing CGRP release using BIBN4098 or specifically activating TREK1/2 using ML67-33 similarly reverses from NO-donors-induced allodynia related to migraine in mice.

ML67-33 increases TREK1 and TREK2 currents in TG sensory neurons

To investigate whether the ML67-33 agonist is specifically regulating TREK1/2 currents in sensory neurons to reduce allodynia, we studied its effects on small and mediumsized TG sensory neurons either from wild-type or double KO animals. These neurons are classically divided within two populations, according to their ability to bind the plant lectin Electronic copy available at: https://ssrn.com/abstract=3850367 isolectin B4 (IB4) from Griffonia simplicifolia [START_REF] Stucky | Isolectin B(4)-positive and -negative nociceptors are functionally distinct[END_REF]. Both IB4 + and IB4 - neurons are involved in nociception but only IB4 -are enriched in neuropeptides, notably CGPR, known for inducing neurogenic inflammation and triggering migraine attacks [START_REF] Frederiksen | Perivascular neurotransmitters: Regulation of cerebral blood flow and role in primary headaches[END_REF][START_REF] Khan | Meningeal contribution to migraine pain: a magnetic resonance angiography study[END_REF].

On primary cultured TG neurons from wild-type mice, perfusion of ML67-33 increased the amplitude of a sustained K + current in IB4 + and IB4 -neurons (3.18 ± 0.46 pA/pF and 7.66 ± 1.80 and pA/pF before and after exposure to ML67-33, respectively for IB4 + neurons, P < 0.05; 2.19 ± 0.30 pA/pF and 8.12 ± 1.62 pA/pF before and after exposure to ML67-33, respectively for IB4 -neurons, P < 0.01, at -25 mV) (Figures 2A and2B). However, no significant difference was detected in the currents obtained from Trek1 -/--Trek2 -/-TG neurons before and after treatment with the TREK agonist (4.71 ± 1.75 pA/pF and 5.05 ± 0.76 and pA/pF before and after exposure to ML67-33, respectively for IB4 + , P > 0.3; 2.77 ± 0.31 pA/pF and 3.60 ± 0.29 pA/pF before and after exposure to ML67-33, respectively for IB4 -, P > 0.05, at -25 mV) (Figures 2C and2D). Therefore, ML67-33 specifically activates TREK1 and/or TREK2 currents in TG sensory neurons.

TREK current activation reduces intrinsic excitability in TG neurons related to migraine

We next determined the functional consequences of the TREK1-TREK2 current on TG sensory neuron excitability. First, our results showed that Trek1 and Trek2 genetic deletion increased both IB4 + and IB4 -neurons excitability (Figures 3 A-D, white points and grey traces; 1.70 ± 0.33 Hz vs 5.12 ± 1.52 Hz for IB4 + from wild-type and Trek1 -/--Trek2 -/-, respectively, P < 0.05; 5.12 ± 1.52 Hz vs 10.89 ± 1.57 Hz for IB4 -from wild-type and Trek1 -/--Trek2 -/- respectively, P < 0.01, for a 150 pA current injection stimulus), indicating that TREK1/2 activation is required to reduce sensory neuron excitability.

Second, in wild-type IB4 + and IB4 -sensory neurons, the TREK current increased by ML67-33 led to a non-significant decrease of the spike frequency for IB4 + neurons (1.70 ± 0.33 Hz vs 1.20 ± 0.36 Hz, P > 0.2) (Figure 3A) and an important reduction of the spike frequency in IB4 -neurons (4.80 ± 0.90 Hz vs 1.00 ± 0.36 Hz, P > 0.001) (Figure 3B). The ML67-33induced firing inhibition might not be detected in IB4 + neurons because at rest their firing rate is already low (Guo and Cao, 2014;Guo et al., 2014). The reduction of the IB4 -excitability is specific to the activation of TREK channels since ML67-33 was not effective in sensory Electronic copy available at: https://ssrn.com/abstract=3850367 neurons from Trek1 -/--Trek2 -/-(IB4 + : 5.12 ± 1.51 Hz vs 4.50 ± 1.19 Hz before and after ML67-33 perfusion, respectively, P > 0.3; IB4 -: 10.89 ± 1.57 Hz and 9.67 ± 3.03 Hz before and after ML67-33 perfusion, respectively; P > 0.6) (Figures 3C and3D). Furthermore, the rheobase (i.e. the lowest current intensity necessary for triggering action potentials (APs) was significantly enhanced by ML67-33 only in wild-type sensory neurons (Figure S2). Together, this indicates that activating TREK1/2 using ML67-33 specifically reduces IB4 -neuronal excitability.

Excitability is closely linked to the resting membrane potential (RMP). Because K2P channels serve as a hub for RMP maintenance at negative values [START_REF] Enyedi | Molecular background of leak K+ currents: two-pore domain potassium channels[END_REF], we evaluated the effect of ML67-33 on this parameter. We found that ML67-33 induced hyperpolarization of TG neurons from wild-type animals (Figures 3E andS3, -51.32 ± 2.40 mV and -62.13 ± 2.84 mV, before and after perfusion of ML67-33, respectively for IB4 + , P < 0.01; -53.96 ± 0.91 mV and -61.46 ± 2.21 mV, before and after perfusion of ML67-33, respectively for IB4 -, P < 0.01) mainly through TREK1 and TREK2 activation since the slight RMP decrease observed for Trek1 -/--Trek2 -/-TG neurons was not statistically significant (-49.94 ± 1,42 mV and -53.94 ± 2.28 mV before and after perfusion of ML67-33, respectively for IB4 + , P > 0.2; -45.70 ± 1,12 mV and -50.70 ± 1.81 mV before and after perfusion of ML67-33, respectively for IB4 -, P > 0.06). In addition, we noticed that the basal value of the RMP of peptidergic neurons from Trek1 -/--Trek2 -/-animals was statistically higher than those from wildtype mice (-45.70 ± 1,12 mV vs -53.96 ± 0.91 mV for Trek1 -/--Trek2 -/-and wild-type peptidergic neurons, respectively, P < 0.001) (Figure 3G). These results confirm that, at rest, TREK1 and TREK2 control the RMP basal value.

Altogether, these data show that TREK1 and TREK2 play a crucial role in the excitability of TG neurons, and particularly of peptidergic IB4 -nociceptive neurons, by determining the threshold for the generation of APs and the spike frequency and by notably setting up the RMP at values close to the K + equilibrium potential. Thus, ML67-33 increases TREK current to reduce the TG excitability, leading to a decrease of the migraine phenotype observed in wild-type animals treated with NO-donors.

Activation of TREK1 and TREK2 abrogates migraine-like facial allodynia in rats

To validate that injection of ML67-33 decreases TG sensory neuronal excitability and suppresses NO-migraine phenotype in vivo, we conducted behavioral experiments in rats. This

Electronic copy available at: https://ssrn.com/abstract=3850367 model allows to measure the mechanical allodynia of the face which is related to TG excitability and therefore constitutes one of the most reliable and quantifiable readout of TG excitability underlying migraine phenotype [START_REF] Harris | The effects of repeated nitroglycerin administrations in rats; modeling migraine-related endpoints and chronification[END_REF][START_REF] Pradhan | Characterization of a novel model of chronic migraine[END_REF][START_REF] Royal | Migraine-Associated TRESK Mutations Increase Neuronal Excitability through Alternative Translation Initiation and Inhibition of TREK[END_REF].

The protocol used to induce chronic migraine was similar to the one used in mice despite the use of the facial sensitivity instead of the paw withdrawal threshold as a readout. After a week of habituation, the animals were tested for facial mechanical sensitivity with von Frey filaments.

Migraine-like phenotype was induced by daily intraperitoneally (i.p.) injections of ISDN (10 mg/kg) for four consecutive days and facial mechanical threshold was measured before each injection over the four days. At day 5, either ML67-33, BIBN4096 or vehicle was administrated and their effects were followed for 3 hours, every 30 minutes (Figure 4A).

As previously described [START_REF] Verkest | Effects of systemic inhibitors of acid-sensing ion channels 1 (ASIC1) against acute and chronic mechanical allodynia in a rodent model of migraine[END_REF], ISDN induced a strong significant reduction of the face withdrawal threshold compared to normal sensitivity (P < 0.001 for the three groups) (Figure 4B). This reduction of the face withdrawal threshold is associated with the development of chronic mechanical allodynia that reflects the induction of trigeminal hyperexcitability and migraine [START_REF] Royal | Migraine-Associated TRESK Mutations Increase Neuronal Excitability through Alternative Translation Initiation and Inhibition of TREK[END_REF]. As shown in Figure 4C, both ML67-33 and BIBN4096 treatments increased in a similar manner the mechanical threshold previously lowered by ISDN injections (5.7 ± 0.77 g vs 6.7 ± 0.39 g for rats injected with ML67-33 and BIBN4096 respectively P > 0.6) (Figures 4C and4D).

Thus, in rodent models of migraine, activation of TREK1 and TREK2 channels reverses from chronic allodynia related to migraine pain as efficiently as antagonizing CGRP with BIBN4096.

