Perspectives on the Two-Pore Domain Potassium Channel TREK-1

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Perspective

Perspectives on the two-pore domain potassium channel TREK-1 (TWIK-Related K+ channel 1), a novel therapeutic target? Delphine VIVIER, Khalil BENNIS, Florian Lesage, and Sylvie Ducki J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00671 • Publication Date (Web): 20 Nov 2015 Downloaded from http://pubs.acs.org on November 28, 2015

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Journal of Medicinal Chemistry

Perspectives on the two-pore domain potassium channel TREK-1 (TWIK-Related K+ channel 1), a novel therapeutic target?

Delphine Vivier, 1,2 Khalil Bennis, 1,2 Florian Lesage,3 Sylvie Ducki* 1,2 (1) Université Clermont Auvergne, ENSCCF, Institut de Chimie de Clermont-Ferrand, BP 10448, F-63000 CLERMONT-FERRAND. (2) CNRS, UMR6296, ICCF, F-63171 AUBIERE (3) Labex ICST, Institut de Pharmacologie Moléculaire et Cellulaire, UMR CNRS 7275, Université de Nice Sophia Antipolis, F-06560 VALBONNE

ACS Paragon Plus Environment

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KEYWORDS: Two-pore domain potassium (K2P) channel, TREK-1 channel, pain, analgesia, depression, therapeutic target,

ABSTRACT. Potassium (K+) channels are membrane proteins expressed in most living cells that selectively control the flow of K+ ions. More than 80 genes encode the K+ channel subunits in the human genome. The TWIK-Related K+ channel (TREK-1) belongs to the two-pore domain K+ channels (K2P) and displays various properties including sensitivity to physical (membrane stretch, acidosis, temperature) and chemical stimuli (signaling lipids, volatile anesthetics). The distribution of TREK-1 in the central nervous system, coupled with the physiological consequences of its opening and closing, lead to the emergence of this channel as an attractive therapeutic target. This perspective reviews the TREK-1 channel, its structural and functional properties, and the pharmacological agents (agonists and antagonists) able to modulate its gating.


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INTRODUCTION Selective diffusion of ions through ion channels is essential for electrolyte homeostasis, cell development and excitability. Potassium (K+) channels allow K+ ions to exit the cell, creating an excess of negative charges inside the cell and driving the electrical membrane potential to negative values. Opening of calcium (Ca2+) and sodium (Na+) channels has the opposite effect, allowing an inward rush of positive charges, which promote depolarization. Thus, at any moment, the cell excitability relies directly on the activated ion channels, Ca2+ and Na+ influx favoring excitability and a K+ efflux opposing it. With over 80 genes in the human genome, K+ channels are one of the largest and most structurally diverse families of ion channels.1 K+ channels are assemblies of transmembrane proteins that selectively control the flow of K+ ions between the extracellular and intracellular compartments. They are known to regulate a wide array of neuronal functions, from resting membrane potential and intrinsic excitability to action potential repolarization and propagation. Among the three main classes of K+ channels, the twopore domain (K2P) K+ channel family includes 15 mammalian members (KCNK genes).2 Although the K2P channels show low sequence similarity (28% sequence similarity between TWIK-1 and TREK-1), they are characterized by the same general 3D architecture. The K2P channel subunits are defined by four transmembrane helices (M1-M4) and two pore domains (P1-P2) which dimerize to form the K+ selectivity filter. One of the most studied members of this family is the TWIK-Related K+ channel (TREK-1), discovered in 1996.3,4 This perspective presents an overview of the TREK-1 channel; its structure, its function, its physiological role and pathological implication. We review the ligands that have been reported to modulate the TREK-1 channel (activators/agonists and inhibitors/antagonists) and their potential application in drug therapy.


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STRUCTURE AND FUNCTION OF TREK-1 The longest isoform of the human TREK-1 subunit (gene: KCNK2, FASTA: O95069-1) is composed of 426 amino acids (same for the mouse protein, gene Kcnk2, FASTA: P97438-1). The protein is composed of four helical transmembrane segments (M1-M4), two pore domains (P1-P2), an extracellular 60-residue cap (consisting of two cap helices C1 and C2) and intracellular N- and C-termini (Figure 1). Both P domains are conventional since they possess the signature sequence GFG (159-161 and 268-270). The active TREK-1 channel is a homodimer of TREK-1 subunits (Figure 2A) which assemble in an anti-parallel manner : the first pore domains (M1-C1-C2-P1-M2) of both subunits are opposite to one another (Figures 2B and 2C). The opposite pore helices and corresponding pore loops thus form the K+ ion selectivity filter.

Human TREK-1 (hTREK-1) is predominantly expressed in the central nervous system (CNS); its expression is abundant in tissues such as the brain, the spinal cord, the heart, the kidneys, the ovaries and the small intestine. Most specifically, TREK-1 is strongly expressed in the amygdala, basal ganglia, cortex, dorsal root ganglia (DRG) and hippocampus. hTREK-1 is found to a lesser extent in the skin, the muscles, the testes and the prostate.5, 6 TREK-1 is also found in peripheral tissues such as the gastrointestinal tract, and in mechanosensitive neurons innervating the bladder and colon where it is involved in detection and transduction of skin and organ deformation. Finally TREK-1 is weakly expressed in human myometrium and upregulated during pregnancy,


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suggesting that TREK-1 may assist with the maintenance of a negative cell membrane potential prior to labour. 7

Functionally, TREK-1 acts as a background (aka baseline or leak) K+ channel that requires physical or chemical stimuli to open.8 TREK-1 shows a strong outward rectification, it maintains the resting potential and controls membrane excitability by opposing depolarization. It also contributes to diverse sensory transduction processes and metabolic regulation. TREK-1 is modulated by various stimuli including mechanical (membrane stretch induces channel opening), temperature (temperatures between 32-37 °C induce channel activation), and intracellular pH (intracellular acidosis leads to channel opening). Besides activation by physical stimuli, TREK-1 can be modulated by various chemical agents such as polyunsaturated fatty acids [PUFA such as arachidonic acid (AA) or docosahexaenoic acid open TREK-1], and phospholipids (phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine and phosphatidic acid stimulate TREK-1 currents).9,10

The complex gating of TREK-1 is regulated by various receptors and second messenger pathways.9 TREK-1 is down-regulated by the stimulation of both Gs- and Gq-coupled membrane receptors.11 Several second messenger pathways are involved in this regulation: activation of protein kinases A and C (PKA, PKC) results in the phosphorylation of the cytoplasmic Cterminus of TREK-1, serotonin via activation of the 5-hydroxytryptamine 4 receptor (5-HT4R), the A-kinase-anchoring protein (AKAP150) and the metabotropic glutamate receptors mGluR1 or mGluR5 lead to inhibition of TREK-1 opening.


