INCLUDING THEM

FIELD OF THE INVENTION:

The invention is in the field of neurology more particularly, the invention relates to methods and composition for treating migraine.

BACKGROUND OF THE INVENTION:

Worldwide, migraines affect nearly 15% or approximately one billion people. According to International Headache Society's ICHD-3 classification system, there are seven types of Migraine, but the two major types of migraine are: migraine without Aura (formerly called Common Migraine) and migraine with Aura (formerly called Classic or Complicated Migraine). Migraine without Aura is the most frequent type of migraine. Symptoms include moderate to severe pulsating headache pain that occurs without warning and is usually felt on one side of the head. It comes along with nausea, confusion, blurred vision, mood changes, fatigue, and increased sensitivity to light, sound, or smells. Migraine with Aura is a type of migraine includes visual disturbances and other neurological symptoms that appear about 10 to 60 minutes before the actual headache and usually last no more than an hour.

Currently, there is not any medication to treat the migraine, there is only the medication to prevent migraine or reduce the frequency of migraine. Accordingly, there is a need to understand the mechanism of the migraine and identify new targets to treat migraine.

Activation and sensitization of primary afferent neurons within the trigeminal (TG) sensory system is likely a key step in the initiation of migraine headache attacks (1). It has been proposed that the underlying pathophysiology of migraine is in part due to ion channel dysfunction (2), including most recently the linking of TRESK, a two-pore-domains K+ (K2P) channel, to inherited migraine with aura (MA) (3, 4) . In humans, 2 types of TRESK channel mutations have been found to produce a dominant negative for TRESK: TRESK-MT, a 2 bp frameshift mutation (F139WfsX24) identified in the KCNK18 gene which causes premature truncation of TRESK(3), and TRESK-C110R (5), a missense variant. Despite the fact that both mutants are able to strongly inhibit TRESK current, only TRESK-MT produces an increase in TG neuron excitability and is associated with an MA phenotype (6, 7) . Thus, there is a need to study the molecular mechanisms which link TRESK-MT.

SUMMARY OF THE INVENTION:

The invention relates to a method for treating migraine in a subject in need thereof comprising a step of administering the subject with a therapeutically effective amount of agonists of: TREKl, TREK2, or agonists of heteromers TRESK-TREKl, TRESK-TREK2 or TREKl -TREK2. In particular, the invention is defined by claims.

DETAILED DESCRIPTION OF THE INVENTION:

Inventors have found that the MT mutation puts an alternative start codon in frame which leads to the translation of a second TRESK fragment. Surprisingly, the 2 gene products, termed MT1 and MT2, have differential dominant negative effects: MT1 targets TRESK while MT2 targets TREKl and TREK2, members of another subfamily of K2P channels. Furthermore, they have shown that by co-assembling with and inhibiting TREKl and TREK2, MT2 increases TG excitability. This resolves the contradictory lack of effects of TRESK- C110R which targets only TRESK and not TREKl or TREK2. Together their results demonstrate that alternative translation initiation is a mechanism initiated by the TRESK-MT mutation which leads to two protein fragments with dominant negative effects on distinct channel targets. This work supports a role for regulation of leak potassium channels as a key part of the underlying cellular mechanism of MA and identifies TREKl, TREK2 and TREKl - TREK2, TREK-TRESK heteromers as novel potential targets for treatment of this disorder. Inventors have also identified a new migraine-related TRESK mutant, Y121LfsX44, which also leads to the production of two TRESK fragments.

Accordingly, the invention relates to a method for treating migraine in a subject in need thereof comprising a step of administering the subject with a therapeutically effective amount of agonists of TREKl , TREK2, or agonists of heteromers TRESK-TREKl , TRESK-TREK2 or TREKl -TREK2.

As used herein, the terms "treating" or "treatment" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

As used herein, the term "migraine" refers to a disabling neurological disorder with an annual prevalence estimated at ~20 which is characterized by attacks of severe, throbbing headaches. According to the International Headache Society's ICHD-3 classification system, there are seven types of Migraine: 1) Migraine without Aura (formerly called Common Migraine). This is the most frequent type of Migraine. Symptoms include moderate to severe pulsating headache pain that occurs without warning and is usually felt on one side of the head It comes along with nausea, confusion, blurred vision, mood changes, fatigue, and increased sensitivity to light, sound, or smells; 2) Migraine with Aura (formerly called Classic or Complicated Migraine). This type of Migraine includes visual disturbances and other neurological symptoms that appear about 10 to 60 minutes before the actual headache and usually last no more than an hour; 3) Migraine without Headache is characterized by visual problems or other aura symptoms, nausea, vomiting, and constipation, but without head pain. Technically, this is known as Typical Aura without Headache; 4) Migraine with Brainstem Aura (formerly called Basilar-Type Migraine) mainly affects children and adolescents, this includes Migraine with Aura symptoms that originate from the brainstem, but without motor weakness. It occurs most often in teenage girls and may be associated with their menstrual cycles. Symptoms include partial or total loss of vision or double vision, dizziness and loss of balance (vertigo), poor muscle coordination, slurred speech, a ringing in the ears (tinnitus), and fainting; 5) hemiplegic Migraine (a sub-type of Migraine with Aura) is a rare but severe form of Migraine that causes temporary paralysis, sometimes lasting several days, on one side of the body prior to or during a headache. Symptoms such as vertigo, a pricking or stabbing sensation, and problems seeing, speaking, or swallowing may begin prior to the headache pain and usually stop shortly thereafter; 6) Retinal Migraine is a very rare type of Migraine characterized by attacks of visual loss or disturbances in one eye; 7) Chronic Migraine is characterized by headaches occurring on 15 or more days per month for more than 3 months, which have the features of Migraine headache on at least 8 days per month. They can be with or without aura, they usually require preventative medications and behaviours to control, and they are often disabling. The two major types of migraine are: migraine without Aura and migraine with Aura. In a particular embodiment, the method according to the invention, wherein the migraine is migraine without Aura. In another particular embodiment, the method according to the invention, wherein the migraine is Migraine with Aura.