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DISCUSSION

Migraine is a neurological disease caused by the combination of environmental, hormonal and genetic components. Genome-wide association studies have revealed the importance of genetic mutations in ion channels involved in synaptic transmission in migraine predisposition [START_REF] Gormley | Meta-analysis of 375,000 individuals identifies 38 susceptibility loci for migraine[END_REF][START_REF] Nyholt | A high-density association screen of 155 ion transport genes for involvement with common migraine[END_REF]. Particularly, K2P channels such as TRESK were shown to play a fundamental role in this disease. The frameshift mutation TRESK-MT perfectly segregates with migraine phenotype in large pedigree [START_REF] Andres-Enguix | Functional analysis of missense variants in the TRESK (KCNK18) K channel[END_REF][START_REF] Lafrenière | A dominant-negative mutation in the TRESK potassium channel is linked to familial migraine with aura[END_REF]. The direct causality linking TRESK-MT to migraine was recently demonstrated by showing that using CRISPR-Cas9 to fix the MT mutation restores a normal nociceptor excitability [START_REF] Pettingill | A causal role for TRESK loss of function in migraine mechanisms[END_REF] and that the TRESK-MT mutation generated altered proteins which affect TREK1 and TREK2 channel function. The TRESK-MT fragment binds and inhibits TREK1 and TREK2 in TG sensory neurons, leading to neuronal hyperexcitability and migraine [START_REF] Royal | Migraine-Associated TRESK Mutations Increase Neuronal Excitability through Alternative Translation Initiation and Inhibition of TREK[END_REF]. This makes TREK1 and TREK2 good candidates for regulating TG excitability and subsequently migraine.

To analyze the potential of modulating TREK channel activity as a novel target to treat this disease, we combined a pharmacological approach using the TREK agonist ML67-33 with mice models deficient for both Trek1 and Trek2. We found that i) in wild-type mice, ML67-33 reverses from NO-donors-induced allodynia and is as efficient as the CGRP antagonist BIBN4096, and that ii) in Trek1 -/--Trek2 -/-mice, ML67-33 has no effect. This shows that reduction of allodynia induced by ML67-33 is due to TREK activation. Furthermore, BIBN4096 and ML67-33 do not trigger an additive effect in wild-type mice, while BIBN4096 alone suppresses the allodynia induced by Trek1/2 genetic deletion. Together, these observation suggests that deleting Trek1/2 promotes an increase of CGRP release that generates a migrainelike phenotype whereas that their activation reduces CGRP release and suppresses migraine.

We confirmed this model by using a rat model allowing to measure the mechanical allodynia pain threshold of the face, which is directly linked to TG neuronal excitability and represents a good index of migraine state [START_REF] Harris | The effects of repeated nitroglycerin administrations in rats; modeling migraine-related endpoints and chronification[END_REF][START_REF] Pradhan | Characterization of a novel model of chronic migraine[END_REF][START_REF] Royal | Migraine-Associated TRESK Mutations Increase Neuronal Excitability through Alternative Translation Initiation and Inhibition of TREK[END_REF]. Accordingly, rats injected with NO-donors recovered from induced allodynia after either BIBN4096 or ML67-33 treatment in a similar manner confirming the TREK1 and TREK activation may prevent CGRP release to suppress migraine phenotype. At the cellular level, activation of TREK1 and TREK2 dramatically decreased the excitability of TG sensory neurons by notably lowering their RMP value. This reduction of neuronal excitability by ML67-33, only

Electronic copy available at: https://ssrn.com/abstract=3850367 observed in wild-type animals, confirms the role of TREK channels in excitability regulation and consequently in pain transmission.

In conclusion, our results provide evidence of the key role of TREK1 and TREK2 channels in migraine induction by regulating TG excitability. Whereas their genetic invalidation induces a neuronal hyperexcitability leading to migraine-like phenotype, their activation suppresses NO-donor induced-migraine phenotype as efficiently as current antimigraine drugs targeting neuropeptide release. Therefore, targeting TREK channel intrinsic activity to reduce TG neuron excitability and CGRP release should be considered as an alternative strategy to treat migraine.

FIGURE LEGENDS

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Mean ± SEM.

Electronic copy available at: https://ssrn.com/abstract=3850367 Electronic copy available at: https://ssrn.com/abstract=3850367 on the occurrence and development of cutaneous chronic allodynia (facial mechanical threshold, g) in rats before treatment with different drugs. One-way ANOVA and Sidak post hoc vs pre-treatment day 1 (* p < 0.5, ** p < 0.01, *** p < 0.001). (C) ML67-33 is as efficient as the CGRP receptor antagonist BIBN4096 to reverse migraine like phenotype: time-evolution of von Frey delta facial withdrawal thresholds in rats injected with a vehicle solution (saline + DMSO 0.1% i.p.), BIBN4096 (1 mg/kg i.p.) and ML67-33 (1 mg/kg i.p.). Two-way ANOVA and Sidak post hoc vs vehicle treatment (* p < 0.05, *** p < 0.001); One-way ANOVA and Dunett's post hoc vs time -0.5 h (# p < 0.5, ## p < 0.01, ### p < 0.001). (D) Variation of the face withdrawal threshold before and 3 hours after compound administration. Kruskal-Wallis and Student-Newman-Keuls post hoc (** p < 0.01). Mean ± SEM.

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SUPPLEMENTAL FIGURES AND LEGENDS

STAR★Methods

RESOURCE AVAILABILITY

Lead Contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Guillaume Sandoz (sandoz@unice.fr).

Materials Availability

This study did not generate new unique reagents.

Data and Code Availability

Source data for the main and supplemental figures in the paper is available at Mendeley doi:10.17632/4vr86gr5ch.1

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Mice

All mouse experiments were conducted according to national and international guidelines and have been approved by the local ethical committee and authorized the French Ministry of Research according to the European Union regulations and the Directive 2010/63/EU (APAFIS#21943-2019073017246158). The C57BL/6J breeders were maintained on a 12 h light/dark cycle with constant temperature (23-24 ºC), humidity (45%-50%), and food and water ad libitum at the animal facility of Institut de Biologie de Valrose. Knock-out mice lacking Trek1 and Trek2 were generated as described [START_REF] Guyon | Glucose inhibition persists in hypothalamic neurons lacking tandem-pore K+ channels[END_REF]. Null mutations were backcrossed against the C57BL/6J inbred strain for more than 10 generations prior to establishing the breeding cages to generate subjects for this study. Age-and sex-matched C57BL/6J wild-type mice, aged 9-12 weeks, were obtained from Charles River Laboratories (Wilmington, MA). Behavioral experiments were performed on 7 to 13-weeks old male mice weighting 20-30 g.

Electronic copy available at: https://ssrn.com/abstract=3850367

Rats

Experiments were performed on 6 to 9 weeks old male Sprague Dawley rats (Janvier Labs) weighing 250 to 400 g. Animals were housed in a 12 hours light-dark cycle with food and water available ad libitum. Animal procedures were approved by the Institutional Local Ethical Committee and authorized by the French Ministry of Research according to the European Union regulations and the Directive 2010/63/EU (Agreements 01550.03). Animals were sacrificed at experimental end points by CO2 euthanasia.

Primary culture of mouse TG neurons

Trigeminal ganglion tissues were collected from postnatal day 2-8 mice of both sex in L15 Leibovitz medium (Sigma) and treated with 2 mg/ml collagenase type II (Worthington) and BSA (Euromedex) for 2 hours, followed by 2.5 mg/ml trypsin (Sigma) for 15 min. Neurons were dissociated in DMEM/F12 medium (Gibco) by triturating with fire-polished and coated glass pipettes and seeded on poly-L-lysine (Sigma) coated coverslips. The DMEM-based culture medium contained 10% fetal bovine serum (Dutscher) and 2 mM Glutamine (Gibco).

HEK293T cells

HEK293T (ATCC, #CRL11268) cells were maintained in DMEM (Gibco) supplemented with 10% FBS (Dutscher) in 35 mm dishes. At 70%-80% confluency they were transiently co-transfected using the calcium phosphate method with a total amount of 3.5 µg of DNA and seeded on 18 mm diameter poly-L-lysine (Sigma) coated glass coverslips in 12 well plates.

METHOD DETAILS

Molecular biology and gene expression

Clones used were mTREK1 (RefSeq: NP_001153322), mTREK2 (NP_084187.2) and mTRESK (RefSeq: NP_997144). All channel DNA was used in the pIRES2eGFP vector.

Heterodimers between TREK1, TREK2 and TRESK were generated by PCR.

Electronic copy available at: https://ssrn.com/abstract=3850367 Electrophysiology HEK293T electrophysiology was performed 24 -48 h after transfection. For whole-cell patch-clamp experiments, cells were recorded in a bath solution containing (in mM) 145 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2 and 10 HEPES, pH 7.4. The glass pipettes (2-5 MΩ of resistance) were filled with (in mM) 140 KCl, 3 MgCl2, 5 EGTA, 10 HEPES, pH 7.3. ML67-33 was perfused at 10 µM for 2-5 min in the bath solution. Whole-cell currents were elicited by voltageramps (from -100 to +100 mV, 1 s) holding the cells at -80 mV. Current densities were measured at 0 mV.