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Several models have been proposed to explain the gating mechanism of TREK-1.12 From the primary sequence of TREK-1 (Figure 1) and known structures of related channels (bacterial KcsA shares 70% homology with TREK-1), 3D models were predicted and compared with experimental data (mutation studies, truncated proteins).13 The homology models combined with molecular simulations converged towards likely “opened” and “closed” conformations (Figure 3). Although the mechanism that controls channel gating is not fully elucidated, the crucial roles of the C-terminus domain as well as the extracellular selectivity filter-based C-type gate have been exposed for the different K2P channels (TREK-1,14 TREK-2,15 and TRAAK16). The Cterminal tail of TREK-1 influences the channel response to a wide range of signals by controlling the extracellular C-type gate, via the transmembrane helix M4 (Figure 3).17 For example, the glutamic acid E306 (intracellular C-terminus) or the tryptophane W275 (extracellular end of the M4 helix close to the selectivity filter) are key residues in transducing channel gating after the action of PUFA (Figure 3A). Mutations of these residues (E306A, E306G or W275S) produce a gain of function phenotype that mimics acidosis. These studies concur that the primary gating occurs at the selectivity filter.18, 19


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CHEMICAL MODULATION OF TREK-1 MODULATORS The physiology and pharmacology of TREK-1 channels have been intensively studied over the past two decades. Several TREK-1 phenotypes have been linked to the regulation or the pharmacological properties of the inactivated channel. TREK-1 KO mice appear healthy and display a normal phenotype (skin color, body tone, weight),20 yet they are less sensitive to general volatile anesthetics,21 lose neuroprotection by PUFA,22 are more resistant to depression,23 are extremely sensitive to kainic acid-induced epileptic seizures and are more perceptive to painful sensations.24-27 These facts suggest that TREK-1 may play a role in the sleep-inducing activity of anesthetics,28 in cerebral ischemia, in mood disorders such as depression, in epilepsy and in pain perception.29, 30 The involvement of TREK-1 in various pathological states has led researchers to consider the channel as a novel therapeutic target.1, 31 Researchers have been using known therapeutic agents as probes to study the involvement of TREK-1 in various pathologies,24, 32 while others are developing selective TREK-1 agonists/antagonists.33-35

TREK-1 activators Mechanical activation of TREK-1 has been observed with molecules that induce morphological changes to the cell membrane. Membrane stretch prevents the intracellular C-terminus of TREK1 from interacting with the cell membrane; ultimately resulting in opening of the TREK-1 channel. (Figure 3A) For example, colchicine, latrunculin A and cytochalasin D prevent the polymerization of cytoskeletal proteins (tubulin/actin) resulting in deformation of the lipid bilayer. The cytoskeleton thus exerts a continuous negative regulatory effect on TREK-1. Amphipathic

phospholipids

(phosphatidylinositol,

phosphatidylserine,

and

phosphatidylethanolamine) interact with the hydrophobic membrane (inner leaflet), changing its curvature and leading to TREK-1 opening.10 Crenators (trinitrophenol, lysolecithin) can also


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insert into the external membrane leaflet, altering membrane curvature, ultimately resulting in activation of TREK-1 currents.36 Genetic knock-out (TREK-1 -/-) confer anesthetic resistance consistent with the reports that volatile anesthetics (chloroform, diethyl ether, halothane and isoflurane) partly rely on their ability to activate TREK-1 to be effective (Figure 3A).21 While chloroform and diethyl ether slightly depressed TASK, halothane and isoflurane activated TREK-1; none of the four compounds affected TRAAK.21 Several studies combining chimeric constructs and site-directed mutagenesis led to the identification of regions within the channel that are affecting anesthetic binding. Although the exact binding site has not been determined, it has been shown that the Cterminus region of TREK-1 is critical for activation by anesthetics (Figure 3A). Recently molecular modeling studies validated these findings by proposing a putative binding site for the general anesthetics.37

The neuroprotective drug riluzole 1 (anti-convulsive and anti-ischemic) currently used to treat amyotrophic lateral sclerosis (ALS) potentiates both TREK-1 and TRAAK currents in a dosedependent manner (Figure 4, Table 1). The activation of TRAAK is rapid, stable and reversible while the activation of TREK-1 is transient, and followed by a K+ current decline.38 This dual effect on TREK-1 was associated with the ability of 1 to increase intracellular cAMP resulting in PKA activation, finally leading to TREK-1 inhibition. Activation of TREK-1 by 1 was confirmed to be direct,39 and 1 was reported to display in vivo antinociceptive activity inhibiting 64% of writhes in the acetic acid-writhing test, which could be related to its ability to modulate TREK-1.34


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Table 1. Pharmacology of TREK-1 activators Compounds Activation of TREK-1 channels 100 µM elicited a rapid and significant stimulation (2-fold increase at +50 mV) 1 of whole-cells TREK-1 currents expressed on COS-7 cells, followed by inhibition through activation of cAMP/PKA.38 EC50 of 139.4 µM.39 10 µM induced a 1.4-fold activation of the current in hTREK-1 transfected CHO cells 40 2

10 µM increased by 2.9-fold the whole-cell current in TREK-1 transfected COS-7 cells.41

3

2.6-fold increase at 20 µM (TREK-1 transfected Xenopus oocyte)34 10 µM increased the current density by 6-fold, 20 µM produced a 8-fold increase (TREK-1 transfected bAZF cells)42 6.2-fold increase with 100 µM in TREK-1 transfected HEK293 cells 10 µM induced a 1.8-fold activation of the current at +50 mV in hTREK-1 transfected CHO cells40

4

40 µM increased by 6-fold the whole-cell current density at +60mV in bTREK-1 transfected CHO-K1 cells. 42

5

2.8-fold increase at 20 µM (TREK-1 transfected Xenopus oocyte) 34

6

10 µM induced a 1.4-fold activation of the current at +50 mV in hTREK-1 transfected CHO cells 40

7

1.3-fold increase at 20 µM (TREK-1 transfected Xenopus oocyte) 34 6.5-fold increase at 40 µM (TREK-1 transfected bAZF cells) 42


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10 µM and 100 nM induced respectively a 3.5- and 1.7-fold activation of the current at +50 mV in hTREK-1 transfected CHO cells. (EC50 = 3 µM) 40

9

2.9-fold increase at 20 µM (TREK-1 transfected Xenopus oocyte) 34

10

7-fold increase at 100 µM (TREK-1 transfected HEK293 cells)

11

EC50 213 µM (TREK-1 transfected Xenopus oocytes) with max 11-fold increase (100 µM). 35

12

EC50 125 µM (TREK-1 transfected Xenopus oocytes) with max 18-fold increase (100 µM). 35

13

EC50 36 µM (TREK-1 transfected Xenopus oocytes) , EC50 9.7 µM (TREK-1 transfected HEK293 cells) with max ~11-fold increase (100 µM).35

