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Migraine: Role of the TRESK two-pore potassium channel
The International Journal of Biochemistry & Cell Biology 43 (2011) 1533–1536
Contents lists available at ScienceDirect
The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Medicine in focus
Migraine: Role of the TRESK two-pore potassium channel Ronald G. Lafrenière a,∗ , Guy A. Rouleau a,b a b
Center of Excellence in Neuroscience of the Université de Montréal (CENUM), Montreal, Quebec, Canada Centre de Recherche du Centre Hospitalier de l’Université de Montréal (CRCHUM), and Department of Medicine, University of Montreal, Montreal, Quebec, Canada
a r t i c l e
i n f o
Article history: Received 30 May 2011 Received in revised form 1 August 2011 Accepted 2 August 2011 Available online 9 August 2011 Keywords: Migraine TRESK Calcineurin Therapeutic Genetics
a b s t r a c t Migraine is a severe episodic headache disorder affecting one in five people. Genetic studies have identified mutations in the CACNA1, ATP1A2 and SCN1A genes in the rare familial hemiplegic migraine. Recently, a mutation in the KCNK18 gene, encoding the TRESK two-pore domain potassium channel, was described in a large family with migraine with aura. This review will elaborate on the possible role of the TRESK channel in regulating neuronal excitability, its role in migraine pathogenesis, and on promising therapeutic opportunities targeting this channel. Crown Copyright © 2011 Published by Elsevier Ltd. All rights reserved.
1. Introduction Migraine consists of recurrent and disabling headaches, usually accompanied by nausea, vomiting, phonophobia or photophobia, or other autonomic symptoms (International Headache Society, 2004). Often the attacks are precipitated by external triggers such as alcohol, lack of sleep, or stress. In ∼20% of cases, attacks are preceded by visual disturbances known as aura that may take the form of scintillating shapes, hallucinations, or black spots. The aura is thought to be associated with the phenomenon of cortical spreading depression (CSD), a wave of neuronal depolarization that slowly propagates over the cortical regions of the brain. Migraine is the most common neurological disorder, affecting ∼12% of the population, and occurs three times more often in women than men. 2. Pathogenesis Prevailing models of migraine pathogenesis invoke neural hyperexcitation as a key mechanism. The hyperexcitation can activate CSD, which in some migraine sufferers is experienced as an aura. The CSD has been shown to directly activate nerves in the trigeminal ganglion leading to release of pro-inflammatory peptides (such as CGRP and substance P) in the meninges, the thin
∗ Corresponding author at: Center of Excellence in Neuroscience of the Université de Montréal (CENUM), CHUM Research Center, De-Seve Pavilion, Room Y-3633, Notre-Dame Hospital, 1560 Sherbrooke Street East, Montréal, Québec, Canada, H2L 4M1. Tel.: +1 514 890 8000x24753; fax: +1 514 412 7602. E-mail address: [email protected] (R.G. Lafrenière).
membranes covering the surface of the brain (Zhang et al., 2011, 2010). Unlike the brain, the meninges and cerebral blood vessels are pain-sensitive structures heavily innervated by the trigeminal nerve. Inflammation in the meninges further activates the trigeminal nerve and produces a painful throbbing headache. Hence migraine is a culmination of inappropriate neuronal hyperactivity leading to localized inflammation in the meninges and painful headache. The associated nausea and aversion to bright lights and loud noises is due to sensitization of neurons in several brain stem regions that modulate these sensory modalities. Over 70% of migraine sufferers have a family history of migraine, suggesting a strong genetic predisposition. Linkage studies in families affected with familial hemiplegic migraine (FHM), a heterogeneous headache disorder that also involves partial limb paralysis during an attack, have identified mutations in three different genes: CACNA1A, ATP1A2, and SCN1A (reviewed in de Vries et al., 2009). These FHM genes encode proteins involved in regulating membrane potential by controlling the flow of ions (calcium, sodium, potassium) in and out of cells. 2.1. Discovery of TRESK as a migraine target We asked whether other brain-expressed ion channel genes might be mutated in migraine patients by screening 150 such genes in a collection of 110 unrelated migraine patients. We previously published a detailed description of the discovery of a frameshifting mutation in the KCNK18 gene, encoding the TRESK two-pore potassium channel protein, in a patient suffering from migraine with aura (Lafreniere et al., 2010). This mutation was present in 7 of the patient’s relatives who also suffered from migraine with