For TG neuron electrophysiology, medium and small sized cells (< 25 µm) were recorded in an extracellular solution containing (in mM): 135 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 5 HEPES, 10 glucose, pH 7.4. In order to distinguish between peptidergic and non-peptidergic, neurons were first incubated with an isolectine-IB4 marker (Invitrogen) for 30 min at 5 µg/mL.

The pipette solution contained the following (in mM): 140 K-gluconate, 10 NaCl, 2 MgCl2, 5 EGTA, 10 HEPES, 2 ATP-Mg, 0.3 GTP-Na, 1 CaCl2. Neurons were excluded from analysis when the RMP was higher than -40 mV. To test neuronal excitability, neurons were held at RMP in current-clamp mode and injected with 1 s depolarizing current in 10 pA incremental steps. The voltage-clamp ramp protocol for TREK channels consisted in voltage-ramps from -25 to -135 mV during 600 ms, holding the cells at -60 mV. ML67-33 was perfused at 10 µM for 2-5 min in the extracellular solution.

All cells were recorded at room temperature using an Axopatch 200B (Molecular Devices) amplifier. Signals were filtered at 10 kHz and digitalized at 20 kHz. Cell recordings, data acquisition and analysis of electrophysiology were performed using pClamp software (Molecular Devices).

Migraine model rodents

The rodent models of NO-induced migraine were induced by i.p. injection of ISDN (Sanofi) at 10 mg/kg, a long-lasting NO donor in both rats and mice.

The mice hindpaw mechanical sensitivity was evaluated with a dynamic plantar aesthesiometer (Ugo Basile). Unrestrained mice were placed in twelve individual plastic boxes on top of a wire surface. The mouse hindpaw was subjected to an increasing force ramp (0 to 7.5 g in 10 s), the paw withdrawal force threshold (g) was measured in quadriplate on each hind Electronic copy available at: https://ssrn.com/abstract=3850367 paw, and the mean force was calculated. For 2 days, mice were habituated to repeated (every 30 min) measurements of hindpaw mechanical sensitivities, and basal values were determined 2 days before the experiments. ISDN was injected each day for 4 days, and the mechanical threshold was measured each day before the next ISDN injection to follow the settlement of chronic allodynia. Normal mechanical sensitivity and allodynia thresholds were taken as the measurement values of the first day before treatment with ISDN and the last measurement before the injection of drugs on wild-type mice, respectively. On the fifth day, BIBN4096 (Tocris) (1 mg/kg i.p.) and ML67-33 (1 mg/kg, i.p.) were administrated as well as their vehicle solution: 0.9% saline and 0.1% DMSO (Sigma). The hindpaw mechanical sensitivity was measured every 30 min before (basal value) and every 30 min after the i.p. injections.

The face mechanical sensitivity of rats was measured using calibrated von Frey filaments (Bioseb, France). Unrestrained rats placed in individual plastic boxes on top of a wire surface were trained over 1 week; a stimulus was applied to the periorbital area, following a progressive protocol, starting with non-noxious filaments during the first days of training. The face withdrawal force threshold (g) was determined by the filament evoking at least three responses over five trials, starting with lower force filaments. ISDN (10 mg/Kg, Sanofi) was injected each day for 4 days, and the mechanical threshold was measured each day before the next ISDN injection to follow the settlement of chronic allodynia. To test the effect of drugs, face mechanical sensitivity was measured every 30 min before (basal value) and for 3 h after compound or vehicle injection.

QUANTIFICATION AND STATISTICAL ANALYSIS

Analysis of whole-cell currents was performed using ClampFit software. For analysis of voltage-ramp traces cursors were set at 0 mV for HEK293T cells, to extract the average current. Cursors were set at 355 ms for TG neurons. Spike frequency was determined as the number of APs triggered in every current step. The rheobase was taken as the minimal current injected to generate a first AP. When neurons did not trigger any AP even for current injections >500 pA, the rheobase value was considered as 0 pA. Signals were filtered at 10 kHz and digitalized at 20 kHz. For electrophysiology and behavioral experiments, statistical details and n values can be found in the figure legends and in the main text.

Figure 1 .

 1 Figure 1. ML67-33 reverses NO induced migraine-like-phenotype through TREK channel activation. (A) Migraine behavioral test timetable. (B) Effect of repeated administration of systemic ISDN NO-donors (10 mg/kg i.p.) on the mechanical threshold (g) and the development of cutaneous allodynia related to migraine in wild-type and Trek1 -/--Trek2 -/-mice. Two-way ANOVA and Sidak post hoc vs wild-type (* p < 0.5, *** p < 0.001); One-way ANOVA and Dunett's post hoc vs pre-treatment day 1 (# p < 0.5, ### p < 0.001). (C) Time-course of cutaneous mechanical threshold (g) after injection of a vehicle solution (saline + DMSO 0.1% i.p.), BIBN4096 (1 mg/kg i.p.) and ML67-33 (1 mg/kg i.p.) in wild-type mice. Two-way ANOVA and Dunett's post hoc vs vehicle (* p < 0.5, ** p < 0.01, *** p < 0.005); One-way ANOVA and Dunett's post hoc vs time -0.5 h (# p < 0.5, ## p < 0.01, ### p < 0.005). (D) Bar graph representing mechanical threshold (g) of the three groups of animals in this study after 2h of the second injection of BIBN4096, vehicle and BIBN4096 respectively. Student's t-test (* p < 0.5, ** p < 0.01). (E-F) Same as C-D for Trek1 -/--Trek2 -/-mice.

Figure 2 .

 2 Figure 2. ML67-33 potentiates TREK channels in TG neurons. (A-D) Representative current traces of IB4 + and IB4 -neurons of small TG neurons obtained from wild-type (A and B) and Trek1 -/--Trek2 -/-(C and D) mice, before and during perfusion of ML67-33 (10 µM). Insets Bar graphs representing current densities at -25 mV (pA/pF). Currents were elicited by voltage-ramps (from -25 to -135 mV, 300 ms duration). Paired t-test (* p < 0.05, ** p < 0.01).

Figure 3 .

 3 Figure 3. ML67-33 reduces TG neuron excitability through TREK channels activation. (A-D) Input-output plots of spike frequency in response to 1-s depolarizing current injection in small IB4 + and IB4 -small TG neurons from wild-type (A and B) and Trek1 -/--Trek2 -/-(C and D) mice before and after perfusion with ML67-33 (10 µM). At right, Representative traces of

Figure 4 .

 4 Figure 4. ML67-33 abrogates mechanical facial allodynia related to migraine. (A) Migraine behavioral test timetable. Green arrows represent the injection of ISDN, a known migraine trigger. Magenta arrows represent the injection of either saline or BIBN4096 or ML67-33 solution injection. Blue arrows represent the measurement of the facial withdrawal mechanical threshold (B) Effect of repeated administration of systemic ISDN (10 mg/kg i.p.)

Figure S1 .

 S1 Figure S1. ML67-33 activates TREK1 homodimers and TRESK/TREK1 heterodimers but not TRESK homodimers, Related to Figure 2 and 3. (A-C) Representative current traces obtained from HEK293T cells expressing either TREK1 (A) or TRESK (B) homodimers or TRESK/TREK1 (C) heterodimers before and after application of ML67-33 (10 µM). Currents were elicited by voltage-ramps (from -100 to +100 mV from a holding potential at -80 mV, 1s duration). Insets show a summary of current densities obtained at 0 mV. Paired t-test (** p < 0.01). (D) Bar graph showing the fold increase after perfusion with ML67-33. Kruskal-Wallis and Student-Newman-Keuls post hoc (*** p < 0.001). Mean ± SEM.

Figure S2 .

 S2 Figure S2. ML67-33 increases rheobase of TG neurons through activation of TREK1 and TREK2 channels, Related to Figure 3. (A-D) Bar graphs showing representing rheobase obtained from IB4 + and IB4 -TG neurons obtained from wild-type (A and B) and Trek1 -/--Trek2 - /-(C and D) mice before and after perfusion with ML67-33 (10 µM). Rheobase was determined by incremental depolarizing current injections in steps of + 10 pA in small-diameter TG neurons. Paired t-test (** p < 0.01). Mean ± SEM.

Figure

  Figure S3. ML67-33 hyperpolarizes TG neurons from wild-type mice through activation of TREK channels, Related to Figure 3. (A-B) Bar graphs showing representing the RMP value obtained from small IB4 + and IB4 -TG neurons before and after perfusion with ML67-33 (10 µM) from wild-type (A) and Trek1 -/--Trek2 -/-(B) mice. Paired t-test (** p < 0.01, ** p < 0.001). Mean ± SEM.