14

EC50 ~ 100 µM (TREK-1 transfected COS-7 cells)32 3.5-fold increase of TREK-1 currents at 100 µM (TREK-1 transfected HEK293 cells) 43

15

2.8-fold increase of TREK-1 currents at 100 µM (TREK-1 transfected HEK293 cells) 43

16

1 µM produced 2.3-fold increase and 3 µM produced 5.1-fold increase of TREK1 currents (TREK-1 transfected HEK293 cells) 43

17


18



10

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The ethacrynic acid derivative DCPIB 2, a selective blocker of volume-regulated anion channels (VRAC), was reported to reversibly activate TREK-1 and TREK-2, partially explaining its neuroprotective action (Figure 4, Table 1).41 The high hydrophobicity of 2 (cLogP 6.3) would suggest that the agonist interacts with the intracellular membrane preventing its interaction of the C-terminus of TREK-1. However, the rapid action of 2 and absence of activation upon intracellular application would contradict this hypothesis. The fact that the parent ethacrynic acid had no effect on TREK-1 suggests that perhaps the functionalities of 2 would be part of the pharmacophore for TREK-1 activation. Cinnamyl 1–3.4-dihydroxy-α-cyanocinnamate 3 (CDC) was reported to act externally since it increased the TREK-1 currents in excised outside-out patch recordings (6-fold increase in TREK-1 current at 10 µM) but failed to activate the channel when applied in excised inside-out patches (Figure 4, Table 1).42 Other caffeate analogues 4-7 were evaluated to identify the structural requirements for TREK-1 activation.42 Although less active than 3, 2-(1-thienyl)ethyl 3,4-dihydroxybenzylidenecyanoacetate 4 (TEDHBCA) and caffeic acid phenylethyl ester 7 (CAPE) effectively activated TREK-1 while caffeic acid (not shown), ethyl 3,4dihydroxybenzylidenecyanoacetate 5 (EDHBCA), and Tyrphostin B46 (not shown) failed to produce any significant increase at 40 µM. Danthi et al. suggested that an aromatic moiety (benzene, furan) was necessary on the ester side chain of the caffeate derivatives for TREK-1 activation while the cyano- group was not essential. Related analogues (tyrphostin 47 6 and ONO-RS-082 8) were reported as TREK-1 agonists (Figure 4, Table 1).40 More recently a range of substituted caffeate esters based on CDC 3 (Figure 4, Table 1) was reported and the caffeic acid 9 (2.9-fold increase at 20 µM) and the furanyl acrylic acid 10 (7-fold increase at 100 µM) were found to enhance TREK-1 currents and displayed potent anti-nociceptive activity in vivo (>30% inhibition of writhes induced by AcOH).34, 44 These results suggest that the ester side


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chain was not part of the pharmacophore for TREK-1 activation, unless the binding sites for the analogues 3-10 are different. The carbazole 11 (ML67) was identified as a weak TREK-1 agonist (EC50 = 213 µM) using high-throughput screening (Figure 4, Table 1).35 A structure-activity relationship study confirmed that the halogen atoms (chlorine) were essential for the drug activity and were kept. Attention hence focused on the N-alkyl side chain. Substitution of the carboxylic acid by the tetrazole bioisostere led to compound 12 with improved activity (ML67-18, EC50 = 125 µM) but the most active analogue was obtained by ring expansion to the acridine analogue 13 (ML67-33, EC50 = 36 µM). The latter was not TREK-1-selective since it equally activated TREK-2 (EC50 = 30 µM) and TRAAK (EC50 = 27 µM) but was ineffective against other K2P channels (TASK, TRESK). Mutation studies (G137I in P1 pore and W275S on M4 helix) demonstrated that 13 activated the extracellular C-type gate of the channel. Fenamates are non-steroid anti-inflammatory drugs used in pain treatment that are reported to reversibly activate TREK-1 (Figure 4, Table 1).43 Flufenamic acid 14 (FFA) produced a 250% enhancement of TREK-1 currents at 100 µM while the structurally-related tetrazole16 (BL-1249) was 100-fold more potent, boosting the activity of TREK-1 currents by 414% at 3 µM. Other fenamates such as mefenamic acid 17 (MFA) and niflumic acid 15 (NFA) also activated TREK-1 (150-180% enhancement of K+ currents at 100 µM), while diclofenac 18 was found to be the least potent TREK-1 agonist. Fenamates are also known to activate other potassium channels including TWIK-related arachidonic acid-stimulated K+ channel (KCNK4/TRAAK), human ether

a-go-go-related

gene

(KCNH2/hERG),

calcium-activated

K+

channels

(KCNMA1/KKCa1.1, KCNT2/KCa4.2), voltage-activated K+ channels (KCNQ/Kv7). Since these agents are known as non-selective cyclooxygenase inhibitors, ibuprofen and indomethacin were also assessed but proved to be weak TREK-1 openers at 100 µM, with only 10% and 24%


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enhancement in TREK-1 currents respectively. The binding of these agents was suggested to involve the C-terminus of TREK-1 however the residues affected in the mutation studies were too far apart to allow the determination of a precise binding site. It may be that the binding interferes with the ability of the C-terminus chain to interact with the membrane (conformational change).

TREK-1 inhibitors TREK-1 was found to play a role in neuroprotection, hence the ability of neuroprotective agents to modulate TREK-1 was assessed. Sipatrigine 19 (Figure 5, Table 2), a blocker of neuronal Na+ and Ca2+ channels, inhibits TREK-1 (IC50 4.0 µM) as well as the related channel TRAAK (45% inhibition at 10 µM) ; the inhibition of both K+ channels was reversible, voltageindependent and dose-dependent.45 Combined with its ability to inhibit glutamate neurotransmission, 19 could have therapeutic application for the treatment of depression.46 The neuroprotective agent 3-n-butylphthalide 20 (NBP) is a natural product extracted from celery seeds and has been used for the treatment of ischemic stroke in China since 2002. (S)-20 displayed dose-dependent and reversible inhibition of TREK-1 currents (IC50 0.06 µM) and was more potent than either the (R)-isomer or the racemate (Figure 5, Table 2).47


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Table 2. Pharmacology of TREK-1 inhibitors Inhibition of TREK-1 channels

Compounds 19

10 µM produced a reversible current depression of 75%, IC50 4.0 µM (50% inhibition of hTREK-1 currents in HEK293 cells) 45

20

10 µM produced a reversible current reduction of 70%, IC50 0.06 µM (50% inhibition of rTREK-1 currents in CHO transfected cells) 47

21

100 µM produced a 84% inhibition of TREK-1 currents, IC50 19 µM (50% inhibition of hTREK-1 currents in tsA201 cells), IC50 14 µM (50% inhibition of hTREK-1 currents in HEK293 cells) 48