As used herein, the term "subject" refers to any mammals, such as a rodent, a feline, a canine, and a primate. Particularly, in the present invention, the subject is a human afflicted with or susceptible to be afflicted with at least one of migraine as described above. In a particular embodiment, the subject is afflicted with or susceptible to be afflicted with Migraine with Aura (MA). In a particular embodiment, the subject has or is susceptible to have a mutation affecting TREKl or TREK2. In another embodiment, the subject has or is susceptible to have a mutation which induce the production of MT2. In a particular embodiment, the subject has or is susceptible to have Y121LfsX44 mutation (TRESK c.361dupTs _TO p) in TRESK.

As used herein, the term "TREKl", also known as potassium channel subfamily K member 2 (KCNK2) refers to Twik-related K+ channel 2. It is a protein that in humans is encoded by the KCNK2 gene. TREK-1 is part of the subfamily of mechano-gated potassium channels that are present in mammalian neurons. TREK-1 channels are important in physiological, pathophysiological, and pharmacological processes, including having a role in electrogenesis, ischemia, and anesthesia. The naturally occurring human TREKl gene has a nucleotide sequence as shown in Genbank Accession numbers: NM 001017424.2, NM 001017425.2 and NM 014217.3. The naturally occurring human TREKl protein has an aminoacid sequence as shown in Genbank Accession numbers: NP 001017424.1 , NP 001017425.2 and NP 055032.1. In a particular embodiment, TREKl is affected by mutation.

As used herein, the term "TREK2", also known as potassium two pore domain channel subfamily K member 10 (KCNK10) refers Twik-related K+ channel 2. It is a protein encoded by KCNK10 gene belongs to the family of potassium channel proteins containing two pore- forming P domains. This channel is an open rectifier which primarily passes outward current under physiological K+ concentrations, and is stimulated strongly by arachidonic acid and to a lesser degree by membrane stretching, intracellular acidification, and general anaesthetics. The naturally occurring human TREK2 gene has a nucleotide sequence as shown in Genbank Accession numbers: NM_021161.4, NMJ38317.2 and NMJ38318.2. The naturally occurring human TREK2 protein has an aminoacid sequence as shown in Genbank Accession numbers: NP 066984.1, NP 612190.1 and NP_612191.1. In a particular embodiment, TREK2 is affected by mutation.

As used herein, the term "TRESK", also known as Potassium channel subfamily K member 18 (KCNK18) or K2P18.1 refers to TWIK-related spinal cord potassium channel. It is a protein that in humans is encoded by the KCNK18 gene. This potassium channel contains two pore-forming P domains. The naturally occurring human TRESK gene has a nucleotide sequence as shown in Genbank Accession number NM l 81840.1 and the naturally occurring human TRESK protein has an aminoacid sequence as shown in Genbank Accession numbers NP 862823.1. In a particular embodiment, TRESK has at least one mutation: F139WfsX24 or Y121LfsX44. As used herein, the term "homodimer" refers to a dimer consisting of two structurally similar monomers joined by bonds that can be either strong or weak, covalent or intermolecular. In the context of the invention, the term "heterodimer" is used when two molecules different structurally and linked together. In the context of the invention, heteromerization of subunits is a widespread mechanism for increasing diversity in voltage- gated (Kv) and inwardly-rectifying (Kir) K+ channel subfamilies. The resulting heteromers are typically characterized by different biophysical and regulatory properties from the homomers of the parent subunits. In particular, the method according to the invention is suitable to treat migraine by administering agonists of heteromers TRESK/TREKl, TRESK/TREK2 or TREK1/TREK2.

As used herein, the term "TRESK-TREK1" refers to a heterodimer. Typically, TRESK and TREK1 are joined each other by bonds that can be either strong or weak, covalent or intermolecular.

As used herein, the term "TRESK-TREK2" refers to a heterodimer. Typically, TRESK and TREK2 are joined each other by strong or weak, covalent or intermolecular.

As used herein, the term "TREK1-TREK2" refers to a heterodimer. Typically, TREK1 and TREK2 are joined each other by strong or weak, covalent or intermolecular.

As used herein, the term "agonist" refers to a compound that binds specifically to

TREKl, TREK2 or to the heteromers TRESK/TREKl, TRESK TREK2 or TREK1/TREK2. Thus, the compound stimulates or promotes the expression and/or the biological activity of TREKl, TREK2 or heteromers TRESK TREKl, TRESK/TREK2 or TREK1/TREK2. The agonist used according to the invention may be any substance, derived from natural sources or from synthesis by chemical and/or genetic engineering methods. Agonists typically include but are not limited to small organic molecule, peptides, polypeptides, antibodies, nucleic acids or ap tamers.

Agonistic activities of a test compound toward TREK1, TREK2, TRESK/TREK1, TRESK/TREK2 or TREK1/TREK2 may be determined by any well-known method in the art. For example, since the agonist of the present invention can promote the function of the TREK1 , TREK2, the agonist can be screened using the natural agonist of TREKl, TREK2 and those which activate their associated receptors. For example, the G protein-coupled receptors (GPCRs) coupled to Gi proteins which lead to the activation of TREKl .

