Academic literature on the topic 'TREK-1 potassium channel'

TREK-1/2 are members of the mechano-gated subfamily of two-pore (K2P) domain potassium channels leaking K+ out of the cell and contributing to the resting membrane potential. In contrast to the classical tetrameric potassium channels, K2P channels are dimeric with an atypical architecture and the structural mechanisms underlying their channel gating are poorly understood. Here we present the crystal structures of human TREK-1 and TREK-2 at resolutions of 2.7 and 3.4Å which provide insights into the basis of intracellular and extracellular gating in this unique family of ion channels. We have solved the structure of TREK-2 in two distinct conformations differing in the orientation of the pore-lining transmembrane helices. The C-terminal M4 helix is hinged at a conserved glycine residue so that it adopts one of two distinct orientations. The M4 helix is either kinked towards the membrane, packing against the M2 inner helix of the adjacent subunit ("M4 up") or straightens and interacts with the M2/M3 helices from the same subunit ("M4 down"). In the M4 down state, a hydrophobic lateral opening runs perpendicular to the ion conductance pathway between M2 and M4 and links the inner vestibule to the membrane-exposed face of the channel. Transition between the "M4 down" and "M4 up" conformations may play a role in channel activation and gating. Cocrystallisation with a TREK-1/2 channel inhibitor promotes the "M4 down" state. The structure of TREK-1 exhibits an "M4-up" conformation but is unusual in that the selectivity filter is significantly distorted with only two correctly-formed potassium sites. The structure also reveals a divalent ion binding site between the extracellular cap and the pore domain loop. The TREK-1 structure illustrates how changes at an extracellular site can affect the pore structure. The structures will be described in detail along with their implications for channel gating in response to intracellular and extracellular stimuli.

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

The rate of aldosterone synthesis by adrenal glomerulosa cells relies on the selective permeability of the glomerulosa cell to K+ ions. In rodent and bovine adrenal glomerulosa cells, this background potassium current is provided by a two-pore loop potassium (K2P) channel: largely TASK-3 in the rat and TREK-1 in the cow. The nature of the K2P channel in the human adrenal cortex is not known, and we have addressed this issue here using the H295R human adrenal cell line. We show that these cells express mRNA and protein for both TASK-3 and TREK-1 K2P channels. Using a potentiometric dye (FMP), we also show that TASK-3 and TREK-1 channel modulators can affect the membrane potential of H295R cells. Transfecting H295R cells with TASK-3 or TREK-1 dominant-negative mutants (TASK-3 G95E or TREK-1 G144E) produced depolarization of H295R cells and altered K-stimulated aldosterone secretion. Finally, transfection of a constitutively active mutant of Gαq into H295R cells (GTPase-deficient Gαq-QL) depolarized them and increased basal aldosterone secretion. Taken together, our data support both TASK-3 and TREK-1 as being functionally operational in the H295R cell line. This suggests that human adrenal glomerulosa cells may utilize both of these K2P channels for their background potassium current.

The tandem of pore domain in a weak inwardly rectifying K+ channel (Twik)-related acid-arachidonic activated K+ channel (TRAAK) and Twik-related K+ channels (TREK) 1 and TREK2 are active as homodimers gated by stretch, fatty acids, pH, and G protein-coupled receptors. These two-pore domain potassium (K2P) channels are broadly expressed in the nervous system where they control excitability. TREK/TRAAK KO mice display altered phenotypes related to nociception, neuroprotection afforded by polyunsaturated fatty acids, learning and memory, mood control, and sensitivity to general anesthetics. These channels have emerged as promising targets for the development of new classes of anesthetics, analgesics, antidepressants, neuroprotective agents, and drugs against addiction. Here, we show that the TREK1, TREK2, and TRAAK subunits assemble and form active heterodimeric channels with electrophysiological, regulatory, and pharmacological properties different from those of homodimeric channels. Heteromerization occurs between all TREK variants produced by alternative splicing and alternative translation initiation. These results unveil a previously unexpected diversity of K2P channels that will be challenging to analyze in vivo, but which opens new perspectives for the development of clinically relevant drugs.