22

IC50 9 µM (50% inhibition of hTREK-1 currents in tsA201 cells) 48

23

IC50 71 nM (50% inhibition of TREK-1 currents measured in presence of 10 µM arachidonic acid in whole COS-7 cells expressing TREK-1) 33

24

IC50 1.8 µM (50% inhibition of hTREK-1 currents in transfected COS cells) 49

25


27


26


28


29

IC50 20 µM (50% inhibition of hTREK-1 currents in transfected COS cells) 49

30

IC50 0.43 µM (50% inhibition of bTREK-1 currents in transfected AZT cells) 50

31


32



14

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33


34


35

IC50 207 µM (50% inhibition of hTREK-1 currents in transfected HEK293 cells) 51

36

IC50 173 µM (50% inhibition of hTREK-1 currents in transfected CHO cells) 52

37

IC50 7.6 µM (50% inhibition of hTREK-1 currents in transfected CHO cells) 52

38

IC50 20.3 µM (50% inhibition of hTREK-1 currents in transfected oocytes), IC50 1.6 µM (50% inhibition of hTREK-1 currents in transfected HEK293 cells) 53

The insensitivity of TREK-1 KO mice to depression23 suggested that antidepressants may modulate TREK-1. The selective serotonin reuptake inhibitor (SSRI) fluoxetine 21 (Prozac®) inhibited TREK-1 currents (IC50 19 µM) in a reversible and voltage-independent manner (Figure 5, Table 2).48 21 was also able to inhibit the related TASK-3 channel, albeit more weakly (31% inhibition at 100 µM in TREK-transfected HEK293 cells). Its active metabolite, norfluoxetine 22, was a more potent TREK-1 antagonist (IC50 9 µM). Mutations studies suggested that the glutamate E306 of the C-terminus and the leucine L320 of the M4 helix could play important roles in the binding of 21, since the mutations E306A and L320W led to the loss of TREK-1 inhibition by 21. The crystal structures of TREK-215 (78% homology with TREK-1) alone (PDB 4BW5) and with 22 (PDB 4XDK) show that the SSRI binds within the hydrophobic fenestration (just below the selectivity filter) and interacts with several hydrophobic residues on the transmembrane helices M2 (I194, P198), M3 (C249, V253) and M4 (F316, L320) and pore helix P2 (V276, L279). These residues being conserved in TREK-1, this work suggests that inhibition of TREK-1 could lead to potentially effective antidepressants.


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The neurotensin receptor 3 (NTSR3 aka sortilin) is a partner of TREK-1 for protein-protein interaction.

A

fragment

of

the

full-length

peptide

sortilin,

spadin

23

(peptide

APLPRWSGPIGVSWGLR, Table 1), was found to be a strong and reversible TREK-1 antagonist (IC50 71 nM in COS-7 cells).33 More importantly 23 was unable to inhibit currents generated by other K2P channels (TREK-2, TRAAK, TASK and TRESK).54 The peptide 23 displayed in vivo efficacy against depression (Forced-swim test and novelty suppressed feeding models) similar to fluoxetine 21, which was directly associated with its ability to inhibit TREK-1 (spadin had no effect on TREK-1 -/- mice). More recently retro-inverso peptides, including AcrlGwsvGipGswrplpa-NH2, were found to be more potent than 23, providing a portfolio for the development of a new family of antidepressants.55 In addition to antidepressants, several antipsychotic drugs significantly inhibit TREK-1 currents (IC50 1-20 µM). Since brain concentrations in antipsychotics are in the range 3-30 µM, their ability to inhibit TREK-1 may be relevant to their therapeutic activity or in their side-effect (Figure 5, Table 2). For example, pimozide 24, flupentixol 25, chlorpromazine 27, fluphenazine 26, haloperidol 28 and loxapine 29 resulted in dose-dependent and reversible inhibition of TREK-1 currents with IC50 in the low micromolar range (1.8-19.7 µM). These drugs also inhibited TREK-2 (IC50 1-10 µM) but not TRAAK.49 In the light of these results, other calcium channel blockers were evaluated for their capacity to antagonize TREK-1.50 Anti-hypertensive dihydropyridines (DHP), amlodipine 30, niguldipine 31 and nifedipine 32 were able to reversibly inhibit TREK-1 with IC50 of 0.43-8.2 µM (Figure 5, Table 2). The calcium antagonists flunarizine 33, a diphenyldiperazine used to treat migraine and epilespy, and anandamide 34, an endogenous cannabinoid, were also found to inhibit TREK-1 (IC50 2.5 and 1.0 µM respectively). Since TREK-1 is expressed in cardiac tissues, several anti-arrhythmic drugs (Figure 5, Table 2) were assessed for their ability to interfere with TREK-1 currents. Lidocaine 35 (Class Ib), a


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common anesthetic, was found to weakly inhibit TREK-1 (IC50 207 µM) in a reversible, concentration-dependent manner.51 The phosphorylation of the serine S348 (C-terminus of TREK-1) was reported to play a regulatory role in lidocaine-mediated inhibition of hTREK1. Mexiletine 36 (Class Ib) and propafenone 37 (Class Ic) blocked hTREK-1 currents (IC50 173 and 7.6 µM respectively) as well as TASK-1 (IC50 97.3 and 5.1 µM respectively).52 Finally carvediol 38 (Class II) was reported to inhibit TREK-1 (IC50 20.3 µM oocytes, 1.6 µM HEK293) as well as TREK-2 channels (IC50 24 µM oocytes,7.6 µM HEK293).53 Targeting of TREK-1 currents by antiarrhythmic drugs may suggest a role of the K+ channel in heart rhythm disorders.56

PERSPECTIVES The K2P channels constitute the youngest K+ channel family discovered nearly 20 years ago. Since their discovery, there has been an increasing interest in this family of K+ channels with reported experimental studies progressively unveiling their functions. The recent elucidation of the crystal structures of TWIK1, TRAAK and TREK-2 has uncovered their unique architecture and has permitted the understanding of their complex gating. Of particular interest in this perspective, the study of the TREK-1 channel has revealed its central role in cerebral ischemia,57 neuroprotection,22 epilepsy,57 depression,54 and pain perception.25, 27, 29 From the understanding of the role of TREK-1 in these pathologies have emerged several modulators, comforting the druggability of this novel target. Effective drug design has so far been hampered by the lack of structural information and limited understanding of the gating mechanism of TREK-1. The recently determined structure of TREK-1 (PDB 4TWK) will certainly increase the interest in this therapeutic target and allow a better understanding of its pharmacology, through the identification of the binding sites (co-crystal with the bound ligands, targeted mutation studies). This could increase the understanding of the modulators binding (agonists as well as antagonists)


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and will contribute to the design of more efficient ligands. The development of specific TREK-1 modulators would allow a better understanding of the implication of TREK-1 in the pathologies (pain, depression) and these modulators could be developed into candidate drugs (analgesic, antidepressants).