Typically, the agonist of the present invention can be obtained using the method screening the substance promoting the function of the TREKl, TREK2, TRESK/TREKl, TRESK/TREK2 or TREK1/TREK2 which comprises direct or indirect agonist. As used herein, a "direct agonist" refers to a compound that has a direct activity on TREK channels, particularly TREKl and/or TREK2 homodimers, and TREKl -TREK2, TREKl -TRESK and TREK2- TRESK heterodimers that do not have an activity on a second protein upstream. As used herein, a "indirect agonist" refers to a compound that has an activity on TREK channels through a second protein (receptor) upstream. For example, activation of Gi protein-coupled receptors such as GABAB receptor or mGluR2 by their respective agonists can lead to TREK channel activation. Example of activators of GABAB receptors are well known in the art (Pin et al 2007) typically, they are selected from the group consisting of: Baclofen, gamma-Hydroxybutyrate (GHB), Phenibut, Isovaline, 3-Aminopropylphosphinic acid, Lesogaberan, SKF-97541, CGP- 44532, CGP-7930, CGP-7930, BHFF, Fendiline, Fasoracetam, BHF-177, BSPP or GS-39783. Example of activators of mGluR2 are well known in the art (Lin Li et al 2015; WO2010130424 ) typically they are selected from the group consisting of: JNJ-46356479, JNJ-40411813, GSK- 1331258, Imidazo[l,2-a]pyridines, 3-Aryl-5-phenoxymethyl-l,3-oxazolidin-2-ones, 3- (Imidazolyl methyl)-3-aza-bicyclo[3.1.0]hexan-6-yl)methyl ethers, BINA, LY-487,379.

In one embodiment, the agonist is a small organic molecule. The term "small organic molecule" refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da. In a particular embodiment, the small molecule is ML67 and its derivate. More particularly, the ML67's derivate is ML67-33 which has the following formula and structure in the art C18H17C12N5 (Sviatoslav N. et al 2013):

In another embodiment the agonist is an aptamer. Aptamers are a class of molecules that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.

In another embodiment the agonist is a polypeptide. The term "polypeptide" refers both short peptides with a length of at least two amino acid residues and at most 10 amino acid residues, oligopeptides (11-100 amino acid residues), and longer peptides (the usual interpretation of "polypeptide", i.e. more than 100 amino acid residues in length) as well as proteins (the functional entity comprising at least one peptide, oligopeptide, or polypeptide which may be chemically modified by being glycosylated, by being lipidated, or by comprising prosthetic groups).

In some embodiments, the agonist is an antibody. As used herein, the term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. The term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting"); small antibody mimetics comprising one or more CDRs and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art (see Kabat et al., 1991, specifically incorporated herein by reference). Diabodies, in particular, are further described in EP 404, 097 and WO 93/1 1 161; whereas linear antibodies are further described in Zapata et al. (1995). Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds- scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et al, 2006; Holliger & Hudson, 2005; Le Gall et al, 2004; Reff & Heard, 2001 ; Reiter et al, 1996; and Young et al, 1995 further describe and enable the production of effective antibody fragments. In some embodiments, the antibody is a "chimeric" antibody as described in U.S. Pat. No. 4,816,567. In some embodiments, the antibody is a humanized antibody, such as described U.S. Pat. Nos. 6,982,321 and 7,087,409. In some embodiments, the antibody is a human antibody. A "human antibody" such as described in US 6,075,181 and 6,150,584. In some embodiments, the antibody is a single domain antibody such as described in EP 0 368 684, WO 06/030220 and WO 06/003388. In a particular embodiment, the inhibitor is a monoclonal antibody. Monoclonal antibodies can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique, the human B- cell hybridoma technique and the EBV-hybridoma technique.

As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., an agonist of TREK1, TREK2, TRESK/TREKl; TRESK/TREK2; TREK1/TREK2) into the subject, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

A "therapeutically effective amount" is intended for a minimal amount of active agent which is necessary to impart therapeutic benefit to a subject. For example, a "therapeutically effective amount" to a subject is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a disorder. It will be understood that the total daily usage of the compounds of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

The agonists as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum- drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1. TRESK heteromerizes physically and functionally with TREKl and

TREK2. Summary bar graph showing pulldown of GFP-TRESK by HA-TRESK, HA-TREK1 or HA-TREK2, but not by HA-TASK1, HA-TASK3, or HA-TRAAK.

Figure 2. TRESK-MT, but not TRESK-C110R, acts as a dominant negative on TREKl and TREK2 channels to increase excitability of TG neurons. (A and B) Representative traces showing the effect of TRESK-Cl 1 OR and TRESK-MT co-expression on TRESK (A) or TREKl (B) on currents elicited by voltage-ramps (from -100 to 100 mV, Is duration). (C) Bar graph summarizing the relative current amplitude at 0 mV for TRESK, TREKl, and TREK2 with or without co-expression of TRESK-Cl 1 OR or TRESK-MT. (D and E) Input-output plots of the spike frequency in response to Is depolarizing current injection in transfected small TG neurons show that an increase in excitability elicited by TRESK-MT is observed in WT, but not T1-/-/T2-/- neurons. The numbers of tested cells are indicated in parentheses. Student's t test (*P< 0.05, **P< 0.01, ***P< 0.001).

Figure. 3. TRESK-MT induces the translation of a second protein, MT2, which mediates TREKl inhibition. (A) Representative traces showing the effect of introduction of a STOP codon at the beginning of the MT2 ORF within the 2-3 loop (TRESK-MTSTOP) on TREKl current. Inset shows a summary of TREKl relative current densities when TRESK- MTSTOP is coexpressed. (B) Representative traces and summary bar graph showing the effect of mutation of candidate alternative start codons (AATGl, AATG2, or AATG3) in TRESK- MT. Currents were elicited by voltage-ramps (from -100 to 100 mV, Is duration). The numbers of cells tested are indicated in parentheses. Student's t test (**P< 0.01, ***P< 0.001) shows the difference between TREKl and TREKl co-expressed with different TRESK-MT constructs.