1357-2725/$ – see front matter. Crown Copyright © 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2011.08.002
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aura, and was absent from 8 relatives who did not have migraine. The frameshift mutation leads to a prematurely truncated and nonfunctional TRESK channel that can also suppress wild-type channel function through a dominant-negative effect. Mutation of TRESK is not a common cause of migraine as we found only one patient with a mutation in our 110 cases and over 500 additional unrelated migraine patients screened. However, one of our collaborators has since identified a second unrelated frameshifting mutation in KCNK18 in a migraine patient of Arab descent (unpublished). As mutations in the SCN9A gene (encoding the Nav1.7 sodium channel alpha subunit) were found to cause the very rare condition of congenital indifference to pain but identified Nav1.7 as a critical component of the pain signaling pathway (Cox et al., 2006; Goldberg et al., 2007), we believe that mutation of TRESK similarly identifies this channel as a critical component of the migraine pain signaling pathway. It is unknown whether the aura is directly related to disruption of TRESK, or due to other factors. Thus far, patients with TRESK mutations suffer from migraine with aura and do not have hemiplegias during an attack. Hence, these subjects have classical migraine. Our discovery therefore links for the first time a common form of migraine to a genetic defect. 2.2. K2P channels TRESK is a member of the two-pore potassium (K2P) channel family, which in human includes 15 related channels (Enyedi and Czirjak, 2010). These channels generate background K+ leak currents controlling the resting membrane potential. K2P channels are modulated dynamically by sensitive chemical and physical triggers, such as pH, cAMP, arachidonic acid, stretch, etc. TRESK is the first member of the family linked to a human disorder. 2.3. TRESK expression pattern Using in situ hybridization in mouse we showed expression of Kcnk18, starting at embryonic day 15.5 and continuing into adult, was highest in dorsal root ganglia (DRG) and in trigeminal ganglia (TG), both neuronal structures important in pain sensation (Fig. 1). There was also expression in several autonomic nervous system ganglia (e.g. stellate ganglia and paravertebral sympathetic ganglia). Small and medium sized sensory neurons were labeled. This is in general agreement with expression studies in rat (Yoo et al., 2009) and mouse (Dobler et al., 2007) where high levels in DRG were noted. Most significantly, high levels of Kcnk18 in DRG and TG neurons link TRESK to neuronal structures that have been directly implicated in migraine or pain signaling circuits. 2.4. TRESK structure The TRESK protein is thought to form homodimers similar to other K2P channels (Lesage et al., 1996). Each subunit contains 4 transmembrane domains (TMDs), and two pore-forming domains. The extracellular domain located between TMD1 and TMD2 contains a conserved Cysteine residue that may form a disulfide bridge to aid channel dimerization, and a conserved N-linked glycosylation site important for surface expression of the channel (Egenberger et al., 2010). TRESK is unique among K2P channels in having a large intracellular regulatory domain located between TMD2 and TMD3. This domain contains two Serine residues that are phosphorylated by kinases to downregulate channel activity, and are dephosphorylated by calcineurin in response to elevated intracellular Ca2+ , thus activating the channel (Czirjak et al., 2004). At least two independent inhibitory pathways converge on these sites: Ser264 in mouse (Ser252 in human) is likely phosphorylated by Protein Kinase A and binds the and ␥ isoforms of 14-3-3 proteins leading to inhibition (Czirjak et al., 2004, 2008) whereas Ser276
Fig. 1. In situ hybridization showing the expression pattern of the Kcnk18 gene specifically in DRG and TG in mouse at embryonic day 18.5 (e18.5). (A) Hematoxylin staining, (B) antisense (as) probe labeling, (C) sense (s) probe labeling. Abbreviations: Br – brain; DRG – dorsal root ganglion; H – heart; K – kidney; Li – liver; Lu – lung; TG – trigeminal ganglion; Th – thymus.