AUTHOR INFORMATION Corresponding Author Prof. Sylvie Ducki. Tel. +33 (0)473407132. Fax. +33 (0)473407008. Email. [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENTS We would like to thank the French National Research Agency (ANR) for funding of the project TREK-ANALGESIA (ANR-12-EMMA-0017-01) ; the French Ministry of Higher Education and Research (MESR) for a doctoral scholarship for DV.

ABBREVIATIONS AKAP150, A Kinase Anchor Protein 150 ;AZF, adrenal zonafasciculata; CAPE, caffeic acid phenethyl ester; CDC, Cinnamyl 1-3,4-dihydroxy-alpha-cyanocinnamate ; CHO, Chinese hamster ovary cells ;DCPIB, 4-(2-Butyl-6,7-Dichlor-2-CycloPentyl-Indan-1-on-5-yl) oxybutyric acid ; DRG, Dorsal Root Ganglia ; EDHBCA, ethyl 3,4-dihydroxybenzylidenecyanoacetate; HEK, Human Embryonic Kidney cells ; 5-HT, 5-hydroxytryptamine or serotonin ; K2P, Two


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Pore Domain. Potassium channel ; KcsA, Potassium Crystallographically-Sited Activation channel ; KO, Knock-Out ; PUFA, PolyUnsaturated Fatty Acid ; TEDHBCA, 2-(1-thienyl)ethyl 3,4,-dihydroxy-benzylidenecyanoacetate ; TRAAK, TWIK-related arachidonic acid-stimulated K+ channel ; TREK, TWIK-RElated K+ channels ; TWIK, Tandem of pore domains in a Weak Inward rectifying K+ channel ; VRAC, volume-regulated anion channels.

BIOGRAPHIES Khalil BENNIS received a PhD in Chemistry in 1988 from the Université Blaise Pascal in Clermont-Ferrand France. He joined the Ecole Nationale Supérieure de Chimie de ClermontFerrand, France in 1989 as a lecturer. His research in organic chemistry focused on carbohydrate chemistry. He recently reoriented his focus on medicinal chemistry with the aim to develop the next generation of analgesic molecules within CESMA. Sylvie DUCKI received a PhD in Chemistry in 1997 from the University of Manchester, UK. After a postdoctoral stay at the Cancer Research Institute (AZ, USA), she joined Pharmacia, Nerviano, Italy. In 2001, she took a lecturership in organic/medicinal chemistry at the University of Salford UK. In 2007, she joined the Ecole Nationale Supérieure de Chimie de ClermontFerrand, France as a professor. Her research in the area of medicinal chemistry focuses on the development of new bioactive molecules for the treatment of cancer and pain. She leads the CESMA team (Design and synthesis of analgesic molecules) within the Institute of Chemistry of Clermont-Ferrand and is vice-president of the Analgesia Institute. Florian LESAGE received a PhD in Life Sciences in 1995 from the University of Nice Sophia Antipolis. In the late 1990s, his has published the original cloning and characterization of many K2P channels including TREK-1. He leads the Laboratoire of Excellence Ion Channel Science


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and Therapeutics (LabEx ICST), a national consortium dedicated to the study of ion channels as targets for the development of new pharmaceutical drugs. Delphine VIVIER is an organic and medicinal chemist. She received a PhD in Chemistry in 2014 from the Université Blaise Pascal in Clermont-Ferrand France. Her doctoral work concerned the development of novel analgesic agents targeting the TREK-1 channel.

REFERENCES 1.

Wickenden, A. D., K+ channels as therapeutic drug targets. Pharmacol. Ther. 2002, 94

(1–2), 157-182. 2.

Lotshaw, D. P., Biophysical, pharmacological, and functional characteristics of cloned

and native mammalian two-pore domain K+ channels. Cell Biochem. Biophys. 2007, 47, 209256. 3.

Fink, M.; Duprat, F.; Lesage, F.; Reyes, R.; Romey, G.; Heurteaux, C.; Lazdunski, M.,

Cloning, functional expression and brain localization of a novel unconventional outward rectifier K+ channel. EMBO J. 1996, 15, 6854-6862. 4.

Lesage, F.; Lazdunski, M., Mapping of human potassium channel genes TREK-1

(KCNK2) and TASK (KCNK3) to chromosomes 1q41 and 2p23. Genomics 1998, 51 (3), 478479. 5.

Chapman, C. G.; Meadows, H. J.; Godden, R. J.; Campbell, D. A.; Duckworth, M.;

Kelsell, R. E.; Murdock, P. R.; Randall, A. D.; Rennie, G. I.; Gloger, I. S., Cloning, localisation and functional expression of a novel human, cerebellum specific, two pore domain potassium channel. Mol. Brain Res. 2000, 82 (1–2), 74-83.


20

Page 21 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


6.

Hervieu, G. J.; Cluderay, J. E.; Gray, C. W.; Green, P. J.; Ranson, J. L.; Randall, A. D.;

Meadows, H. J., Distribution and expression of TREK-1, a two-pore-domain potassium channel, in the adult rat CNS. Neuroscience 2001, 103 (4), 899-919. 7.

Buxton, I. L. O.; Heyman, N.; Wu, Y.-Y.; Barnett, S.; Ulrich, C., A role for stretch-

activated potassium currents in the regulation of uterine smooth muscle contraction. Acta Pharmacol. Sin. 2011, 32, 758-764. 8.

Patel, A. J.; Honoré, E., Properties and modulation of mammalian 2P domain K+

channels. Trends Neurosci. 2001, 24 (6), 339-346. 9.

Honoré, E., The neuronal background K2P channels: focus on TREK-1. Nat. Rev.

Neurosci. 2007, 8, 251-261. 10.

Chemin, J.; Patel, A. J.; Delmas, P.; Sachs, F.; Lazdunski, M.; Honore, E., Chapter 7 -

Regulation of the mechano-gated K2P channel TREK-1 by membrane phospholipids. In Current Topics in Membranes, Owen, P. H., Ed. Academic Press: 2007; Vol. Volume 59, pp 155-170. 11.

Shrestha, S. S.; Parmar, M.; Kennedy, C.; Bushell, T. J., Two-pore potassium ion

channels are inhibited by both Gq/11- and Gi-coupled P2Y receptors. Mol. Cell. Neurosci. 2010, 43 (4), 363-369. 12.

Treptow, W.; Klein, M. L., The membrane-bound state of K2P potassium channels. J.

Am. Chem. Soc. 2010, 132, 8145-8151. 13.

Milac, A.; Anishkin, A.; Fatakia, S. N.; Chow, C. C.; Sukharev, S.; Guy, H. R., Structural

models of TREK channels and their gating mechanism. Channels 2011, 5 (1), 23-33. 14.

Bagriantsev, S. N.; Clark, K. A.; Minor, D. L., Metabolic and thermal stimuli control

K2P2.1 (TREK-1) through modular sensory and gating domains. EMBO J. 2012, 31 (15), 32973308.