Figure. 4. MT2, but not MT1, by acting as a dominant negative on TREKl and TREK2 channels, increases neuronal excitability of WT small TG neurons. (A and B) Representative traces showing the effect of TRESK-MT 1 or TRESK-MT2 co-expression on TRESK (A) or TREKl (B) currents. Currents were elicited by voltage-ramps (from -100 to 100 mV, Is duration). (C) Bar graph summarizing the relative TRESK, TREKl and TREK2 current amplitudes at 0 mV when MT1 or MT2 are co-expressed. Student's t test (***p< 0.001). (D and E) Representative traces and input-output plots of spikes generated by incremental depolarizing current injections in WT small-diameter TG neurons transfected with either GFP ("WT"), the GFP-tagged MT1 subunit ("MT1") or the GFP-tagged MT2 subunit ("MT2"). (F) Input-output plots of spike frequency show a lack of effect of GFP-MT2 expression on TG neurons from TREK1/TREK2 double KO mice (T1-/-/T2-/-). The numbers of cells tested are indicated in parentheses. Student's t test (**P< 0.01, ***P< 0.001).

Figure 5: TREK1-/-/TREK2-/- double knockout animals present a migraine-like hypersensitivity to mechanical stimuli, (a, b) Schematic of experimental behavioral paradigms. Green arrows represent the injection of ISDN, a known migraine trigger. Blue arrows represent the measurement of mechanical sensitivity, (c) Basal mechanical responses, assessed after the first ISDN injection, were significantly decreased in double knockout animals and remained less than WT for the first 1.5 hrs following ISBN injection, (d) Mechanical responses, assessed prior to and after chronic ISDN injections, were significantly decreased in double knockout animals (c, d) Student's t test to compare WT vs TREKl -/-/TREK2-/- mice (*P< 0.05, **P< 0.01, ***P< 0.001). (e) Comparison of the mechanical threshold before and after chronic treatment with ISDN. Topiramate treatment were assessed before and 2 hours after topiramate injection. Numbers of mice tested are indicated in parentheses, Student's t test to compare WT vs TREK1-/-/TREK2-/- mice (*P< 0.05, **P< 0.01, ***P< 0.001).

Figure 6: TRESK-c.361dupT (Y121LfsX44) acts as a dominant negative to reduce both TRESK and TREK1 current. Co-synthesis of mCherry-MTl and MT2-GFP products from the mCherry-TRESK- c.361dupT-GFP cDNA in HEK293T cells. (A) Representative traces showing the effect of TRESK c.361dupT (A) and TRESK c.361dupTSTOP (A) co- expression on TRESK current. Currents were elicited by voltage-ramps (from -100 to 100 mV, Is duration). (B) Same as (A) for TREKl . (C) same as (A) for TREK2. (D) Bar graph summarizing the relative TRESK, TREKl and TREK2 current amplitudes at 0 mV for TRESK, TREKl and TREK2 when TRESK c.361dupT and TRESK c.361dupTSTOP are coexpressed. Student's t test (**P<0.01 and ***P< 0.001).

EXAMPLE:

Material & Methods

Molecular Biology, Cell Culture and Gene Expression

Channel DNA was used in the pIRES2eGFP, pcDNA3.1 and pCMV-HA vectors.

HEK293 cells were maintained in DMEM with 5% FBS on poly-L-lysine-coated glass coverslips in 12 well plates. Cells were transiently co -transfected using Lipofectamine 2000 (Invitrogen) with a total of 1-1.6 μg of DNA total per 18 mm diameter cover slip.

Primary cultures of mouse TG neurons

All mouse experiments were conducted according to national and international guidelines and have been approved by the local ethical committee (CIEPAL NCE). The C57BL/6 breeders were maintained on a 12 h light/dark cycle with constant temperature (23- 24°C), humidity (45-50%), and food and water ad libitum at the animal facility of Valrose. TG tissues were collected from postnatal day 8 mice of either sex and treated with 2 mg/ml collagenase type II (Worthington) for ~2 hours, followed by 2.5 mg/ml trypsin for 15 min. Neurons were dissociated by triturating with fire-polished glass pipettes and seeded on polylysine/laminin coated coverslips. The DMEM-based culture medium contained 10% fetal bovine serum and 2mM GlutaMAX (Invitrogen). Neurons were transfected at 1 d in vitro (DIV) using Lipofectamine 2000 (Invitrogen). Transfected neurons were identified by the green fluorescence and patch clamp recordings were performed between DIV 3 and 5.

Knock-out mice

Mice lacking Trekl and Trek2 were generated as described (19). Null mutations were backcrossed against the C57BL/6J inbred strain for 10+ generations prior to establishing the breeding cages to generate subjects for this study. Age- and sex-matched C57bl/J6 WT mice, aged 9-12 weeks, were obtained from Charles River Laboratories (Wilmington, MA).

Electrophysiology

HEK293 cell electrophysiology was performed 24-72 h after transfection in solution containing (in mM): 145 mM NaCl, 4 mM KC1, 1 mM MgC12, 2 mM CaC12 and 10 mM HEPES. Glass pipettes of resistance between 3 and 6 ΜΩ were filled with intracellular solution containing (in mM): 140 KC1, 10 Hepes, 5 EGTA, 3 MgC12, pH 7.4. Cells were patch clamped using an Axopatch 200A (Molecular Devices) amplifier in the whole cell mode. Currents were elicited by voltage-ramps (from -100 to 100 mV, Is in duration) and the current density was calculated at 0 mV.