Sour (acid) taste is postulated to result from intracellular acidification that modulates one or more acid-sensitive ion channels in taste receptor cells. The identity of such channel(s) remains uncertain. Potassium channels, by regulating the excitability of taste cells, are candidates for acid transducers. Several 2-pore domain potassium leak conductance channels (K2P family) are sensitive to intracellular acidification. We examined their expression in mouse vallate and foliate taste buds using RT-PCR, and detected TWIK-1 and -2, TREK-1 and -2, and TASK-1. Of these, TWIK-1 and TASK-1 were preferentially expressed in taste cells relative to surrounding nonsensory epithelium. The related TRESK channel was not detected, whereas the acid-insensitive TASK-2 was. Using confocal imaging with pH-, Ca2+-, and voltage-sensitive dyes, we tested pharmacological agents that are diagnostic for these channels. Riluzole (500 μM), selective for TREK-1 and -2 channels, enhanced acid taste responses. In contrast, halothane (≤ ∼17 mM), which acts on TREK-1 and TASK-1 channels, blocked acid taste responses. Agents diagnostic for other 2-pore domain and voltage-gated potassium channels (anandamide, 10 μM; Gd3+, 1 mM; arachidonic acid, 100 μM; quinidine, 200 μM; quinine, 100 mM; 4-AP, 10 mM; and TEA, 1 mM) did not affect acid responses. The expression of 2-pore domain channels and our pharmacological characterization suggest that a matrix of ion channels, including one or more acid-sensitive 2-pore domain K channels, could play a role in sour taste transduction. However, our results do not unambiguously identify any one channel as the acid taste transducer.

Mechanosensitive hTREK-1 two-pore-domain potassium (hK2P2.1) channels give rise to background currents that control cellular excitability. Recently, TREK-1 currents have been linked to the regulation of cardiac rhythm as well as to hypertrophy and fibrosis. Even though the pharmacological and biophysical characteristics of hTREK-1 channels have been widely studied, relatively little is known about their posttranslational modifications. This study aimed to evaluate whether hTREK-1 channels are N-glycosylated and whether glycosylation may affect channel functionality. Following pharmacological inhibition of N-glycosylation, enzymatic digestion or mutagenesis, immunoblots of Xenopus laevis oocytes and HEK-293T cell lysates were used to assess electrophoretic mobility. Two-electrode voltage clamp measurements were employed to study channel function. TREK-1 channel subunits undergo N-glycosylation at asparagine residues 110 and 134. The presence of sugar moieties at these two sites increases channel function. Detection of glycosylation-deficient mutant channels in surface fractions and recordings of macroscopic potassium currents mediated by these subunits demonstrated that nonglycosylated hTREK-1 channel subunits are able to reach the cell surface in general but with seemingly reduced efficiency compared to glycosylated subunits. These findings extend our understanding of the regulation of hTREK-1 currents by posttranslational modifications and provide novel insights into how altered ion channel glycosylation may promote arrhythmogenesis.

Detrusor overactivity (DO) is the abnormal response of the urinary bladder to physiological stretch during the filling phase of the micturition cycle. The mechanisms of bladder smooth muscle compliance upon the wall stretch are poorly understood. We previously reported that the function of normal detrusor is regulated by TREK-1, a member of the mechanogated subfamily of two-pore-domain potassium (K2P) channels. In the present study, we aimed to identify the changes in expression and function of TREK-1 channels under pathological conditions associated with DO, evaluate the potential relationship between TREK-1 channels and cytoskeletal proteins in the human bladder, and test the possibility of modulation of TREK-1 channel expression by small RNAs. Expression of TREK-1 channels in DO specimens was 2.7-fold decreased compared with control bladders and was associated with a significant reduction of the recorded TREK-1 currents. Isolated DO muscle strips failed to relax when exposed to a TREK-1 channel opener. Immunocytochemical labeling revealed close association of TREK-1 channels with cell cytoskeletal proteins and caveolins, with caveolae microdomains being severely disrupted in DO specimens. Small activating RNA (saRNA) tested in vitro provided evidence that expression of TREK-1 protein could be partially upregulated. Our data confirmed a significant downregulation of TREK-1 expression in human DO specimens and provided evidence of close association between the channel, cell cytoskeleton, and caveolins. Upregulation of TREK-1 expression by saRNA could be a future step for the development of in vivo pharmacological and genetic approaches to treat DO in humans.

We tested the hypothesis that TREK-1, a two-pore domain K channel, is involved with dilations in arteries. Because there are no selective activators or inhibitors of TREK-1, we generated a mouse line deficient in TREK-1. Endothelium-mediated dilations were not different in arteries from wild-type (WT) and TREK-1 knockout (KO) mice. This includes dilations of the middle cerebral artery to ATP, dilations of the basilar artery to ACh, and relaxations of the aorta to carbachol, a cholinergic agonist. The nitric oxide (NO) and endothelium-dependent hyperpolarizing factor components of ATP dilations were identical in the middle cerebral arteries of WT and TREK-1 KO mice. Furthermore, the NO and cyclooxygenase-dependent components were identical in the basilar arteries of the different genotypes. Dilations of the basilar artery to α-linolenic acid, an activator of TREK-1, were not affected by the absence of TREK-1. Whole cell currents recorded using patch-clamp techniques were similar in cerebrovascular smooth muscle cells (CVSMCs) from WT and TREK-1 KO mice. α-linolenic acid or arachidonic acid increased whole cell currents in CVSMCs from both WT and TREK-1 KO mice. The selective blockers of large-conductance Ca-activated K channels, penitrem A and iberiotoxin, blocked the increased currents elicited by either α-linolenic or arachidonic acid. In summary, dilations were similar in arteries from WT and TREK-1 KO mice. There was no sign of TREK-1-like currents in CVSMCs from WT mice, and there were no major differences in currents between the genotypes. We conclude that regulation of arterial diameter is not altered in mice lacking TREK-1.