21


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

15.

Page 22 of 41

Dong, Y.; Pike, A.; Mackenzie, A.; McClenaghan, C.; Aryal, P.; Dong, L.; Quigley, A.;

Grieben, M.; Goubin, S.; Mukhopadhyay, S.; Ruda, G.; Clausen, M.; Cao, L.; Brennan, P.; Burgess-Brown, N.; Sansom, M.; Tucker, S.; Carpenter, E., K2P channel gating mechanisms revealed by structures of TREK-2 and a complex with Prozac. Science 2015, 347, 1256-1259. 16.

Lolicato, M.; Riegelhaupt, P.; Arrigoni, C.; Clark, K.; Minor, D., Transmembrane helix

straightening and buckling underlies activation of mechanosensitive and thermosensitive K2P channels. Neuron 2014, 84 (6), 1198-1212. 17.

Schneider, E. R.; Anderson, E. O.; Gracheva, E. O.; Bagriantsev, S. N., Chapter 5 -

Temperature sensitivity of two-pore (K2P) potassium channels. In Current Topics in Membranes, León, D. I.; Feng, Q., Eds. Academic Press: 2014; Vol. Volume 74, pp 113-133. 18.

Piechotta, P. L.; Rapedius, M.; Stansfeld, P. J.; Bollepalli, M. K.; Erhlich, G.; Andres-

Enguix, I.; Fritzenschaft, H.; Decher, N.; Sansom, M. S. P.; Tucker, S. J.; Baukrowitz, T., The pore structure and gating mechanism of K2P channels. EMBO J. 2011, 30, 3607-3619. 19.

Bagriantsev, S. N.; Peyronnet, R.; Clark, K. A.; Honoré, E.; Minor, D. L., Multiple

modalities converge on a common gate to control K2P channel function. EMBO J. 2011, 30, 3594-3606. 20.

Sabbadini, M.; Yost, C. S., Molecular biology of background K+ channels: Insights from

K2P knockout mice. J. Mol. Biol. 2009, 385 (5), 1331-1344. 21.

Patel, A. J.; Honoré, E.; Lesage, F.; Fink, M.; Romey, G.; Lazdunski, M., Inhalational

anesthetics activate two-pore-domain background K+ channels. Nat. Neurosci. 1999, 2 (5), 422426. 22.

Franks, N. P.; Honoré, E., The TREK K2P channels and their role in general anaesthesia

and neuroprotection. Trends Pharmacol. Sci. 2004, 25 (11), 601-608.


22

Page 23 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


23.

Heurteaux, C.; Lucas, G.; Guy, N.; Yacoubi, M. E.; Thümmler, S.; Peng, X.-D.; Noble,

F.; Blondeau, N.; Widmann, C.; Borsotto, M.; Gobbi, G.; Vaugeois, J.-M.; Debonnel, G.; Lazdunski, M., Deletion of the background potassium channel TREK-1 results in a depressionresistant phenotype. Nat. Neurosci. 2006, 9, 1134-1141. 24.

Devilliers, M. l.; Busserolles, J.; Lolignier, S.; Deval, E.; Pereira, V.; Alloui, A.; Christin,

M.; Mazet, B.; Delmas, P.; Noel, J.; Lazdunski, M.; Eschalier, A., Activation of TREK-1 by morphine results in analgesia without adverse side effects. Nat. Commun. 2013, 4, 2941-2948. 25.

Alloui, A.; Zimmermann, K.; Mamet, J.; Duprat, F.; Noel, J.; Chemin, J.; Guy, N.;

Blondeau, N.; Voilley, N.; Rubat-Coudert, C.; Borsotto, M.; Romey, G.; Heurteaux, C.; Reeh, P.; Eschalier, A.; Lazdunski, M., TREK-1, a K+ channel involved in polymodal pain perception. EMBO J. 2006, 25, 2368-2376. 26.

Noel, J.; Zimmermann, K.; Busserolles, J.; Deval, E.; Alloui, A.; Diochot, S.; Guy, N.;

Borsotto, M.; Reeh, P.; Eschalier, A.; Lazdunski, M., The mechano-activated K+ channels TRAAK and TREK-1 control both warm and cold perception. EMBO J. 2009, 28, 1308-1318. 27.

Alloui, A.; Busserolles, J.; Lazdunski, M.; Eschalier, A., The TREK-1 channel: an

attractive target for the development of new analgesics? Douleur et Analgésie 2008, 21, 215-220. 28.

Steinberg, E. A.; Wafford, K. A.; Brickley, S. G.; Franks, N. P.; Wisden, W., The role of

K2P channels in anaesthesia and sleep. Pfluegers Arch. 2015, 467 (5), 907-916. 29.

Mathie, A.; Veale, E. L., Two-pore domain potassium channels: potential therapeutic

targets for the treatment of pain. Pfluegers Arch. 2015, 467 (5), 931-943. 30.

Ehling, P.; Cerina, M.; Budde, T.; Meuth, S. G.; Bittner, S., The CNS under

pathophysiologic attack—examining the role of K2P channels. Pfluegers Arch. 2015, 467 (5), 959-972.


23


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

31.

Page 24 of 41

Renigunta, V.; Schlichthörl, G.; Daut, J., Much more than a leak: structure and function

of K2P-channels. Pfluegers Arch. 2015, 467, 867-894. 32.

Takahira, M.; Sakurai, M.; Sakurada, N.; Sugiyama, K., Fenamates and diltiazem

modulate lipid-sensitive mechano-gated 2P domain K+ channels. Pfluegers Arch. 2005, 451 (3), 474-478. 33.

Mazella, J.; Pétrault, O.; Lucas, G.; Deval, E.; Béraud-Dufour, S.; Gandin, C.; El-

Yacoubi, M.; Widmann, C.; Guyon, A.; Chevet, E.; Taouji, S.; Conductier, G.; Corinus, A.; Coppola, T.; Gobbi, G.; Nahon, J.-L.; Heurteaux, C.; Borsotto, M., Spadin, a sortilin-derived peptide, targeting rodent TREK-1 channels: A new concept in the antidepressant drug design PLoS Biol. 2010, 8 (4), e1000355. 34.

Rodrigues, N.; Bennis, K.; Vivier, D.; Pereira, V.; Chatelain, F. C.; Chapuy, E.; Deokar,

H.; Busserolles, J.; Lesage, F.; Eschalier, A.; Ducki, S., Synthesis and structure-activity relationship study of substituted caffeate esters as antinociceptive agents modulating the TREK-1 channel. Eur. J. Med. Chem. 2014, 75, 391-402. 35.

Bagriantsev, S. N.; Ang, K.-H.; Gallardo-Godoy, A.; Clark, K. A.; Arkin, M. R.; Renslo,

A. R.; Minor, D. L., A high-throughput functional screen identifies small molecule regulators of temperature- and mechano-sensitive K2P channels. ACS Chem. Biol. 2013, 8, 1841-1851. 36.