Neuronal excitability was studied in small-diameter TG neurons transfected with the pCMV-HA-GFP-X constructs (X is in frame with GFP) or the pCMV-HA-GFP control plasmid. Both plasmids derivate from pCMV-HA in which we have subcloned the GFP sequence in frame. Extracellular solution contained (in mM): 135 NaCl, 5 KC1, 2 CaC12, 1 MgC12, 5 HEPES, 10 glucose, pH 7.4 with NaOH, 310 mOsm. The pipette solution contained the following (in mM): 140 K-gluconate, 10 NaCl, 2 MgC12, 5 EGTA, 10 HEPES, 2 ATP-Mg, 0.3 GTP-Na, lCaC12 pH 7.3 with KOH, 290 mOsm. Recording pipettes had < 4.5 ΜΩ resistance. Series resistance (<20 ΜΩ) was not compensated. Signals were filtered at 10 kHz and digitized at 20 kHz. After establishing whole-cell access, membrane capacitance was determined with amplifier circuitry. The amplifier was then switched to current-clamp mode to measure resting membrane potential (Vrest). Neurons were excluded from analysis if the Vrest was higher than -40 mV or if the input resistance was smaller than 200 ΜΩ. To test neuronal excitability, neurons were held at Vrest and injected with 1 s depolarizing currents in 25 pA incremental steps until at least 1 action potential (AP) was elicited.

Western blot analysis

HEK293T cells were homogenized in PBS containing saponin (0.5% w/v), Triton X - 100 (0.5% w/v) and protease inhibitors (Roche Diagnostics, Basel, Switzerland). Lysates were clarified by centrifugation at 20 000 g for 30 min. Proteins were separated on 10% SDS polyacrylamide gel and blotted onto nitrocellulose membrane (Hybond - C extra, Amersham Biosciences, Freiburg, Germany). Detection was carried out using mouse monoclonal antibody clone HA-7 against the HA epitope (Sigma).

Single Molecule Pulldown For SimPull experiments, HEK 239T cells were harvested from coverslips by incubating with Ca2+-free PBS buffer for 20-30 minutes followed by gentle pipetting. Cells were lysed in buffer containing (in mM): 150 NaCl, 1 EDTA, protease inhibitor cocktail (Thermo Scientific) and 1.5% IGEPAL (Sigma). After 30-60 minute incubation at 4°, lysate was centrifuged for 20 minutes at 16,000 g and the supernatant was collected. Coverslips passivated with PEG (~99%)/ biotin-PEG(~l%) and treated with neutravidin were prepared as described(2). 15 nM biotinylated anti-HA antibody (abeam, #ab26228) was applied for 20 minutes and then washed out. Antibody dilutions and washes were done in T50 buffer containing (in mM): 50 NaCl, 10 Tris, pH 7.5. Lysate, diluted in standard patch clamp electrophysiology extracellular recording solutions (see Electrophysiology), was then applied to the chamber and washed away following brief incubation (~2 minutes). Single molecules were imaged using a 488 nm Argon laser on a total internal reflection fluorescence microscope with a 60x objective (Olympus). We recorded the emission light after an additional 3x magnification and passage through a double dichroic mirror and an emission filter (525/50 for GFP) with a back-illuminated EMCCD camera (Andor iXon DV-897 BV). Movies of 500-800 frames were acquired at frame rates of 10-30 Hz. The imaged area was 13 x 13 um2. At least 5 movies were recorded for each condition and data was analyzed using custom software. Multiple independent experiments were performed for each condition. Representative data sets are presented to quantitatively compare conditions tested on the same day.

Results

In this invention, inventors addressed the following question: why is TRESK-MT, but not TRESK-C110R, able to increase TG excitability and, subsequently, induce migraine despite sharing the same dominant-negative functional effect? Despite the fact that K2P channels share a similar architecture and global function, they share a low level of sequence identity, even between members of the same subfamily. Surprisingly, this low level of identity does not preclude heteromerization, as we and others recently showedwithin the TREK subfamily (8- 10). Based on this and the fact that TG neurons express many K2P channels (TREKl, TREK2, TRAAK, TASK1 and TASK3) (11, 12), inventors hypothesized that the difference between TRESK mutants is due to their differential ability to modify the function of other K2P channels through heteromerization. To assess the ability of TRESK to heteromerize with other K2P channels which are expressed in TG neurons, we used the recently-developed single-molecule pull-down ("SiMPull") assay (13, 14) to visualize individual antibody- immobilized protein complexes on polyethylene glycol-passivated glass coverslips. We co-expressed GFP-TRESK with either HA-TRESK, HA-TREK1, HA-TREK2, HA-TRAAK, HA-TASK1, or HA-TASK3 and assessed their ability to co-immunoprecipitate (Co-IP) GFP-TRESK via an anti-HA antibody. HA-TRESK, HA-TREKl, and HA-TREK2, were able to co-IP many fluorescent GFP-TRESK spots (Fig. 1 A whereas no GFP-TRESK spots were observed for HA- TRAAK, HA-TASK1 or HA-TASK3 (Fig. 1A) indicating that TRESK co-assembly with other K2P channels is specific for TREKl and TREK2.

Next, to test the ability of TREKl and TRESK to form a functional complex we developed a TREK functional heterodimerization assay based on an engineered "Photoswitchable Conditional Subunit" (TREKl -PCS) of TREKl . The TREKl -PCS is a TREKl subunit where the C-terminus has been deleted to produce ER retention, which can be rescued through co-assembly with a full-length subunit (15). Following co-assembly and surface targeting, TREKl -PCS can then optically control the channel via a tethered photoswitchable blocker which attaches to a genetically engineered cysteine. Therefore, gain of photosensitivity of an identified co-expressed TREK interacting subunit allows for the verification of a functional heteromer with TREKl . As expected, expression of TREKl -PCS alone did not generate a photoswitchable current (data not shown) but co-expression with either TREKl or TRESK induced a robust photoswitchable current (data not shown), indicating that the TRESK subunit is able to co-assemble with TREKl -PCS to form a heteromeric channel with a common pore. Consistent with SiMPull data (data not shown), no photocurrent was observed when TASK3 was co-expressed with TREKl -PCS. Having found that TREKl and TREK2 can heteromerize with TRESK, we next investigated the ability of TRESK mutants to modify TREKl and TREK2 currents.