in mouse (Ser264 in human) is phosphorylated by an unknown kinase (Czirjak and Enyedi, 2010). This regulatory domain therefore confers upon TRESK a dynamic switch in response to Ca2+ signaling. 2.5. TRESK function Perhaps the greatest insight we have on the function of TRESK is from an ENU-derived mouse knockout (Dobler et al., 2007). A missense (Gly339Arg) which destroys the highly conserved GFGD motif of the second pore domain, leads to a complete loss of TRESK channel function in homozygous mice. The mice are viable and fertile, and show no gross anatomical or behavioral phenotype. Whereas TRESK was demonstrated to be expressed at high levels in DRG, surprisingly the KO has minimal effect on the resting membrane potential. Instead, DRG neurons from TRESK KO mice displayed a lower threshold for activation, reduced action potential duration, and slightly higher amplitudes of after-hyperpolarization, suggesting that DRG neurons from KO mice were more excitable than WT DRG neurons. The authors also discuss the coupling of TRESK to the histamine H1 receptor, mention that histamine can activate TRESK channels, and speculate that the physiological role of TRESK currents is to dampen cellular excitability in inflammatory responses when histamine or other inflammatory response modulators are released into the surrounding tissue. Interestingly, migraine symptoms (frequency, severity, duration, pain intensity)
R.G. Lafrenière, G.A. Rouleau / The International Journal of Biochemistry & Cell Biology 43 (2011) 1533–1536
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increasing the open probability of the channels (Liu et al., 2004), suggesting that they have a direct effect on TRESK. TRESK showed the highest sensitivity to volatile anesthetics of all the K2P channels (Liu et al., 2004). Halothane activation of the related K2P channels TREK1 and TASK1 could be abolished by mutating a 6 amino acid motif located in the C-terminal domain (Talley and Bayliss, 2002), suggesting that halothane interacts directly with the channels. In vivo isoflurane sensitivity was increased in a different TRESK KO mouse model (Chae et al., 2010). These studies therefore identify volatile anesthetics as a class of highly potent TRESK agonists and link TRESK activation to a possible mechanism of action of volatile anesthetics.
3. Therapy
Fig. 2. Modulators of TRESK activity. Elevated intracellular Ca2+ activates calcineurin, which in turn can activate TRESK and NFATs. The immunosuppressant drugs Cyclosporin A and FK506 can block the action of calcineurin. Volatile anesthetics can activate TRESK channels directly, while binding of 14-3-3 proteins can downregulate TRESK activity. TRESK activation should increase the threshold of excitability of trigeminal ganglion neurons, and help prevent or reduce severity of migraines.
were decreased by about 50% in patients using subcutaneous histamine as a migraine prophylactic (Millan-Guerrero et al., 2008). Given that migraine is intimately linked to inflammation of the meninges and associated trigeminal afferents, these studies support a role for TRESK in controlling the excitability of TG neurons during inflammation. A more recent study has also highlighted the role of TRESK in controlling neuronal excitability (Tulleuda et al., 2011). 2.6. TRESK modulators and migraine Dephosphorylation of Ser264 by calcineurin increases TRESK activity. Calcineurin also regulates NFATs (nuclear factor of activated T cells), transcription factors that regulate inducible expression of many cytokines. Cyclosporin A and FK506 (tacrolimus), two potent immunosuppressants in clinical use that specifically inhibit the calcineurin activation of NFATs, are predicted to mimic a TRESK loss of function by keeping the channel insensitive to increases in intracellular Ca2+ (Fig. 2). Is the use of these immunosuppressants associated with migraine? Indeed “Calcineurin inhibitor-induced headache” was described in a cohort of 74 organ transplant patients receiving Cyclosporin and/or tacrolimus: 38% of patients developed a new headache syndrome (mainly migraines or probable migraines) within 3 years of the organ transplantation, while 23% of patients had an increase in the frequency or intensity of a preexisting headache (Ferrari et al., 2005). An earlier study (Steiger et al., 1994) found that out of 34 patients who had undergone orthoptic liver transplantation and were on Cyclosporin, 6 had developed a recurrent headache typical of migraine. In 2 of these patients, the headache pain was improved by reducing the dose of Cyclosporin. Hence, there is indirect evidence that pharmacological inhibition of calcineurin (and presumably inhibition of TRESK activity) can increase the frequency and/or severity of migraine headaches. Volatile anesthetics, such as isoflurane and halothane, produced nearly a doubling of TRESK current at clinically relevant doses by
The most commonly used drugs for acute migraine attacks are the triptans, which mimic the molecular structure of serotonin, and act as selective serotonin (5-HT1B and 5-HT1D) receptor agonists. Activation of the serotonin receptors inhibits release of the pro-inflammatory peptides by the trigeminal neurons. Other drugs used for acute migraines include ergotamines, and non-steroidal anti-inflammatory agents (NSAIDs), but these have higher risks of adverse effects. For migraine prophylaxis, more generally acting agents such as beta-blockers, anticonvulsants, antidepressants, calcium channel blockers, and NSAIDs are used. Some of these can reduce the number of migraine attacks by up to 50% in some individuals. However current migraine therapy is consistently effective in only 19–31% of patients, and only 21–50% of patients are satisfied with their current treatment (MacGregor et al., 2003). Hence, there is a need for more effective migraine treatments. Based on the agonist action of volatile anesthetics, we believe it is possible to develop novel small compound agonists of TRESK that would be highly selective and potent, and could be used as abortive or prophylactic drugs. TRESK is an ion channel expressed at the surface of the neuron, so it may be possible to develop agonists that do not need to get into the cells to work. Based on our in situ hybridization studies in mice, the gene is expressed in a very limited set of neurons, and as such, modulating its activity would be predicted to have minimal effect on tissues where it is not expressed. The trigeminal ganglion also lies outside the blood–brain barrier, and is thus more accessible to small compounds than other structures located in the brain or brainstem. Finally, given that TRESK is distantly related to other K2P channels, it should be possible to develop TRESK agonists that are highly selective. The challenge will be to develop small compound TRESK agonists that can decrease the excitability of TRESK-expressing neurons without increasing unwanted anesthetic or immune system side-effects.