Maingret, F.; Patel, A. J.; Lesage, F.; Lazdunski, M.; Honore, E., Mechano- or acid

stimulation, two interactive modes of activation of the TREK-1 potassium channel. J. Biol. Chem. 1999, 274, 26691-26696. 37.

Bertaccini, E. J.; Dickinson, R.; Trudell, J. R.; Franks, N. P., Molecular modeling of a

tandem two pore domain potassium channel reveals a putative binding site for general anesthetics. ACS Chem. Neurosci. 2014, 5 (12), 1246-1252.


24

Page 25 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


38.

Duprat, F.; Lesage, F.; Patel, A. J.; Fink, M.; Romey, G.; Lazdunski, M., The

neuroprotective agent riluzole activates the two P domain K+ channels TREK-1 and TRAAK. Mol. Pharmacol. 2000, 57, 906-912. 39.

Cadaveira-Mosquera, A.; Ribeiro, S. J.; Reboreda, A.; Perez, M.; Lamas, J. A.,

Activation of TREK currents by the neuroprotective agent riluzole in mouse sympathetic neurons. J. Neurosci. 2011, 31 1375-1385. 40.

Feinmark, S. J.; Robinson, R. B. Method of treating a condition associated with

phosphorylation of TASK-1. WO 2007014347 2007. 41.

Minieri, L.; Pivonkova, H.; Caprini, M.; Harantova, L.; Anderova, M.; Ferroni, S., The

inhibitor of volume-regulated anion channels DCPIB activates TREK potassium channels in cultured astrocytes. Br. J. Pharmacol. 2012, 168, 1240-1254. 42.

Danthi, S.; Enyeart, J. A.; Enyeart, J. J., Caffeic acid esters activate TREK-1 potassium

channels and inhibit depolarization-dependent secretion. Mol. Pharmacol. 2004, 65, 599-610. 43.

Veale, E. L.; Al-Moubarak, E.; Bajaria, N.; Omoto, K.; Cao, L.; Tucker, S. J.; Stevens, E.

B.; Mathie, A., Influence of the N-terminus on the biophysical properties and pharmacology of TREK-1 potassium channels. Mol. Pharmacol. 2014, 85 (5), 671-681. 44.

Ducki, S.; Bennis, K.; Eschalier, A.; Busserolles, J.; Lesage, F.; Rodrigues, N.; Vivier, D.

Pain relief compounds. WO2013098416 A3, December 30, 2011. 45.

Meadows, H. J.; Chapman, C. G.; Duckworth, D. M.; Kelsell, R. E.; Murdock, P. R.;

Nasir, S.; Rennie, G.; Randall, A. D., The neuroprotective agent sipatrigine (BW619C89) potently inhibits the human tandem pore-domain K+ channels TREK-1 and TRAAK. Brain Res. 2001, 892 (1), 94-101.


25


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

46.

Page 26 of 41

Tsai, S.-J., Sipatrigine could have therapeutic potential for major depression and bipolar

depression through antagonism of the two-pore-domain K+ channel TREK-1. Med. Hypotheses 2008, 70 (3), 548-550. 47.

Ji, X.-C.; Zhao, W.-H.; Cao, D.-X.; Shi, Q.-Q.; Wang, X.-L., Novel neuroprotectant

chiral 3-n-butylphthalide inhibits tandem-pore-domain potassium channel TREK-1. Acta Pharmacol. Sin. 2011, 32, 182-187. 48.

Kennard, L. E.; Chumbley, J. R.; Ranatunga, K. M.; Armstrong, S. J.; Veale, E. L.;

Mathie, A., Inhibition of the human two-pore domain potassium channel, TREK-1, by fluoxetine and its metabolite norfluoxetine. Br. J. Pharmacol. 2005, 144 (6), 821-829. 49.

Thümmler, S.; Duprat, F.; Lazdunski, M., Antipsychotics inhibit TREK but not TRAAK

channels. Biochem. Biophys. Res. Commun. 2007, 354 (1), 284-289. 50.

Liu, H.; Enyeart, J. A.; Enyeart, J. J., Potent inhibition of native TREK-1 K+ channels by

selected dihydropyridine Ca2+ channel antagonists. J. Pharmacol. Exp. Ther. 2007, 323, 39-48. 51.

Nayak, T. K.; Harinath, S.; Nama, S.; Somasundaram, K.; Sikdar, S. K., Inhibition of

human two-pore domain K+ channel TREK-1 by local anesthetic lidocaine: Negative cooperativity and half-of-sites saturation kinetics. Mol. Pharmacol. 2009, 76 (4), 903-917. 52.

Schmidt, C.; Wiedmann, F.; Schweizer, P. A.; Becker, R.; Katus, H. A.; Thomas, D.,

Class I antiarrhythmic drugs inhibit human cardiac two-pore-domain K+ (K2P) channels. Eur. J. Pharmacol. 2013, 721 (1–3), 237-248. 53.

Kisselbach, J.; Seyler, C.; Schweizer, P. A.; Gerstberger, R.; Becker, R.; Katus, H. A.;

Thomas, D., Modulation of K2P2.1 and K2P10.1 K+ channel sensitivity to carvedilol by alternative mRNA translation initiation. Br. J. Pharmacol. 2014, 171 (23), 5182-5194.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


54.

Borsotto, M.; Veyssiere, J.; Maati, H. M. o.; Devader, C.; Mazella, J.; Heurteaux, C.,

Targeting two-pore domain K+ channels TREK-1 and TASK-3 for the treatment of depression: a new therapeutic concept. Br. J. Pharmacol. 2015, 172, 771-784. 55.

Veyssiere, J.; Maati, H. M. o.; Mazella, J.; Gaudriault, G.; Moreno, S.; Heurteaux, C.;

Borsotto, M., Retroinverso analogs of spadin display increased antidepressant effects. Psychopharmacology 2015, 232, 561-574. 56.

Schmidt, C.; Wiedmann, F.; Schweizer, P. A.; Katus, H. A.; Thomas, D., Inhibition of

cardiac two-pore-domain K+ (K2P) channels – an emerging antiarrhythmic concept. Eur. J. Pharmacol. 2014, 738, 250-255. 57.

Heurteaux, C.; Guy, N.; Laigle, C.; Blondeau, N.; Duprat, F.; Mazzuca, M.; Lang, L.;

Widmann, C.; Zanzouri, M.; Romey, G.; Lazdunski, M., TREK-1, a K+ channel involved in neuroprotection and general anesthesia. EMBO J. 2004, 23 (13), 2684-2695.


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FIGURES

Figure 1. Primary sequence of TREK-1 channel (FASTA: O95069-1) with main domains highlighted in bold green (N-M1-C1-C2-P1-M2-M3-P2-M4-C) based on the crystal structure of TREK-1 (PDB 4TWK).