As previously shown (7), both TRESK-MT and TRESK-Cl 1 OR exert a dominant- negative effect on whole cell TRESK currents (Fig. 2A, C). Since TREKl can co-assemble with TRESK (Fig. 1), we addressed the impact of the MT and C110R variants on TREKl current. We found that TRESK-Cl 10R co-expression did not modify TREKl current whereas TRESK-MT co-expression induced a near-complete inhibition of TREKl current (Fig. 2B, C). Similar to TREKl , TRESK-MT but not TRESK-C 11 OR strongly inhibited TREK2 current (Fig. 2C). This dominant negative effect is likely dependent on co-assembly since TASK1, TASK3 and TRAAK, which do not co-IP with TRESK (Fig. 1), were not sensitive to TRESK-MT co- expression (data not shown). Together these data show that TRESK-MT can inhibit TRESK, TREKl and TREK2 whereas TRESK-Cl 10R is only able to inhibit TRESK. Based on the fact that TRESK-MT but not TRESK-Cl 1 OR is able to induce TG neuron hyperexcitability (5, 6), we hypothesized that TRESK-MT induces sensory neuron hyper-excitability primarily by acting on TREK1 and TREK2, not TRESK.

To investigate the role of TREK1 and TREK2 in the induction of TG hyperexcitability by TRESK-MT, we tested if overexpression of GFP-TRESK-MT alters the passive and active electrophysiological properties of small-diameter (<25 μιη) TG neurons from wild-type or TREK1/TREK2 double knockout (TREK 1 -/ -/TREK2-/ -) mice. As previously shown, TRESK- MT expression in WT TG neurons led to an increase in excitability (Fig. 2) which included a decrease in the rheobase (74 ± 11 pA vs 47 ± 5 pA, P<0.05 for TG neurons expressing GFP or TRESK-MT, respectively) and an increase in the number of action potentials (APs) evoked by suprathreshold current injections compared to control (Fig. 2D). Notably, neurons from TREK1-/-/TREK2-/- mice were more excitable than WT TG neurons (data not shown). These neurons present a smaller rheobase (55 ± 6 pA, P<0.05) and a significant increase in the number of APs evoked by suprathreshold current injections compare to WT-TG. Consistent with a role for TREKl and 2 in mediating the effects of TRESK-MT, TRESK-MT overexpression did not alter the excitability or rheobase (55 ± 6 pA vs 53 ± 6 pA, for TG neuron expressing GFP or TRESK-MT respectively) of TREKl -/-/TREK2-/- neurons (Fig. 2D). Together these data strongly support the idea that TRESK-MT differs from TRESK- CI 10R by its ability to target TREKl and TREK2 to increase the excitability of TG neurons, likely a crucial step in the induction of migraines.

We next explored how TRESK-MT exerts its effects on TREK channels at the molecular level. The F139WfsX24 frameshift mutation of TRESK-MT results in the premature truncation of human TRESK protein from 384 to 162 aa. The corresponding mutation has very similar effects on the mouse TRESK gene, generating a truncated protein with the first 149 aa of wild- type TRESK and a 50 aa aberrant sequence at the C terminus. We fused a GFP tag to the N- terminus of the MT subunit and tested its ability to be immobilized by HA-TRESK, HA-TREK1 or HA-TREK2 in the SiMPull assay. Surprisingly, only HA-TRESK was able to co-IP GFP- TRESK-MT via an anti-HA antibody (data not shown). This result confirms that TRESK-MT associates with TRESK to induce its dominant negative effect but raises the question of how TRESK-MT is able to inhibit TREKl and TREK2 without direct association.

It has been hypothesized that alternative translation initiation (ATI) of eukaryotic mRNAs, including those that encode K2P channels (16), may be a method to expand the proteome (17). A close examination of the nucleotide sequence of TRESK-MT revealed that the F139WfsX24 frame shift mutation puts two new ATG codons in frame. We hypothesized that one of these codons may serve as an ATI site that can lead to the formation of a second truncated TRESK protein ("MT2") of -28 kDa that would include short (either 2 or 19 aa) N- terminal aberrant sequence followed by the C-terminal part of TM2, the 2-3 intracellular loop, TM3, P2 loop, TM4 and the C terminal domains. To test whether MT2 is co-expressed with MTl, we introduced an N-terminal mCherry-tag in frame with MTl and a C-terminal GFP in frame with MT2 within the mouse TRESK-MT cDNA ("mCherry-TRESK-MT-GFP"; Fig. 3C). Expression of this construct leads to cells with both mCherry and GFP fluorescence. Next, we introduced an N-terminal hemagglutinin (HA) tag in frame with MTl and another one in frame with MT2 within the mouse TRESK-MT cDNA ("HA-TRESK-MT-HA"). Lysate from cells transfected with HA-TRESK-MT-HA was probed in a western blot with anti-HA antibodies and 2 bands, with a similar intensity, corresponding to the expected molecular weight for MTl (~23 kDa) and MT2 (~28 kDa) were detected (data not shown). Together these data show that TRESK-MT leads to the production of two distinct fragments of TRESK that may produce distinct functional effects.