3.1. Conclusions The TRESK K2P channel represents a novel and interesting component of the migraine pathogenesis pathway and may lead to new approaches to treat migraine headaches. Its highly specific expression pattern in TG neurons and presumed role in abating neuronal excitability (Dobler et al., 2007; Tulleuda et al., 2011) under inflammatory conditions make it an excellent target for development of migraine therapeutics. Given the prior knowledge that volatile anesthetics act as agonists on the TRESK channel, and their potential mode of action, it may be possible to test a small collection of compounds with similar structures using a medicinal chemistry approach. Since TRESK is specifically expressed in DRG, TG and parasympathetic neurons, modulators of TRESK could conceivably have use in other pain disorders (especially inflammatory pain), or as medications controlling sweating, nausea, or hypertension.
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Further research will determine the importance of TRESK as a novel therapeutic target for migraine or related pain disorders. References Chae YJ, Zhang J, Au P, Sabbadini M, Xie GX, Yost CS. Discrete change in volatile anesthetic sensitivity in mice with inactivated tandem pore potassium ion channel TRESK. Anesthesiology 2010;113:1326–37. Cox JJ, Reimann F, Nicholas AK, Thornton G, Roberts E, Springell K, et al. An SCN9A channelopathy causes congenital inability to experience pain. Nature 2006;444:894–8. Czirjak G, Enyedi P. TRESK background K(+) channel is inhibited by phosphorylation via two distinct pathways. J Biol Chem 2010;285:14549–57. Czirjak G, Toth ZE, Enyedi P. The two-pore domain K+ channel, TRESK, is activated by the cytoplasmic calcium signal through calcineurin. J Biol Chem 2004;279:18550–8. Czirjak G, Vuity D, Enyedi P. Phosphorylation-dependent binding of 14-3-3 proteins controls TRESK regulation. J Biol Chem 2008;283:15672–80. de Vries B, Frants RR, Ferrari MD, van den Maagdenberg AM. Molecular genetics of migraine. Hum Genet 2009;126:115–32. Dobler T, Springauf A, Tovornik S, Weber M, Schmitt A, Sedlmeier R, et al. TRESK two-pore-domain K+ channels constitute a significant component of background potassium currents in murine dorsal root ganglion neurones. J Physiol 2007;585:867–79. Egenberger B, Polleichtner G, Wischmeyer E, Doring F. N-linked glycosylation determines cell surface expression of two-pore-domain K+ channel TRESK. Biochem Biophys Res Commun 2010;391:1262–7. Enyedi P, Czirjak G. Molecular background of leak K+ currents: two-pore domain potassium channels. Physiol Rev 2010;90:559–605. Ferrari U, Empl M, Kim KS, Sostak P, Forderreuther S, Straube A. Calcineurin inhibitorinduced headache: clinical characteristics and possible mechanisms. Headache 2005;45:211–4. Goldberg YP, MacFarlane J, MacDonald ML, Thompson J, Dube MP, Mattice M, et al. Loss-of-function mutations in the Nav1.7 gene underlie congenital