Figure 2. (A) Topology of the TREK-1 subunit from the intracellular N-terminus to the intracellular C-terminus (including M1, C1, C2, P1, M2, M3, P2 and M4 domains). (B, C) Crystal structure of TREK-1 (PDB 4TWK, resolution 2.6 Å) with chains A and B in blue and green respectively and K+ ions in magenta viewed (B) parallel to the plane of the membrane and (C) from the cytoplasmic side of the membrane.

Figure 3. Model of TREK-1 channel gating highlighting the roles of glutamic acid E306 (intracellular C-terminus) and tryptophane W275 (extracellular end of the M4 helix close to the selectivity filter) as key residues (yellow) in transducing channel gating. (A) TREK-1 is activated by physico-chemical changes (stretch, depolarization, heat, intracellular acidosis), PUFA (arachidonic acid) and volatile anesthetics (chloroform, halothane). Upon activation, the Cterminal tail of TREK-1 associates with the lipid bilayer, and through the M4 helix relays them to the extracellular C-type gate; (B) Activation of the Gs/cAMP/PKA and the Gq/PLC/DAG/PKC signaling pathways inhibit TREK-1 channels by phosphorylation of residues on the C-terminal tail. The C-terminal tail of TREK-1 disssociates from the lipid bilayer and the M4 helix is shifted laterally in the resting state, gating the channel at the selectivity filter (black) hence preventing flow of K+ ions (pink).


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Figure 4. TREK-1 activators

Figure 5. TREK-1 inhibitors


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Figure 1 10 20 30 40 50 MLPSASRERP GYRAGVAAPD LLDPKSAAQN SKPRLSFSTK PTVLASRVES 60 70 DTTINVMKWK TVSTIFLVVV M1 110 120 TFISQHSCVN STELDELIQQ C2 160 170 AGTVITTIGF GNISPRTEGG M2 210 220 GKGIAKVEDT FIKWNVSQTK M3 260 270 WSALDAIYFV VITLTTIGFG P2 310 320 AAVLSMIGDW LRVISKKTKE

80 90 100 LYLIIGATVF KALEQPHEIS QRTTIVIQKQ C1 130 140 150 IVAAINAGII PLGNTSNQIS HWDLGSSFFF P1 180 190 200 KIFCIIYALL GIPLFGFLLA GVGDQLGTIF 230 240 250 IRIISTIIFI LFGCVLFVAL PAIIFKHIEG 280 290 300 DYVAGGSDIE YLDFYKPVVW FWILVGLAYF M4 330 340 350 EVGEFRAHAA EWTANVTAEF KETRRRLSVE

360 370 380 390 400 IYDKFQRATS IKRKLSAELA GNHNQELTPC RRTLSVNHLT SERDVLPPLL 410 420 KTESIYLNGL TPHCAGEEIA VIENIK


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Figure 3


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35

Figure 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


10 20 30 40 50 MLPSASRERP GYRAGVAAPD LLDPKSAAQN SKPRLSFSTK PTVLASRVES 60 70 DTTINVMKWK TVSTIFLVVV M1 110 120 TFISQHSCVN STELDELIQQ C2 160 170 AGTVITTIGF GNISPRTEGG M2 210 220 GKGIAKVEDT FIKWNVSQTK M3 260 270 WSALDAIYFV VITLTTIGFG P2 310 320 AAVLSMIGDW LRVISKKTKE

80 90 100 LYLIIGATVF KALEQPHEIS QRTTIVIQKQ C1 130 140 150 IVAAINAGII PLGNTSNQIS HWDLGSSFFF P1 180 190 200 KIFCIIYALL GIPLFGFLLA GVGDQLGTIF 230 240 250 IRIISTIIFI LFGCVLFVAL PAIIFKHIEG 280 290 300 DYVAGGSDIE YLDFYKPVVW FWILVGLAYF M4 330 340 350 EVGEFRAHAA EWTANVTAEF KETRRRLSVE

360 370 380 390 400 IYDKFQRATS IKRKLSAELA GNHNQELTPC RRTLSVNHLT SERDVLPPLL 410 420 KTESIYLNGL TPHCAGEEIA VIENIK ACS Paragon Plus Environment

Page 36 of 41

Page 37 of2 41 Figure 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


C2

C1

A

B

out

C2 P2

P1 M 1 in

C1

M 2

M 3

M 4

out C

M2

N

P1 P2 M1

C

M3 M4 C

in

N ACS Paragon Plus Environment

Figure 3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


A

Page 38 of 41

B W

W Stretch

HOOC + + +

M4

M4

M4

M4

K+

+ + E+ COOH

HOOC

P P

Acidosis, PUFA anesthetics


K+

P P E COOH Gs/cAMP/PKA Gq/PLC/DAG/PKC

Page 39 of4 41 Figure 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


O HOOC

N H2 N S

O

O

CF3

HO

C 4H 9 O

Cl

3, R = 4, R = 5, R = 6, R =

2 Cl O

O O

HO

HO

O

Cl

O

NH3 + Cl-

C 5H 11

8 Cl

OH

OH

HO 9

Cl

O

Cl N

N

CN

O-CH 2-CH=CH-Ph 0-CH 2-CH 2-(2-thienyl) OEt NHBn O

HO

N H

7

CN

HO

Cl

1

HO

R

R

10 11, R = COOH 12, R = tetrazole

F3 C

N N N NH

COOH

H N

N NH N N

F3 C

H N

H N

X 14, X = CH 15, X = N

16

17


COOH

13

Cl

COOH H N Cl 18

Cl

Figure 5

Cl

N N

C 4H 9

Journal of Medicinal Chemistry R

H N

O

Page 40 of 41

O

N

CF 3

N

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

NH 2

O

Cl

21, R = CH 3 22, R = H

20

19

Ala-Pro-Leu-Pro-Arg-Trp-Ser-Gly-Pro-Ile-Gly-Val-Ser-Trp-Gly-Leu-Arg F

23

OH

N

N

N N

NH

X

CF3

O

N

S

Cl

S

25, X = C= 26, X = N-

24

F

N

27

N Cl

N

O

OH

MeOOC

N

Cl

COOEt

Cl N

F

N H

O 29

28

30 F

NO 2 N

O

Ph

COOMe

O

Ph

NO 2 MeOOC

COOMe N

N H

31

N H 32

O HO

NH 2 O

33 NH2

N

N H

O

NH

O OH N H

N H

O

36

O

ACS Paragon Plus Environment O

OH

O

34 35

N

F

O

37

H N

Page 41 of 41 TOC 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


TREK-1

Agonists

Antagonists

Anesthesia Neuroprotection Inflammation Pain

Depression Epilepsy Arrhythmia


Perspectives on the Two-Pore Domain Potassium Channel TREK-1

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