To probe the function of MT2, we introduced a stop codon into the MT2 ORE of TRESK-MT at the beginning of the 2-3 loop. The introduction of the stop codon in MT2 ORE did not change the ability of TRESK-MT to inhibit TRESK current (data not shown), this stop codon abolished the ability of TRESK-MT to produce a dominant negative functional effect on TREK1 (Fig. 3A). We next confirmed the importance of this second ORE by mutating, in TRESK-MT, the putative ATI one by one. Mutation of the first ATG abolished the ability of TRESK-MT to inhibit TREK1 but not TRESK (Fig. 3B) whereas mutation of the second ATG did not alter the ability of TRESK-MT to inhibit TREK1 current (Fig. 3B). This data indicates that the ATI site is the first internal ATG. As a control, we mutated a third ATG, which is also present in the WT-TRESK sequence, and found that it did not change the ability of TRESK- MT to inhibit both TRESK and TREK1 currents (Fig. 3B)

To independently express MTl and MT2 for functional characterization, we subcloned both ORFs into separate mammalian expression vectors. Co-expression of MT2 with TRESK did not modify TRESK current (Fig. 4A, C), while MTl co-expression induced a ~3-fold decrease of the current which was similar to what was observed for the co-expression of the full TRESK-MT construct (Fig. 4A, C). On the contrary, co-expression of MTl did not modify TREK1 current (Fig. 4B, C) but co-expression of MT2 induced a ~4-fold decrease of the current, similar to what was observed with co-expression of the full TRESK-MT construct (Fig. 4B, C). Similar results were obtained for TREK2 (Fig. 4C). Consistent with the functional data, we found that GFP-MT2 is co-immunoprecipitated with TREKl and TREK2 (data not shown). Finally, to validate the physiological role of interaction between TREK1, TREK2 and MT2, we tested the functional effect of MT2 in TG neurons. Whereas MT1 expression did not alter the excitability of TG neurons (Fig. 4D), MT2 increased it significantly (Fig. 4D,E). We confirmed that this effect is linked to TREK1 and TREK2 since MT2 overexpression failed to increase the excitability of TREK1-/-/TREK2-/- TG neurons (Fig. 4F).

An increase in TG neuron excitability is a crucial step in the induction of migraines. Having found that expression of the TRESK-MT mutant increases TG excitability through TREK1-TREK2 inhibition, we hypothesized that TREK1-TREK2 KO mice would show an increased susceptibility to a migraine-related phenotype. Migraine is associated with increased sensitivity to all sensory modalities and it appears that cutaneous allodynia can be used as a quantifiable marker of migraine disorderl . One approach to model acute and chronic migraine is the quantification of this increase in response to known migraine triggers such as Isosorbide Dinitrate (ISDN). We quantified ISDN-evoked mechanical hyperalgesia in TREK1-/-/TREK2- /- and wild-type controls in acute and chronic conditions. In a first experiment mechanical nociception thresholds were determined with a dynamic von Frey aesthesiometer before and during a 3-hour period after intraperitoneal injection of ISDN (10 mg/kg) (Fig. 5a). In a second experiment, we assessed mechanical nociception thresholds in both TREK1-/-/TREK2-/- and wild-type littermate controls, by intraperitoneally injecting ISDN every day for four days as a model of chronic migraine-associated pain (Fig. 5b)2. We found that, at rest, TREK1-/- /TREK2-/- mice showed a decreased mechanical threshold compared to WT mice (2.6 ± O.lg vs 3.9 ± O.lg; PO.001). Notably, the basal threshold of TREK1-/-/TREK2-/- mice is similar to both the threshold observed 1.5 hours after acute ISDN injection (P=0.831) (Fig. 5c) and after 4 days of chronic injection (Fig. 5c) in WT mice. In the acute model experiment, during the first 1.5- hour following ISDN injection, the mechanical thresholds remained significantly lower in TREK1-/-/TREK2-/- mice than in wild-type controls (P < 0.001 after 30 minutes and P < 0.01 after 1 hour with a linear mixed-effects model). In the chronic migraine-associated pain assay, the mechanical thresholds remained significantly lower for TREK1-/-/TREK2-/- mice compared to wild-type controls over the four day period despite a strong reduction in the threshold for WT mice over the course of treatment.

Having found that ISDN did not change the mechanical threshold in both acute and chronic migraine-associated pain models in TREK1-/-/TREK2-/- mice, we wondered if a treatment used in prophylaxis in migraine patient, topiramate, could reverse this observed migraine-like phenotype. We assessed the mechanical nociception threshold in TREKl-/- /TREK2-/- mice before and 2 hours following the intraperitoneal injection of 30 mg/kg of topiramate. Treatment with topiramate inhibited the chronic basal hyperalgesia seen in TREKl - /-/TREK2-/- mice (1.2 ± 0.2g; Fig. le), as was previously observed for a nitroglycerin-evoked form of hyperalgesia2. As a control, we tested WT mice and did not observe any significant shift of the mechanical threshold following topiramate treatment (Fig. 5e).

MT2-producing alternative translation initiation is found in other migraine-associated

TRESK mutants. Having found that MT2 is responsible for the migraine-associated increase in TG excitability through the inhibition of TREKl and TREK2, we anticipated that other frameshift mutations may exist which place the ATG at position +356 in-frame with the reference open reading frame of TRESK. Such mutations would lead to the formation of MT2. This mutation could be either a 2 bp deletion or 1 bp insertion in the region between the ATG at position +356 and the TGA at position +427 (data not shown). We used the Exome Aggregation Consortium (Ex AC) database (Lek et al, 2016) and found one variant (Y121LfsX44) with a T duplication (+1 pb, c.361dupT) that places the ATI site in frame with the TRESK ORE (data not shown). We introduced this insertion into the mCherry-TRESK- GFP (mCherry-TRESK-c.361dubT-GFP) sequence and found, similar to mCherry-TRESK- MT-GFP (data not shown), that this construct led to HEK293T cells with both mCherry and GFP fluorescence (data not shown) showing the co -translation of both MT1 and MT2 proteins. Similar to TRESK-MT, this mutant is able to inhibit both TRESK, TREKl and TREK2 (Figure 6). As was seen for TRESK-MT (data not shown), introduction of a stop codon into the MT2 ORE (TRESK c.361dupTSTOP) of this mutant abolished its ability to inhibit TREKl and TREK2 (Figure 6), but not TRESK. Since this Y121LfsX44 mutation leads to the same molecular effects as TRESK-MT on TREK function, we hypothesized that it may be associated with a migraine phenotype. To address this, we looked in the ClinVar database (Landrum et al, 2016) and found that this mutant has been correlated with a migraine phenotype (RCV000490385.1).