Electronic copy available at: https://ssrn.com/abstract=3850367

ACKNOWLEDGMENTS

We thank Pr Daniel L. Minor for providing us the selective activator of TREK channels ML67-33. We thank the Dr Franck Chatelain and Dr Florian Lesage for providing us the TREK1 -/--TREK2 -/-mice. This work was supported by a grant to G.S. by the Fondation pour la Recherche Medicale (Equipe labellisée FRM 2017, FRM DEQ20170336753) the Agence Nationale pour la Recherche (AT2R-TRAAK-Bioanalgesics ANR-17-CE18-0001 and ANR-17-ERC2-0023), the Laboratory of Excellence "Ion Channel Science and Therapeutics" (grant ANR-11-LABX-0015-01) and the French government, through the UCAJEDI Investments in the Future project managed by the National Research Agency (ANR) with the reference number ANR-15-IDEX-01.

DECLARATION OF INTERESTS

The authors declare no competing interest Electronic copy available at: https://ssrn.com/abstract=3850367


Clément Verkest

Stephanie Häfner

Pablo Ávalos Prado

Anne Baron

Guillaume Sandoz
email: sandoz@unice.fr

Migraine and Two-Pore-Domain Potassium Channels

Keywords: Migraine, trigeminal sensory neurons, K 2P channels, KCNK, heterodimerization, frameshift mutation-induced alternative translation initiation (fsATI), allodynia, Kozac sequence, excitability

come

Introduction

Migraine is one of the most common diseases world-wide, affecting nearly 15% of the global population, with a women to men ratio of 3:1. Its societal and economic burden is considerable (Burch and others 2015). Migraine manifests as a unilateral throbbing headache and is accompanied by multiple symptoms such as nausea, vomiting, photophobia, phonophobia or cutaneous allodynia. The migraine attack is often split into four different phases following a temporal pattern that can last for several days: premonitory, aura, headache and postdrome phase (Goadsby and others 2017) (Fig. 1). The premonitory phase can begin as early as three days before the headache and is characterized by fatigue, mood changes, food craving and yawning [START_REF] Ashina | Erenumab (AMG 334) in episodic migraine: Interim analysis of an ongoing open-label study[END_REF]. Around less than one third of patients will develop an aura before the headache, which consists of visual and sensory disturbances that will last from several minutes to a few hours. The underlying event is thought to be a cortical spreading depression (CSD), a wave of brief depolarization followed by a long-lasting depression of neuronal activity that propagates slowly in the brain [START_REF] Charles | Cortical spreading depression and migraine[END_REF]. Then, the headache phase will start, lasting between 4 to 72 hours. The migraine attack resolves with a postdrome phase, lasting from hours to days, and consisting of residual headache, fatigue and impaired cognitive functions (Bose and others 2018). In addition to the presence or not of aura, the frequency of headaches is used to further differentiate episodic migraine (headache with or without aura that occurs 1 to 14 days per month, for at least three months) or chronic migraine patients (at least 15 days of headache per month for at least three months) [START_REF] Adams | The impact of chronic migraine: The Chronic Migraine Epidemiology and Outcomes (CaMEO) Study methods and baseline results[END_REF]. A broad array of symptoms contributes to the disability associated with migraine attacks and indicates involvement of brain regions in homeostatic, autonomic, emotional and sensory processing, thus highlighting a wide involvement of the nervous system in migraine (Vila-Pueyo 2018; [START_REF] Ashina | Erenumab (AMG 334) in episodic migraine: Interim analysis of an ongoing open-label study[END_REF]. Imaging studies have confirmed that these non-pain symptoms and changes in the brain are not simply responses to pain but can exist in the absence of headache and produce functional imaging changes on their own (Denuelle and others 2007;[START_REF] Schulte | Functional Neuroimaging in Migraine: Chances and Challenges[END_REF].

Therefore, migraine can be considered as a broad sensory disorder. Migraine patients display abnormal responses toward pain (Nahman-Averbuch and others 2019) or light [START_REF] Bernstein | The migraine eye: distinct rod-driven retinal pathways' response to dim light challenges the visual cortex hyperexcitability theory[END_REF]. Other differences lie on responses that should be adaptive but become impaired or maladaptive, such as altered brainstem processing (Vila-Pueyo and others 2019), changes in gray matter volume [START_REF] Coppola | Cerebral gray matter volume in patients with chronic migraine: correlations with clinical features[END_REF], impaired adaptive cerebral hemodynamic mechanisms (Frederiksen and others 2019), habituation deficiency [START_REF] Coppola | Somatosensory evoked high-frequency oscillations reflecting thalamo-cortical activity are decreased in migraine patients between attacks[END_REF] or metabolic impairment (Gross and others 2019). Due to the nature of the triggers reported by patients (stress, sleep disorder etc.) [START_REF] Karsan | Biological insights from the premonitory symptoms of migraine[END_REF][START_REF] Turner | Influences on headache trigger beliefs and perceptions[END_REF] and a clear involvement of the hypothalamus) in the early phases of the attack, migraine is considered as a deregulated response toward various stressful stimuli, highlighting the difficulty of the brain to keep a physiological and stable state. Thus, migraine has been described as a pathophysiology of allostasis (i.e. the responses that help to maintain physiological stability) (Borsook and others 2012). Despite the broad impact of migraine, the understanding of several aspects of its pathophysiology and underlying mechanisms remain unclear.

Initiation of migraine attacks: triggering factors and genetic aspects

Triggering factors

Among various external factors able to trigger migraine attacks, stress is the most reported [START_REF] Turner | Influences on headache trigger beliefs and perceptions[END_REF]. In clinical and preclinical context, methods for inducing a migraine attack include the injection of triggering molecules, like the calcitonin gene-related peptide (CGRP), for which elevated levels have been measured in patients during migraine attacks (Edvinsson and others 2018), nitric oxide (NO) donors or pituitary adenylate-cyclase activating peptide (PACAP) (Al-Karagholi and others 2019; Ashina and others 2017b;Ashina and others 2017a;[START_REF] De Felice | Triptan-induced latent sensitization: a possible basis for medication overuse headache[END_REF][START_REF] De Felice | Opiate-induced persistent pronociceptive trigeminal neural adaptations: potential relevance to opiate-induced medication overuse headache[END_REF]Guo and others 2016;[START_REF] Liu | Functional analysis of a migraineassociated TRESK K+ channel mutation[END_REF]. However, NO donors, for example, are triggers in migraine patients but poorly affect healthy volunteers (Schytz and others 2010). Nevertheless, in certain experimental conditions (doses and combined use of provocative drugs, duration of treatment), a higher proportion of healthy individuals may develop a genuine migraine attack, suggesting the existence of a putative "migraine threshold" [START_REF] Olesen | Can nitric oxide induce migraine in normal individuals?[END_REF].

Genetic aspects

Early studies on the genetics of migraine have led to the identification of several genes responsible for rare cases of familial hemiplegic migraine (FHM). This includes FHM1, a gain-of-function mutation affecting the alpha subunit of the voltage-gated calcium channel Ca v 2.1 (CACNA1A) leading to an increase in synaptic transmission (van den Maagdenberg and others 2004), FHM2, a loss-of-function mutation targeting the alpha 2 subunit of the astrocytic Na + /K + ATP-dependent pump (ATP1A2) and resulting in an impaired clearance of synaptic glutamate [START_REF] Fusco | Haploinsufficiency of ATP1A2 encoding the Na+/K+ pump alpha2 subunit associated with familial hemiplegic migraine type 2[END_REF] and FHM3, impairing the alpha subunit of the voltage-gated sodium channel Na v 1.1 (SCN1A) [START_REF] Dichgans | Mutation in the neuronal voltage-gated sodium channel SCN1A in familial hemiplegic migraine[END_REF]Zerimech and others 2020). However, those mutations are also linked to other diseases such as dyskinesia, ataxia or mental retardation. Furthermore, they only account to a minor proportion of migraine patients (around 1 of 10000) [START_REF] Ducros | Genetics of migraine[END_REF]. Another gene whose loss of function has been recently associated with migraine is KCNK18, which encodes the Two-Pore-Domain (K 2P ) potassium channel TRESK (Twik-related spinal cord K + channel) [START_REF] Lafrenière | A dominant-negative mutation in the TRESK potassium channel is linked to familial migraine with aura[END_REF]. The mutation in this gene was the only one which has been linked to a "common" migraine phenotype. Nevertheless, the latter was a matter of controversy until recently [START_REF] Royal | Migraine-Associated TRESK Mutations Increase Neuronal Excitability through Alternative Translation Initiation and Inhibition of TREK[END_REF]Pettingill and others 2019, see below).