indifference to pain in multiple human populations. Clin Genet 2007;71: 311–9. International Headache Society. The International Classification of Headache Disorders: 2nd edition. Cephalalgia 2004;24(Suppl. 1):9–160. Lafreniere RG, Cader MZ, Poulin JF, Andres-Enguix I, Simoneau M, Gupta N, et al. A dominant-negative mutation in the TRESK potassium channel is linked to familial migraine with aura. Nat Med 2010;16:1157–60. Lesage F, Reyes R, Fink M, Duprat F, Guillemare E, Lazdunski M. Dimerization of TWIK-1 K+ channel subunits via a disulfide bridge. EMBO J 1996;15:6400–7. Liu C, Au JD, Zou HL, Cotten JF, Yost CS. Potent activation of the human tandem pore domain K channel TRESK with clinical concentrations of volatile anesthetics. Anesth Analg 2004;99:1715–22. MacGregor EA, Brandes J, Eikermann A. Migraine prevalence and treatment patterns: the global Migraine and Zolmitriptan Evaluation survey. Headache 2003;43:19–26. Millan-Guerrero RO, Isais-Millan R, Barreto-Vizcaino S, Gutierrez I, Rivera-Castano L, Trujillo-Hernandez B, et al. Subcutaneous histamine versus topiramate in migraine prophylaxis: a double-blind study. Eur Neurol 2008;59:237–42. Steiger MJ, Farrah T, Rolles K, Harvey P, Burroughs AK. Cyclosporin associated headache. J Neurol Neurosurg Psychiatry 1994;57:1258–9. Talley EM, Bayliss DA. Modulation of TASK-1 (Kcnk3) and TASK-3 (Kcnk9) potassium channels: volatile anesthetics and neurotransmitters share a molecular site of action. J Biol Chem 2002;277:17733–42. Tulleuda A, Cokic B, Callejo G, Saiani B, Serra J, Gasull X. TRESK channel contribution to nociceptive sensory neurons excitability: modulation by nerve injury. Mol Pain 2011;7:30. Yoo S, Liu J, Sabbadini M, Au P, Xie GX, Yost CS. Regional expression of the anestheticactivated potassium channel TRESK in the rat nervous system. Neurosci Lett 2009;465:79–84. Zhang X, Levy D, Kainz V, Noseda R, Jakubowski M, Burstein R. Activation of central trigeminovascular neurons by cortical spreading depression. Ann Neurol 2011;69:855–65. Zhang X, Levy D, Noseda R, Kainz V, Jakubowski M, Burstein R. Activation of meningeal nociceptors by cortical spreading depression: implications for migraine with aura. J Neurosci 2010;30:8807–14.
Contents lists available at ScienceDirect
The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Medicine in focus
Migraine: Role of the TRESK two-pore potassium channel Ronald G. Lafrenière a,∗ , Guy A. Rouleau a,b a b
Center of Excellence in Neuroscience of the Université de Montréal (CENUM), Montreal, Quebec, Canada Centre de Recherche du Centre Hospitalier de l’Université de Montréal (CRCHUM), and Department of Medicine, University of Montreal, Montreal, Quebec, Canada
a r t i c l e
i n f o
Article history: Received 30 May 2011 Received in revised form 1 August 2011 Accepted 2 August 2011 Available online 9 August 2011 Keywords: Migraine TRESK Calcineurin Therapeutic Genetics
a b s t r a c t Migraine is a severe episodic headache disorder affecting one in five people. Genetic studies have identified mutations in the CACNA1, ATP1A2 and SCN1A genes in the rare familial hemiplegic migraine. Recently, a mutation in the KCNK18 gene, encoding the TRESK two-pore domain potassium channel, was described in a large family with migraine with aura. This review will elaborate on the possible role of the TRESK channel in regulating neuronal excitability, its role in migraine pathogenesis, and on promising therapeutic opportunities targeting this channel. Crown Copyright © 2011 Published by Elsevier Ltd. All rights reserved.