Together these results show that, unexpectedly, the strongly MA-associated TRESK mutations F139WfsX2 and Y121LfsX44 lead to the production of two protein fragments that can either target wild-type TRESK or wild-type TREKl and TREK2 potassium channels. This resolves the contradictory lack of effects of TRESK-Cl 1 OR which also serves as a dominant negative of TRESK but, as we show here, has no effect on TREKl nor TREK2. These findings suggest that ATI may be a more prevalent phenomenon than previously thought which can lead to unexpected effects in a protein's function, regulation and role in disease. Future work will be needed to elucidate the molecular, cellular, and circuit mechanisms of heteromeric K2P channel modulation in MA to gain a deeper understanding of the underlying pathophysiology and to devise a means of targeting this complex for treatment. The results with using ISDN show that TREK1-/-/TREK2-/- mice present a phenotype at rest which is similar to the phenotype observed in ISDN-treated animals in which migraine have been induced and this phenotype is partially reversed by topiramate, a drug used in the clinic to treat chronic migraine.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

1 Noseda, R. & Burstein, R. Migraine pathophysiology: anatomy of the trigemino vascular pathway and associated neurological symptoms, CSD, sensitization and modulation of pain. Pain 154 Suppl 1, doi: 10.1016/j.pain.2013.07.021 (2013).

2 Yan, J. & Dussor, G. Ion channels and migraine. Headache 54, 619-639, doi: 10.1111/head. l2323 (2014).

3 Lafreniere, R. G. et al. A dominant-negative mutation in the TRESK potassium channel is linked to familial migraine with aura. Nat Med 16, 1157-1160, doi: 10.1038/nm.2216

(2010).

4 Wood, H. Migraine: Familial migraine with aura is associated with a mutation in the TRESK potassium channel. Nat Rev Neurol 6, 643, doi: 10.1038/nrneurol.2010.166 (2010).

5 Andres-Enguix, I. et al. Functional analysis of missense variants in the TRESK (KCNK18) K channel. Sci Rep 2, 237, doi: 10.1038/srep00237 (2012).

6. Liu, P. et al. Functional analysis of a migraine-associated TRESK K+ channel mutation. J Neurosci 33, 12810-12824, doi:10.1523/JNEUROSCI.1237-13.2013 (2013).

7 Guo, Z., Liu, P., Ren, F. & Cao, Y. Q. Nonmigraine-associated TRESK K+ channel variant CI 10R does not increase the excitability of trigeminal ganglion neurons. JNeurophysiol 112, 568-579, doi: 10.1152/jn.00267.2014 (2014).

8 Talley, E. M., Solorzano, G., Lei, Q., Kim, D. & Bayliss, D. A. Cns distribution of members of the two-pore-domain (KCNK) potassium channel family. J Neurosci 21, 7491- 7505 (2001).

9 Levitz, J. et al. Heterodimerization within the TREK channel subfamily produces a diverse family of highly regulated potassium channels. Proc Natl Acad Sci U S A 113, 4194-

4199, doi: 10.1073/pnas.1522459113 (2016).

10 Hwang, E. M. et al. A disulphide- linked heterodimer of TWIK-1 and TREK-1 mediates passive conductance in astrocytes. Nat Commun 5, 3227, doi: 10.1038/ncomms4227 (2014). 11 Blin, S. et al. Mixing and matching TREK/TRAAK subunits generate heterodimeric K2P channels with unique properties. Proc Natl Acad Sci U S A 113, 4200-4205, doi: 10.1073/pnas.1522748113 (2016).

12 Bautista, D. M. et al. Pungent agents from Szechuan peppers excite sensory neurons by inhibiting two-pore potassium channels. Nat Neurosci 11, 772-779, doi: 10.1038/nn.2143

(2008).

13 Yamamoto, Y., Hatakeyama, T. & Taniguchi, K. Immunohistochemical colocalization of TREK- 1, TREK-2 and TRAAK with TRP channels in the trigeminal ganglion cells. Neurosci Lett 454, 129-133, doi: 10.1016/j.neulet.2009.02.069 (2009).

14 Jain, A., Liu, R., Xiang, Y. K. & Ha, T. Single-molecule pull-down for studying protein interactions. Nat Protoc 7, 445-452, doi: 10.1038/nprot.2011.452 (2012).

15 Jain, A. et al. Probing cellular protein complexes using single-molecule pull-down. Nature 473, 484-488, doi: 10.1038/naturel0016 (2011).

16 Sandoz, G., Levitz, J., Kramer, R. H. & Isacoff, E. Y. Optical control of endogenous proteins with a photoswitchable conditional subunit reveals a role for TREK1 in GABA(B) signaling. Neuron 74, 1005-1014, doi: 10.1016/j.neuron.2012.04.026 (2012).

17 Thomas, D., Plant, L. D., Wilkens, C. M., McCrossan, Z. A. & Goldstein, S. A. Alternative translation initiation in rat brain yields K2P2.1 potassium channels permeable to sodium. Neuron 58, 859-870, doi: 10.1016/j.neuron.2008.04.016 (2008).

18 Kochetov, A. V. Alternative translation start sites and hidden coding potential of eukaryotic niRNAs. Bioessays 30, 683-691, doi: 10.1002/bies.20771 (2008).

19 Guyon, A. et al. Glucose inhibition persists in hypothalamic neurons lacking tandem- pore K+ channels. J Neurosci 29, 2528-2533, doi:10.1523/JNEUROSCI.5764-08.2009 (2009).