Due to their contribution to neuronal excitability, a genomic study has explored the potential involvement of hundreds of ion transport genes in migraine pathology [START_REF] Nyholt | A high-density association screen of 155 ion transport genes for involvement with common migraine[END_REF]. However, a significant gene variant has not been identified. In the last years, larger scale genome-wide association studies (GWAS) have made important contributions to identify genetic components involved in migraine, resulting in new hypotheses regarding migraine pathophysiology (Nyholt and others 2017). Further studies provided evidence that common types of migraine are genetically complex: multiple genetic variants with small effect sizes act together with environmental factors to confer migraine susceptibility [START_REF] Gormley | Common Variant Burden Contributes to the Familial Aggregation of Migraine in 1,589 Families[END_REF]. Interestingly, those studies have identified other variants in genes encoding K 2P channels, namely KCNK5 (TASK2, Twik-related acid sensing K + channel) (Gormley and others 2016) and KCNK4 (TRAAK, Twik-related arachidonic acid stimulated K + channel). It is also of note that migraine has a genetic overlap with psychiatric disorders, especially depression, in which K 2P channels have been also shown to play a role [START_REF] Heurteaux | Deletion of the background potassium channel TREK-1 results in a depression-resistant phenotype[END_REF]Djillani and others 2019).

Anatomy -Focus on the migraine pain pathway

Multiple sensory symptoms arise during a migraine attack, being the headache the most prominent one. It is now well established that migraine headache is the result of the activation and sensitization (decreasing response threshold and increasing response strengths) of trigeminal neurons and, to a lesser extent, cervical nociceptive C (non-myelinated) and Aδ (myelinated) fibers that innervate cranial meninges and their blood vessels [START_REF] Khan | Meningeal contribution to migraine pain: a magnetic resonance angiography study[END_REF]Strassman and others 1996). This ensemble is commonly referred to the trigeminovascular system (Fig. 2). The pathway starts in trigeminal sensory neurons whose cell bodies are located in the trigeminal ganglia (TG) and send peripheral axons to the meningeal dura mater and the surrounding blood vessels through the ophthalmic branch of the trigeminal nerve (V1). Central axons from TG neurons project to the trigeminal nucleus caudalis (TNC) (Uddman and others 1985). This nucleus integrates signals from projections coming from periorbital skin and pericranial muscles. The ascending axon projections of TNC transmit nociceptive information to diverse hypothalamic, brainstem and basal ganglia nuclei that could explain different migraine related symptoms such as vomiting, nausea and yawning (Burstein and others 2015). Additionally, trigeminovascular nociceptive information is subject to extensive modulation by multiple descending neurons from different brain regions [START_REF] Ashina | Erenumab (AMG 334) in episodic migraine: Interim analysis of an ongoing open-label study[END_REF], including various brainstem nuclei, notably the Locus coeruleus and Periaqueductal Grey [START_REF] Edelmayer | Medullary pain facilitating neurons mediate allodynia in headache-related pain[END_REF]Vila-Pueyo and others 2019), the hypothalamus [START_REF] Ashina | Erenumab (AMG 334) in episodic migraine: Interim analysis of an ongoing open-label study[END_REF] or the cerebral cortex (Noseda and others 2010).

Activation of trigeminal and innervating fibers in meninges is thought to initiate a neurogenic and sterile inflammation, owing to the dense population of various vasoactive neuropeptides (i.e. CGRP, PACAP and substance P) within sensory neurons and to the proximity of blood vessels, immune cells (Edvinsson and others 2018;Levy and others 2019;[START_REF] Rua | Advances in Meningeal Immunity[END_REF] and autonomic neuron fibers [START_REF] Harriott | Contribution of primary afferent channels to neuropathic pain[END_REF]. Meningeal sensory neurons are equipped with a rich set of receptors and ion channels, allowing them to be exquisitely sensitive and to respond to a broad range of chemical and mechanical stimuli (Burgos-Vega and others 2016; Levy and others 2019; Bonnet and others 2019). There is now growing evidence that an inflammatory component might sustain nociceptor activation and sensitization, through the action of protons, cytokines or neurotrophic factors and their downstream targets (van Dongen and others 2017).

However, the initiating event leading to the activation of meningeal sensory neurons in the context of a migraine attack remains unclear. Regarding migraine with aura, CSD was shown to activate meningeal nociceptors, being also linked to a meningeal inflammation (Bolay and others 2002;[START_REF] Karatas | Spreading depression triggers headache by activating neuronal Panx1 channels[END_REF][START_REF] Hadjikhani | Extra-axial inflammatory signal in parameninges in migraine with visual aura[END_REF]. A more likely explanation combines extensive modulation of trigeminovascular information, predetermined hyperexcitable migraine brain and increase of nociceptive information by external triggering factors (Kunkler and others 2018;[START_REF] Bree | Development of CGRP-dependent pain and headache related behaviours in a rat model of concussion: Implications for mechanisms of post-traumatic headache[END_REF]. Nowadays, it is well-established that trigeminal neuron hyperexcitability is a key factor for migraine headache generation and some associated symptoms (throbbing headache, cutaneous allodynia, some autonomic symptoms) through its role in the development of peripheral and central sensitization at various levels of the migraine pain network (Strassman and others 1996;Olesen and others 2009).

K 2P channels and pain

K 2P channels constitute one of the major families of K + channels. Its members share a dimeric structure with four transmembrane domains and two pore loops per subunit. Because of the two pore loops per subunit, these channels are not active as tetramer but as dimer (Fig. 3). K 2P channels exhibit a "background" K + conductance that is time-and voltageindependent and brings the resting membrane potential of cells toward the K + potential equilibrium. Therefore, they serve as a hub for the regulation of neuronal excitability by setting the resting membrane potential below the firing threshold in excitable cells. Lafrenière and others 2010). This group mainly includes TWIK1, the members of the TREK subfamily (TREK1, TREK2 and TRAAK), the TASK subfamily members (TASK1 and TASK3) and TRESK [START_REF] Li | Role of leak potassium channels in pain signaling[END_REF]. They are all expressed throughout the nervous system along the pain pathway in various populations of dorsal root ganglia (DRG) or TG sensory neurons. It is worth to note that they are enriched in small and unmyelinated neurons that are mainly involved in noxious stimuli detection (e.g. peptidergic, non-peptidergic, itch or cold-sensitive) (Acosta and others 2014;[START_REF] Alloui | TREK-1, a K+ channel involved in polymodal pain perception[END_REF][START_REF] Pereira | Role of the TREK2 potassium channel in cold and warm thermosensation and in pain perception[END_REF].

K 2P channels and migraine

With the identification of the FHM genes in the late 90s and in the mid-2000s, a subsequent large-scale study was conducted by Nyholt and colleagues investigating the potential contribution to migraine pathology of common variations affecting several ion transport-related genes. Though none of the studied variants reached a statistical threshold, two K 2P channels (TREK1 and THIK2) were preliminarily selected as promising candidates in a Finnish cohort [START_REF] Nyholt | A high-density association screen of 155 ion transport genes for involvement with common migraine[END_REF]. A pioneer study clearly linking K 2P channels and migraine led to the identification of a frameshift mutation in the gene encoding for TRESK channel, the F139WfsX24 mutation (TRESK-MT). This mutation, segregating perfectly with migraine with aura phenotype (and sometime without aura), was shown to produce a non-functional protein that served as a dominant-negative form of the wild-type (WT) TRESK channel (Lafrenière and others 2010) explaining its genetic dominant role.

TRESK-MT has been shown to induce hyperexcitability of TG neurons (Liu and others 2013;

Guo and others 2014), which likely underscores its role in migraine. Furthermore, the causative role of this mutation was emphasized by an elegant study with human nociceptive neurons derived from induced pluripotent stem cells from migraine patients. In fact, these neurons are hyperexcitable and CRISPR-Cas9 engineering, for correcting the F139WfsX24 mutation, reversed the neuronal hyperexcitability [START_REF] Pettingill | A causal role for TRESK loss of function in migraine mechanisms[END_REF]. However, in a subsequent genetic screening study, another missense TRESK variant, C110R, was identified (Andres-Enguix and others 2012). The TRESK-C110R mutation, similarly to TRESK-MT, was shown to produce a non-functional channel which exerts a dominant negative effect on the WT-TRESK channel in heterologous cells, but unexpectedly this mutant was found to have no effect on TG excitability when overexpressed (Guo and others 2014) and in human derived C110R mutant neurons [START_REF] Pettingill | A causal role for TRESK loss of function in migraine mechanisms[END_REF]. This absence of effect elucidates why this mutant was found in both migraine and control subjects. We have recently shown in rodent that, even though both mutations lead to the same apparent deleterious effect on TRESK function, only TRESK-MT is able to functionally inhibit two other members of the K 2P channel family (Fig. 5). Inhibition of TREK1 and TREK2 channels activity then induces TG hyperexcitability and subsequent migraine pathophysiology (Fig. 6) (Royal and others 2019; see below).

K 2P channels, heteromerization and TG excitability

Even though K 2P channels share a low level of sequence identity, it has been recently shown that heteromerization is not rare between K 2P members, increasing functional diversity [START_REF] Levitz | Heterodimerization within the TREK channel subfamily produces a diverse family of highly regulated potassium channels[END_REF][START_REF] Blin | Mixing and matching TREK/TRAAK subunits generate heterodimeric K2P channels with unique properties[END_REF]others 2016). Heteroassembly can occur within the same subfamily but also between different subfamily members, as was shown for the dimerization of TRESK with TREK1 and TREK2 in TG neurons [START_REF] Royal | Migraine-Associated TRESK Mutations Increase Neuronal Excitability through Alternative Translation Initiation and Inhibition of TREK[END_REF]. The question, why only TRESK-MT, and not TRESK-C110R leads to an inhibition of TREK1 and TREK2, was solved by the discovery of a new mechanism allowing transmission of inherited diseases through an alternative translation initiation induced by a frameshift mutation in TRESK-MT called frameshift mutation induced Alternative Translation Initiation (fsATI, Box 2; Royal and others 2019).