1. Introduction Migraine consists of recurrent and disabling headaches, usually accompanied by nausea, vomiting, phonophobia or photophobia, or other autonomic symptoms (International Headache Society, 2004). Often the attacks are precipitated by external triggers such as alcohol, lack of sleep, or stress. In ∼20% of cases, attacks are preceded by visual disturbances known as aura that may take the form of scintillating shapes, hallucinations, or black spots. The aura is thought to be associated with the phenomenon of cortical spreading depression (CSD), a wave of neuronal depolarization that slowly propagates over the cortical regions of the brain. Migraine is the most common neurological disorder, affecting ∼12% of the population, and occurs three times more often in women than men. 2. Pathogenesis Prevailing models of migraine pathogenesis invoke neural hyperexcitation as a key mechanism. The hyperexcitation can activate CSD, which in some migraine sufferers is experienced as an aura. The CSD has been shown to directly activate nerves in the trigeminal ganglion leading to release of pro-inflammatory peptides (such as CGRP and substance P) in the meninges, the thin
∗ Corresponding author at: Center of Excellence in Neuroscience of the Université de Montréal (CENUM), CHUM Research Center, De-Seve Pavilion, Room Y-3633, Notre-Dame Hospital, 1560 Sherbrooke Street East, Montréal, Québec, Canada, H2L 4M1. Tel.: +1 514 890 8000x24753; fax: +1 514 412 7602. E-mail address: [email protected] (R.G. Lafrenière).
membranes covering the surface of the brain (Zhang et al., 2011, 2010). Unlike the brain, the meninges and cerebral blood vessels are pain-sensitive structures heavily innervated by the trigeminal nerve. Inflammation in the meninges further activates the trigeminal nerve and produces a painful throbbing headache. Hence migraine is a culmination of inappropriate neuronal hyperactivity leading to localized inflammation in the meninges and painful headache. The associated nausea and aversion to bright lights and loud noises is due to sensitization of neurons in several brain stem regions that modulate these sensory modalities. Over 70% of migraine sufferers have a family history of migraine, suggesting a strong genetic predisposition. Linkage studies in families affected with familial hemiplegic migraine (FHM), a heterogeneous headache disorder that also involves partial limb paralysis during an attack, have identified mutations in three different genes: CACNA1A, ATP1A2, and SCN1A (reviewed in de Vries et al., 2009). These FHM genes encode proteins involved in regulating membrane potential by controlling the flow of ions (calcium, sodium, potassium) in and out of cells. 2.1. Discovery of TRESK as a migraine target We asked whether other brain-expressed ion channel genes might be mutated in migraine patients by screening 150 such genes in a collection of 110 unrelated migraine patients. We previously published a detailed description of the discovery of a frameshifting mutation in the KCNK18 gene, encoding the TRESK two-pore potassium channel protein, in a patient suffering from migraine with aura (Lafreniere et al., 2010). This mutation was present in 7 of the patient’s relatives who also suffered from migraine with
1357-2725/$ – see front matter. Crown Copyright © 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2011.08.002
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aura, and was absent from 8 relatives who did not have migraine. The frameshift mutation leads to a prematurely truncated and nonfunctional TRESK channel that can also suppress wild-type channel function through a dominant-negative effect. Mutation of TRESK is not a common cause of migraine as we found only one patient with a mutation in our 110 cases and over 500 additional unrelated migraine patients screened. However, one of our collaborators has since identified a second unrelated frameshifting mutation in KCNK18 in a migraine patient of Arab descent (unpublished). As mutations in the SCN9A gene (encoding the Nav1.7 sodium channel alpha subunit) were found to cause the very rare condition of congenital indifference to pain but identified Nav1.7 as a critical component of the pain signaling pathway (Cox et al., 2006; Goldberg et al., 2007), we believe that mutation of TRESK similarly identifies this channel as a critical component of the migraine pain signaling pathway. It is unknown whether the aura is directly related to disruption of TRESK, or due to other factors. Thus far, patients with TRESK mutations suffer from migraine with aura and do not have hemiplegias during an attack. Hence, these subjects have classical migraine. Our discovery therefore links for the first time a common form of migraine to a genetic defect. 