The TRESK-MT mutation leads, through fsATI, to the production of two protein fragments instead of one, as expected (Box 1): TRESK-MT1, which corresponds to the expected C-terminal truncated TRESK channel and inhibits TRESK, and TRESK-MT2, which corresponds to an N-terminal truncated TRESK channel. TRESK-MT2, by coassembling and inhibiting TREK1 and TREK2, induces TG sensory neuron hyperexcitability (Fig. 5). Overexpression of TRESK-MT2 in rat TG neurons, using virus, is sufficient to induce a chronic facial mechanical allodynia related to chronic migraine, an effect which has not been seen with MT1 (Royal and others 2019). Interestingly, fsATI was also found for another TRESK mutant (Y121LfsX44), which is the only other TRESK mutation related to migraine in human [START_REF] Royal | Migraine-Associated TRESK Mutations Increase Neuronal Excitability through Alternative Translation Initiation and Inhibition of TREK[END_REF]Domitrz and others 2016;[START_REF] Rainero | KCNK18 (TRESK) genetic variants in Italian patients with migraine[END_REF]. This mutation also leads to the production of a similar TRESK-MT2 [START_REF] Royal | Migraine-Associated TRESK Mutations Increase Neuronal Excitability through Alternative Translation Initiation and Inhibition of TREK[END_REF]. The fundamental role of TREK1 and TREK2 in migraine induction has been further supported by TREK1/TREK2 double knock-out (KO) mice (Fig. 5). They exhibit a chronic migraine-like cutaneous mechanical allodynia phenotype which can be reversed by Topiramate, a drug used in clinic to treat migraine [START_REF] Royal | Migraine-Associated TRESK Mutations Increase Neuronal Excitability through Alternative Translation Initiation and Inhibition of TREK[END_REF]. This suggests, that, in human, migraine is due to the dysfunction of TREK1 and TREK2 and their respective heteromers with mutated TRESK, but not due to TRESK invalidation alone (Fig. 6).

In parallel, other preclinical studies in rodents have further confirmed a key involvement of K 2P channels in trigeminal nociception and migraine-related headache.

Recently, TRESK invalidation was shown to increase peptidergic trigeminal neurons excitability (Guo and others 2019) and to enhance headache behavior induced by application of inflammatory mediators in the meninges [START_REF] Pettingill | A causal role for TRESK loss of function in migraine mechanisms[END_REF]. These results would favor that TRESK is sufficient to generate migraine. Nevertheless, the finding by Guo and others. and Pettingill and others. also fits with TREK1 and TREK2 implication. In fact, by only targeting TRESK, TRESK-MT1 and C110R, the sensory neuron excitability is not altered (Pettingill and others 2019; Guo and others 2014; Royal and others 2019), whereas genetic invalidation affects TRESK homodimers as well as heterodimers (TRESK-TREK1, TRESK-TREK2) and therefore also TREK1 and TREK2 expression and composition at the cell surface (the equilibrium is modified). These differences between gene invalidation and dominant negative (affecting only TRESK) may explain why TRESK invalidation modifies TG excitability and induces a migraine phenotype. All in all, migraine-like behavior in rodents seems to involve both TRESK, TREK1 and TREK2 monomeric channels and TRESK-TREK heterodimeric channels.

Finally, in addition to these channels, TASK3 is also predominant in TG neurons, where it co-localizes with TRESK, TREK1 and TREK2 (Yamamoto and others 2009).

Nevertheless, there is no evidence of TASK3 participation in migraine attacks. It is worth to mention, that TASK3 was not found to heteromerize with TRESK, TREK1 and TREK2 [START_REF] Levitz | Heterodimerization within the TREK channel subfamily produces a diverse family of highly regulated potassium channels[END_REF][START_REF] Royal | Migraine-Associated TRESK Mutations Increase Neuronal Excitability through Alternative Translation Initiation and Inhibition of TREK[END_REF].

Toward new treatments for migraine involving K 2P channels targeting Migraine therapy aims to terminate or to relief acute attacks, but also to prevent the evolution from episodic toward chronic migraine. Several drugs are used but present several side effects (Table1 and 2), for example, Topiramate may be used to treat chronic migraine but it has severe well-known side effects including ataxia, confusion, diplopia, dizziness, drowsiness, dysphasia, fatigue, memory impairment and others. Given the importance of CGRP as a migraine trigger, the use of monoclonal antibodies against this peptide represents one of the most effective treatments against the disease. Up to now, three different humanized monoclonal antibodies against CGRP pathway have been tested and exhibited excellent results: Fremanezumab and Galcanezumab, which target CGPR, and Erenumab, which targets CGRP receptors (Fig. 7). All three products have demonstrated efficacy for the preventive treatment of chronic and episodic migraine and can be periodically self-administered [START_REF] Spindler | Recent Medications Approved for Preventing Migraine Headaches[END_REF]. In addition, they support the fact that an intervention at the level of the peripheral nervous system is sufficient to target migraine headache. Nevertheless, general side effects caused by monoclonal antibody drugs cannot be excluded such as flu-like symptoms and allergic reactions in addition to the "specific" side effects such as dizziness, joint pain or pruritus and erythema.

Given their abundant expression in DRG, TG sensory and primary afferent neurons, drugs targeting K 2P channels constitute a promising therapy for the treatment of migraine and its associated pain, but also of inflammatory, neuropathic or cancer pains [START_REF] Pereira | Role of the TREK2 potassium channel in cold and warm thermosensation and in pain perception[END_REF][START_REF] Noël | The mechanoactivated K+ channels TRAAK and TREK-1 control both warm and cold perception[END_REF][START_REF] Alloui | TREK-1, a K+ channel involved in polymodal pain perception[END_REF][START_REF] Descoeur | Oxaliplatin-induced cold hypersensitivity is due to remodelling of ion channel expression in nociceptors[END_REF]. This idea is strengthened by the fact that K 2P channels have been shown to be involved in other diseases such as depression [START_REF] Heurteaux | Deletion of the background potassium channel TREK-1 results in a depression-resistant phenotype[END_REF] which severely affect 10% of the population suffering from migraine disorders, and that some drugs targeting K 2P channels have been proved to be effective against migraine, such as cloxyquin through TRESK activation [START_REF] Pettingill | A causal role for TRESK loss of function in migraine mechanisms[END_REF].

As previously shown, at least three K 2P channels subunits and their respective heteromers have been proved to be involved in the normal physiology and pathophysiology of migraine. TRESK, TREK1 and TREK2 containing channels seem to serve as a hub to set the negative resting potential of TG neurons [START_REF] Dobler | TRESK twopore-domain K+ channels constitute a significant component of background potassium currents in murine dorsal root ganglion neurones[END_REF][START_REF] Lafrenière | A dominant-negative mutation in the TRESK potassium channel is linked to familial migraine with aura[END_REF][START_REF] Royal | Migraine-Associated TRESK Mutations Increase Neuronal Excitability through Alternative Translation Initiation and Inhibition of TREK[END_REF]. Thus, specific pharmacology targeting these channels should be potentially relevant for the development of anti-migraine treatment. Furthermore, targeting heterodimers would allow to reach higher specificity. Currently, only a small number of agonists of the aforementioned channels have been studied to be effective for migraine treatment. Among them, the anti-leukotriene agent Montelukast for asthma management has been demonstrated to act as a well-tolerated and prophylactic agent against acute migraine episodes at 10-20 mg dose per day (Sheftell and others 2000). Its efficacy may lie in its ability to specifically activate TRESK and TRESK heteromer channels (Wright and others 2019).

During migraine, experimental studies have demonstrated an increase of prostaglandin 2 (PGE2) in the trigeminovascular system, where their receptors are distributed along TG and TNC neurons and induce vasodilation of cranial vessels (Antonova and others 2013). PGE2 is also known to be a potent inhibitor of TREK1 channels [START_REF] Honoré | The neuronal background K2P channels: focus on TREK1[END_REF]. Non-steroidal antiinflammatory drugs (NSAIDs), which represent another widespread treatment to manage headache in acute migraine, may eventually avoid TREK1 inhibition.

Finally, although they have not yet been clinically tested, a number of selective activators of K 2P channels should also be considered for chronic migraine prevention. Among them, it is worth to mention ML67-33, a well-known specific activator of TREK1, TREK2 and TRAAK channels, that exhibit an EC 50 in micromolar range [START_REF] Bagriantsev | A highthroughput functional screen identifies small molecule regulators of temperature-and mechano-sensitive K2P channels[END_REF]. Further development of selective agents targeting specific heterodimers involving TRESK may be considered to increase specificity and to reduce secondary drug effects.

Concluding remarks

Migraine is a common and disabling disease associated with disorders in neuronal excitability and cerebral blood flow. The complexity of the underlying processes which are not yet fully understood still render migraine treatment difficult. Currently, it is assumed that migraine headache is triggered by TG neuron activation. Hyperexcitability of TG fibers evokes release of vasoactive neuropeptides that enhance vasodilation causing neurogenic inflammation. This leads to a pain response to be conveyed to higher brain centers including cortex, hypothalamic, brainstem and basal ganglia nuclei. The mechanisms leading to trigeminal hyperexcitability have not been fully elucidated until very recently. K 2P channels, namely homo-and heterodimers of TRESK, TREK1 and TREK2, seem to play a key role in TG neuronal excitability and are therefore promising targets for migraine treatment. The discovery of a new type of translation initiation, generating several protein forms that can regulate other channels by heteromerization, clarified the understanding of K 2P channel implication in this disease. Henceforth, the novel role of K 2P channels in migraine, as well as their ability to generate functional and non-functional heterodimers, provides motivation for developing a deeper understanding of their function and organization within the plasma membrane and demonstrates that they have to be now taken into consideration for the development of highly specific pharmacological agents to treat migraine. Heteromultimerization is a classical mechanism commonly used to increase the diversity of protein complexes. Despite the fact that K 2P channels share rather low sequence identity, it has been demonstrated that heteromerization is possible between different K 2P members. The first hints that pointed to the general possibility of a heteroassembly of K 2P members were reported in 2002 [START_REF] Czirják | Formation of Functional Heterodimers between the TASK-1 and TASK-3 Two-pore Domain Potassium Channel Subunits[END_REF] when co-expression of TASK1 and TASK3 in Xenopus oocytes led to functional channels with intermediate channel properties. After, the existence of native TASK1/TASK3 dimers was proven in mammalian motoneurons (Berg and others 2004). Later, it was shown that these channels also dimerize Box 2 -Frameshift-mutation induced alternative translation initiation

The TRESK-MT mutation, induced by the frameshift F139WfsX24, is expected to encode for a non-functional channel, as it is truncated by its C-terminal part. The WT-TRESK channel was shown to interact with TREK1 and TREK2 in SiMPull assays, whereas the TRESK-MT channel was not [START_REF] Royal | Migraine-Associated TRESK Mutations Increase Neuronal Excitability through Alternative Translation Initiation and Inhibition of TREK[END_REF]. If the inhibition of the TREK1/2 current by co-expression of TRESK-MT had not its origin in a co-assembly with this truncated channel form, there must be another explanation.

It has been assumed for a long time that one eukaryotic mRNA leads to the production of one single protein and that the variety of protein isoforms derived from one gene is evoked by alternative splicing or transcription from alternative promoters. The dogma of the existence of a unique translation start site was rebutted by Marilyn Kozak, who described that in certain cases, the rule of one mRNA -one protein is not followed as strictly [START_REF] Kozak | Initiation of translation in prokaryotes and eukaryotes[END_REF]. In fact, ribosomes can recognize an alternative initiation site and induce the generation of a second protein coming from the same RNA.

The canonical translation usually starts by binding of the 40S ribosomal subunit, initiator tRNA and several initiating factors (e.g. elF4E) at the 5' cap structure, a 7methylguanosine (m 7 G) which precedes all eukaryotic mRNAs. The formation of this socalled pre-initiation complex is followed by linear scanning of the mRNA for an AUG initiation codon that is located in the right context which is in most eukaryotic mRNAs given by the Kozak sequence (A/G-XX-ATGG [START_REF] Kozak | An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs[END_REF]. Subsequent binding of the 60S ribosomal subunit to the translation initiation site (TIS) then initiates the protein translation.

However, if the context of the first AUG on the mRNA is not optimal (i.e. not fully matching the Kozak sequence), the codon might be skipped, and the translation will start at a downstream AUG which has a more optimal context. This mechanism is also referred to as leaky scanning [START_REF] Kochetov | Alternative translation start sites and hidden coding potential of eukaryotic mRNAs[END_REF].

Such mechanism increases the diversity of proteins derived from one gene and has been already nicely described for K 2P channels in 2008 (Thomas and others 2008). In this study, an alternative translation initiation site in the TREK1 channel expressed in rat brain has

Furthermore

  , they were recently shown to contribute to neuronal high firing frequency and high-speed conduction along myelinated axons by driving action potential repolarization (Brohawn and others 2019; Kanda and others 2019; Ávalos Prado and Sandoz 2019). 15 genes have been found to encode for this channel family that can be subdivided into six subfamilies depending of their sequence homology and biophysical properties (Box 1). K 2P channels play a central and active role in the response of cells to extracellular and intracellular signals as diverse as G-Protein Coupled Receptor (GPCR) signaling, pH and membrane stretch. They have been shown to participate in multiple physiological and pathological aspects of peripheral pain perception. Notably, they have been implicated in thermal and mechanical nociception (Fig. 4, Noël and others 2009) and also in inflammatory, neuropathic, cancer, and recently, migraine pain (Alloui and others 2006; Descoeur and others 2011;

  with a member of a different subfamily, namely TWIK1(Plant and others 2012), and several studies reported potential dimers of TWIK1/TREK1 (Mi Hwang and others 2014), THIK1/THIK2 (Blin and others 2014), TREK1/TREK2/TRAAK[START_REF] Blin | Mixing and matching TREK/TRAAK subunits generate heterodimeric K2P channels with unique properties[END_REF][START_REF] Levitz | Heterodimerization within the TREK channel subfamily produces a diverse family of highly regulated potassium channels[END_REF], TASK1/TALK2 (Suzuki and others 2017) and finally, TREK1/TREK2/TRESK[START_REF] Royal | Migraine-Associated TRESK Mutations Increase Neuronal Excitability through Alternative Translation Initiation and Inhibition of TREK[END_REF]. Nevertheless, not all K 2P subunits of the K 2P family are able to dimerize to form functional channels, but the compilation of a full K 2P interactome is impeded by the great variety of channel properties as well as the absence of specific pharmacology. The toolbox to study K 2P channel dimerization ranges from electrophysiology recordings of co-expressed or tandem-linked channels to fluorescence resonance energy transfer (FRET) assays or fluorescence imaging of co-localization experiments. The use of such various technical approaches has led to controversies in the field, for example for the TREK1-TWIK1 dimers, which has been described as the main potassium conductance in astrocytes[START_REF] Hwang | A disulphide-linked heterodimer of TWIK-1 and TREK-1 mediates passive conductance in astrocytes[END_REF], whereas this dimer was shown not to exist in a previous study[START_REF] Plant | A Role for K2P Channels in the Operation of Somatosensory Nociceptors[END_REF]. To address the question of protein assembly, a new technology allowing direct visualization at single molecule level has been developed, the so-called Single Molecule Pulldown (SiMPull) assay (Jain and others 2011), based on antibody-mediated immunoprecipitation of protein subunits carrying an affinity tag (e.g. HA) on passivated coverslips (Fig.B1-1). These proteins serve as bait molecules which can capture their respective fluorescence tagged protein partners. The visualization of this interaction is then carried out by total internal fluorescence (TIRF) microscopy. The combination of biochemical immunoprecipitation with single molecule fluorescence microscopy allows to systematically test all K 2P interactions among the 120 that can be expected from the combination of the 15 members of the K 2P channel family (Fig.B1-2).

Figure B1- 1 :

 1 Figure B1-1: Principle of the Single Molecule Pulldown assay. (A) To study the interaction of two proteins of interest, a protein with a fluorescence tag like the green fluorescent protein (GFP) is co-expressed with a second protein exhibiting an affinity tag like HA. (B) After lysis of the cells, the lysate is applied in flow chambers on passivated coverslips containing surface tethered antibodies. (C) The presence of GFP-tagged proteins allows the visualization of single protein complexes by TIRF microscopy and subunit counting by observation of bleaching events.

Figure B1- 2 :

 2 Figure B1-2: Interactome study of K 2P channels in TG neurons. Green lines indicate a confirmed interaction between the respective subunits, while red lines connect subunits which are not interacting. The dotted line indicates the controversial interaction between TREK1 and TWIK1.

  been found to lead to a second truncated channel isoform missing the first 56 amino acids. This Δ1-56 TREK1 channel is able to form functional homodimers as pseudo-heterodimers with the full-length protein which leads to various channel isoforms with alternate open probability, conductance and permeability properties. Here, the second AUG was embedded in a stronger Kozak sequence which is recognized more efficiently as the first AUG. The study impressively demonstrated, how leaky scanning of ion channel genes like KCNK2 can effectively contribute to the regulation of CNS excitability.