2.2. K2P channels TRESK is a member of the two-pore potassium (K2P) channel family, which in human includes 15 related channels (Enyedi and Czirjak, 2010). These channels generate background K+ leak currents controlling the resting membrane potential. K2P channels are modulated dynamically by sensitive chemical and physical triggers, such as pH, cAMP, arachidonic acid, stretch, etc. TRESK is the first member of the family linked to a human disorder. 2.3. TRESK expression pattern Using in situ hybridization in mouse we showed expression of Kcnk18, starting at embryonic day 15.5 and continuing into adult, was highest in dorsal root ganglia (DRG) and in trigeminal ganglia (TG), both neuronal structures important in pain sensation (Fig. 1). There was also expression in several autonomic nervous system ganglia (e.g. stellate ganglia and paravertebral sympathetic ganglia). Small and medium sized sensory neurons were labeled. This is in general agreement with expression studies in rat (Yoo et al., 2009) and mouse (Dobler et al., 2007) where high levels in DRG were noted. Most significantly, high levels of Kcnk18 in DRG and TG neurons link TRESK to neuronal structures that have been directly implicated in migraine or pain signaling circuits. 2.4. TRESK structure The TRESK protein is thought to form homodimers similar to other K2P channels (Lesage et al., 1996). Each subunit contains 4 transmembrane domains (TMDs), and two pore-forming domains. The extracellular domain located between TMD1 and TMD2 contains a conserved Cysteine residue that may form a disulfide bridge to aid channel dimerization, and a conserved N-linked glycosylation site important for surface expression of the channel (Egenberger et al., 2010). TRESK is unique among K2P channels in having a large intracellular regulatory domain located between TMD2 and TMD3. This domain contains two Serine residues that are phosphorylated by kinases to downregulate channel activity, and are dephosphorylated by calcineurin in response to elevated intracellular Ca2+ , thus activating the channel (Czirjak et al., 2004). At least two independent inhibitory pathways converge on these sites: Ser264 in mouse (Ser252 in human) is likely phosphorylated by Protein Kinase A and binds the and ␥ isoforms of 14-3-3 proteins leading to inhibition (Czirjak et al., 2004, 2008) whereas Ser276
Fig. 1. In situ hybridization showing the expression pattern of the Kcnk18 gene specifically in DRG and TG in mouse at embryonic day 18.5 (e18.5). (A) Hematoxylin staining, (B) antisense (as) probe labeling, (C) sense (s) probe labeling. Abbreviations: Br – brain; DRG – dorsal root ganglion; H – heart; K – kidney; Li – liver; Lu – lung; TG – trigeminal ganglion; Th – thymus.
in mouse (Ser264 in human) is phosphorylated by an unknown kinase (Czirjak and Enyedi, 2010). This regulatory domain therefore confers upon TRESK a dynamic switch in response to Ca2+ signaling. 2.5. TRESK function Perhaps the greatest insight we have on the function of TRESK is from an ENU-derived mouse knockout (Dobler et al., 2007). A missense (Gly339Arg) which destroys the highly conserved GFGD motif of the second pore domain, leads to a complete loss of TRESK channel function in homozygous mice. The mice are viable and fertile, and show no gross anatomical or behavioral phenotype. Whereas TRESK was demonstrated to be expressed at high levels in DRG, surprisingly the KO has minimal effect on the resting membrane potential. Instead, DRG neurons from TRESK KO mice displayed a lower threshold for activation, reduced action potential duration, and slightly higher amplitudes of after-hyperpolarization, suggesting that DRG neurons from KO mice were more excitable than WT DRG neurons. The authors also discuss the coupling of TRESK to the histamine H1 receptor, mention that histamine can activate TRESK channels, and speculate that the physiological role of TRESK currents is to dampen cellular excitability in inflammatory responses when histamine or other inflammatory response modulators are released into the surrounding tissue. Interestingly, migraine symptoms (frequency, severity, duration, pain intensity)
R.G. Lafrenière, G.A. Rouleau / The International Journal of Biochemistry & Cell Biology 43 (2011) 1533–1536
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increasing the open probability of the channels (Liu et al., 2004), suggesting that they have a direct effect on TRESK. TRESK showed the highest sensitivity to volatile anesthetics of all the K2P channels (Liu et al., 2004). Halothane activation of the related K2P channels TREK1 and TASK1 could be abolished by mutating a 6 amino acid motif located in the C-terminal domain (Talley and Bayliss, 2002), suggesting that halothane interacts directly with the channels. In vivo isoflurane sensitivity was increased in a different TRESK KO mouse model (Chae et al., 2010). These studies therefore identify volatile anesthetics as a class of highly potent TRESK agonists and link TRESK activation to a possible mechanism of action of volatile anesthetics.
3. Therapy
Fig. 2. Modulators of TRESK activity. Elevated intracellular Ca2+ activates calcineurin, which in turn can activate TRESK and NFATs. The immunosuppressant drugs Cyclosporin A and FK506 can block the action of calcineurin. Volatile anesthetics can activate TRESK channels directly, while binding of 14-3-3 proteins can downregulate TRESK activity. TRESK activation should increase the threshold of excitability of trigeminal ganglion neurons, and help prevent or reduce severity of migraines.
were decreased by about 50% in patients using subcutaneous histamine as a migraine prophylactic (Millan-Guerrero et al., 2008). Given that migraine is intimately linked to inflammation of the meninges and associated trigeminal afferents, these studies support a role for TRESK in controlling the excitability of TG neurons during inflammation. A more recent study has also highlighted the role of TRESK in controlling neuronal excitability (Tulleuda et al., 2011). 2.6. TRESK modulators and migraine Dephosphorylation of Ser264 by calcineurin increases TRESK activity. Calcineurin also regulates NFATs (nuclear factor of activated T cells), transcription factors that regulate inducible expression of many cytokines. Cyclosporin A and FK506 (tacrolimus), two potent immunosuppressants in clinical use that specifically inhibit the calcineurin activation of NFATs, are predicted to mimic a TRESK loss of function by keeping the channel insensitive to increases in intracellular Ca2+ (Fig. 2). Is the use of these immunosuppressants associated with migraine? Indeed “Calcineurin inhibitor-induced headache” was described in a cohort of 74 organ transplant patients receiving Cyclosporin and/or tacrolimus: 38% of patients developed a new headache syndrome (mainly migraines or probable migraines) within 3 years of the organ transplantation, while 23% of patients had an increase in the frequency or intensity of a preexisting headache (Ferrari et al., 2005). An earlier study (Steiger et al., 1994) found that out of 34 patients who had undergone orthoptic liver transplantation and were on Cyclosporin, 6 had developed a recurrent headache typical of migraine. In 2 of these patients, the headache pain was improved by reducing the dose of Cyclosporin. Hence, there is indirect evidence that pharmacological inhibition of calcineurin (and presumably inhibition of TRESK activity) can increase the frequency and/or severity of migraine headaches. Volatile anesthetics, such as isoflurane and halothane, produced nearly a doubling of TRESK current at clinically relevant doses by
The most commonly used drugs for acute migraine attacks are the triptans, which mimic the molecular structure of serotonin, and act as selective serotonin (5-HT1B and 5-HT1D) receptor agonists. Activation of the serotonin receptors inhibits release of the pro-inflammatory peptides by the trigeminal neurons. Other drugs used for acute migraines include ergotamines, and non-steroidal anti-inflammatory agents (NSAIDs), but these have higher risks of adverse effects. For migraine prophylaxis, more generally acting agents such as beta-blockers, anticonvulsants, antidepressants, calcium channel blockers, and NSAIDs are used. Some of these can reduce the number of migraine attacks by up to 50% in some individuals. However current migraine therapy is consistently effective in only 19–31% of patients, and only 21–50% of patients are satisfied with their current treatment (MacGregor et al., 2003). Hence, there is a need for more effective migraine treatments. Based on the agonist action of volatile anesthetics, we believe it is possible to develop novel small compound agonists of TRESK that would be highly selective and potent, and could be used as abortive or prophylactic drugs. TRESK is an ion channel expressed at the surface of the neuron, so it may be possible to develop agonists that do not need to get into the cells to work. Based on our in situ hybridization studies in mice, the gene is expressed in a very limited set of neurons, and as such, modulating its activity would be predicted to have minimal effect on tissues where it is not expressed. The trigeminal ganglion also lies outside the blood–brain barrier, and is thus more accessible to small compounds than other structures located in the brain or brainstem. Finally, given that TRESK is distantly related to other K2P channels, it should be possible to develop TRESK agonists that are highly selective. The challenge will be to develop small compound TRESK agonists that can decrease the excitability of TRESK-expressing neurons without increasing unwanted anesthetic or immune system side-effects.
3.1. Conclusions The TRESK K2P channel represents a novel and interesting component of the migraine pathogenesis pathway and may lead to new approaches to treat migraine headaches. Its highly specific expression pattern in TG neurons and presumed role in abating neuronal excitability (Dobler et al., 2007; Tulleuda et al., 2011) under inflammatory conditions make it an excellent target for development of migraine therapeutics. Given the prior knowledge that volatile anesthetics act as agonists on the TRESK channel, and their potential mode of action, it may be possible to test a small collection of compounds with similar structures using a medicinal chemistry approach. Since TRESK is specifically expressed in DRG, TG and parasympathetic neurons, modulators of TRESK could conceivably have use in other pain disorders (especially inflammatory pain), or as medications controlling sweating, nausea, or hypertension.
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