Figure B2- 1 :

 1 Figure B2-1: Two common examples of eukaryotic translation initiation. (A) In the linear scanning model, the 40S ribosomal subunit binds to the 5' m 7 G cap followed by subsequent scanning of the mRNA for an AUG codon, ideally embedded in a Kozak sequence (A/G-XX-AUGG), which serves as TIS. After assembly with the 60S ribosomal subunit, translation is initiated. (B) If the first scanned AUG is embedded in a suboptimal Kozak sequence (Y-XX-AUGZ, Y and Z representingKozak-deviant bases), it is not efficiently recognized, and the scanning will proceed until the next suitable TIS is detected.

Figure B2- 2 :FiguresFigure 1 .

 21 Figure B2-2: Principle of the frameshift-induced alternative translation initiation. (A) Organization of ORF1 and ORF2 on mRNA of WT-TRESK. (B) The deletion of two base pairs by the c.410_411delCT mutation results to a premature translation stop in ORF1(TRESK-MT1). Coincidentally, the start codon of ORF2 at +356 is put in frame with the ORF of TRESK, inducing the translation of MT2.

Figure 2 .

 2 Figure 2. Migraine pathophysiology: Anatomy of the trigeminovascular pathway. The migraine attack begins in trigeminal ganglion (TG), whose axons (red lines) project to meningeal dura mater blood vessels through the ophthalmic branch stimulating the production of vasoactive neuropeptides. Nociceptive information is then carried from meninges to the rest of the brain though sensory and autonomic neurons. A second group of axons from TG targets the trigeminal nucleus caudalis(TNC), which also receives information from anterior and posterior parts of head and upper neck (red lines). TNC conveys then the inputs to diverse nuclei at limbic system, basal ganglia, hypothalamus, cortex and thalamus (blue lines). The thalamus also integrates stimuli detected from the outside and from internal organs (green lines). Additional nuclei are believed to contribute to migraine headache. Activation of the sphenopalatine ganglion (SPG) (orange lines) may stimulate antidromic sensory fibers converging to the superior salivatory nucleus (SUS), which contains cell bodies involved in the parasympathetic autonomic vasodilator pathway.

Figure 3 .

 3 Figure 3. Membrane topology of Potassium channels. Voltage-gated channels (red) possess six transmembrane domains and one pore domain (P). Some of them also present a seventh transmembrane segment. The inward rectifier channels (green) consist of two transmembrane domains and one pore domain (P), like the voltage-dependent channels they are active as tetramers. The two-pore-domain potassium channels (blue) possess four transmembrane domains and two pore domains (P1 and P2) and are active as dimers.

Figure 4 .

 4 Figure 4. TREK channel members are involved in several polymodal ways of pain perception. Thermal and mechanical stimuli are detected by several TREK channels expressed in nociceptors (along with other ASIC, TRP and piezo channels…). They are primary sensors of a nervous signal that is sent to the cortex where it is integrated as a pain signal. DRG for dorsal root ganglion.

Figure 5 .

 5 Figure 5. TREK1 -/-/TREK2 -/-double knockout animals present a migraine-like phenotype. (A) Schematic of experimental behavioral paradigms. Green arrows represent the injection of ISDN, an established migraine trigger. Blue arrows represent measurements of mechanical sensitivity. (B) Bar graph representing the paw withdrawal mechanical thresholds assessed 1.5 hours after ISDN injection. The WT mice present a significant decreased paw withdrawal, whereas no modification was observed for double knockout animals (T1 -/-/T2 -/-). (C) Mechanical responses, assessed prior to and after chronic ISDN injection (4 days), were significantly decreased in WT animals, whereas no significant change of allodynia was observed in T1 -/-/T2 -/-animals. (D) Variation ΔThreshold (g) of the paw withdrawal mechanical threshold induced by ISDN chronic treatment or topiramate injection. Mechanical responses were assessed before and after chronic ISDN treatment (4 days, left bars), and before and two hours after topiramate injection (right bars) in ISDN-non treated WT and T1 -/-/T2 -/-mice (from Royal and others 2019).

Figure 6 :

 6 Figure 6: TRESK-MT causes migraine via inhibition of TREK1 and TREK2 by TRESK-MT2. Top: TRESKC110R targets only TRESK, which is not sufficient to induce an increase of trigeminal sensory neuron excitability and therefore does lead to migraine. Middle: the excitability of trigeminal neurons in the presence of the wild version of TRESK is normal. Bottom: the TRESK-MT mutation generates an ATI (see Box1) resulting in the formation of two proteins, TRESK-MT1 and TRESK-MT2. TRESK-MT1 inhibits TRESK, while TRESK-MT2 targets TREK1 and TREK2. Inhibition of TREK1 and TREK2 leads to neuronal hyperexcitability causing a migraine phenotype.

Figure 7 .

 7 Figure 7. Main targets of currently used and potential future drugs for treating migraine headache. The analgesic action is mainly exerted by influencing several classes of receptors (ion channels, GPCRs etc.) at multiple levels of the migraine pain pathway (peripheral trigeminal sensory neurons, TNC second order and higher neurons in the brain). They are principally acting on the neuronal hyperexcitability and the sensitization associated to migraine headache. Multiple drugs will aim at limiting the excessive depolarization through the blockade of inward cation flux (e.g antiepileptics) or, on the opposite, through the activation of hyperpolarizing potassium channels (e.g potential K 2P agonists). Other classes act on the neuronal modulation (e.g CGRP antibodies) or neurotransmitters release (e.g Triptans).

Table 1 . Current preventive treatments of migraine, their targets and the proposed 1 mechanism of action.

 1

	2
	Preventive treatment	Targets	Mechanism of action
	Beta blockers (Propranolol)	β1-2 receptor antagonist	Stress reduction, neuronal modulation (descending controls), vascular effects?
		Valproate: Na v antagonist	Decrease in neuronal excitation
	Antiepileptics (Valproate, Topiramate, Levetiracetam, Flunarizine, Gabapentine)	Topiramate: acts on Na v , Ca v and carbonic anhydrase, modulates GABA-A and kainate receptors Levetiracetam: acts on SV2A (vesicular protein), Ca v Flunarizine: calmodulin blocker, H1R, 5HTR and D2R antagonist	Decrease in neuronal excitation Decrease in neuronal excitation, modulation of neuronal activity Reduces neuronal excitation, modulation of neuronal activity?
		Gabapentine: Ca v (α2δ) antagonist	Reduces neuronal excitation
		Amitriptyline: prevents	Multiple actions: Decrease in
	Antidepressors (tricyclic: Amitriptyline, SNRIs:	monoamine reuptake, multiple potential secondary targets (5HTR, H1-2R, α 1R, Ca v , Na v , K v etc.)	neuronal excitation, modulation of neuronal activity
	Venlafaxine)	Venlafaxine: prevents monoamine	Modulation of neuronal
		reuptake	activity?
	5HTR antagonists (Pizotifen, Methysergide)	5HT2R antagonists	Modulation of neuronal activity?
			Multiple actions: muscle
	Botulinum toxin	Exocytosis of neurotransmitter vesicle	contraction, neuronal modulation (TRP trafficking,
			synaptic transmission)?
		Lisinopril: ACE blocker (reduces
		production of angiotensine II)
	ACE inhibitors
	(Lisinopril,		Multiple actions: Modulation
	Candesartan)	Candesartan: AT1R antagonist	of neuronal activity (autonomic and sensory),
			vascular?
	Anti-CGRP		Modulation of neuronal
	(Erenumab,	Erenumab: antibody directed	function (neurotransmission,
	Fremanezumab)	against CGRP receptor	neuromodulation)

Table 2 . Current treatments of acute migraine, their targets and the proposed mechanism of action.

 2

	Acute treatment	Targets	Mechanism of action
	First line analgesics (Paracetamol, NSAID)	Paracetamol: multiple targets (modulation of K v 7, TRPV1, Ca v 3.2, COXs…)	Multiple actions: Decrease in neuronal excitation, modulation of neuronal activity? Reduction of the production of
		NSAID: inhibition COXs	inflammatory molecules
			(prostaglandines etc.)
			Modulation of neuronal
	Triptans (Sumatriptan)	5HT1B/5HT1D agonists	function, other cells (vascular
			for 5HT1B)?
	Dihydroergotamine	5HT1B/5HT1D agonists	Like Triptans, with a potential central and vascular action
	CGRP antagonists		Modulation of neuronal
	(Rimegepant,	CGRP receptor antagonist	function (neurotransmission,
	Ubrogepant)		neuromodulation)
			Modulation of neuronal
	Ditans (Lasmiditan)	5HT1F/5HT1D agonist	function (neurotransmission,
			neuromodulation)

Funding

This work was supported by a grant to G.S. by the Fondation pour la Recherche Medicale (Equipe labellisée FRM 2017, FRM DEQ20170336753 and ANR AT2R-TRAAK-Bioanalgesics ANR-17-CE18-0001) as well as Laboratory of Excellence "Ion Channel Science and Therapeutics," grant ANR-11-LABX-0015-01 and the French government, through the UCAJEDI Investments in the Future project managed by the National Research Agency (ANR) with the reference number ANR-15-IDEX-01. The work was also supported by a grant to P.A.P. by the Fondation pour la Recherche Medicale (FRM 2019, FDT201904008083).