Currently, TREK-1 is considered to be the main mechanosensitive channel in detrusor smooth muscle (DSM) cells. The aim of our study was to detect the functioning of the K+-conducting mechanosensitive TREK-1 channel in rat DSM cells using the patch-clamp technique in response to hydrodynamic stimulation (shear stress) and to determine the effects of a TREK-1 agonist – arachidonic acid (AA) and an antagonist – L-methionine. Mechanical stimulation of DSM cells using hydrodynamic stress led to the appearance of a membrane current with signs of pronounced outward rectification at positive membrane potentials, which is typical of TREK-1 activation. The application of AA (50 mcmol/l) activated a current with similar characteristics of the outward rectification to the shear stress-activated one. L-methionine (10 mcmol/l) almost completely prevented the generation of an outwardly rectifying current in response to shear stress stimulation. DSM cells also retained the ability to generate a mechanoactivated current with a more pronounced inward component when extracellular and intracellular K+ were replaced by Cs+. It was concluded that the dominant mechanoactivated current in rat DSM cells is carried by K+-selective TREK-1 channels, but a small portion of this current can also be carried by other nonselective mechanosensitive cation channels.

Nitrogen supersaturation and bubble formation can occur in the vascular system after diving, leading to death and nervous disorders from decompression sickness (DCS). Bubbles alter the vascular endothelium, activate platelets, and lead to focal ischemia with neurological damage mediated by the mechanosensitive TREK-1 neuronal potassium ion channel that sets pre- and postsynaptic resting membrane potentials. We report a neuroprotective effect associated with TREK-1. C57Bl6 mice were subjected to decompression from a simulated 90 msw dive. Of 143 mice that were wild type (WT) for TREK-1, 51.7% showed no DCS, 27.3% failed a grip test, and 21.0% died. Of 88 TREK-1 knockouts (KO), 26.1% showed no DCS, 42.0% failed a grip test, and 31.8% died. Mice that did not express TREK-1 had lower DCS resistance and were more likely to develop neurological symptoms. We conclude that the TREK-1 potassium channel was neuroprotective for DCS.

Les cancers de la prostate (CaP), du sein et des poumons sont souvent de diagnostic difficile notamment en raison de leur caractère initial asymptomatique. Rarement douloureux aux stades primaires, ces cancers ont une forte propension à métastaser vers le microenvironnement osseux. Cliniquement, à ce stade, cela se traduit par des douleurs qui sont à la fois très invalidantes et résistantes aux traitements antalgiques de référence, y compris aux morphiniques. La difficulté provient en partie du caractère multifactoriel de ces douleurs qui associent composantes nociceptive et neuropathique. Parmi les acteurs de cette douleur, un rôle essentiel est attribué au dérèglement du système nerveux (périphérique et central) et à la croissance tumorale qui affecte tous deux le microenvironnement osseux.Le glutamate, neurotransmetteur excitateur majeur du système nerveux, est récemment apparu comme une cible potentielle dans la prise en charge de certains cancers, dont le CaP. Le riluzole, une molécule anti glutamatergique, possédant déjà une autorisation de mise sur le marché dans le traitement de la SLA, a démontré un effet antalgique dans plusieurs modèles de douleur inflammatoire et neuropathique et un effet antiprolifératif in vitro. C’est pourquoi, nous avons recherché si cette molécule pouvait avoir des effets bénéfiques dans le traitement des douleurs osseuses induites par le CaP, et comment il pouvait influer sur le développement du CaP métastatique osseux. Nous avons utilisé un modèle de douleur osseuse par injection intratibiale de cellules humaines de CaP, les cellules PC3-luc, et administré de riluzole dans l’eau de boisson. Chez ce modèle, le riluzole a démontré un effet antalgique significatif impliquant le canal TREK-1, canal sélectif pour l’ion potassium. De plus, le riluzole diminue significativement la viabilité des cellules in vitro et ralentit la croissance tumorale in vivo sans affecter le remodelage osseux. L’effet antiprolifératif du riluzole impliquerait une augmentation de l’expression des canaux TREK-1 dans les cellules PC3 participant à leur hyperpolarisation.En conclusion, ce travail permet de mettre en évidence l’intérêt du riluzole en tant que molécule d’intérêt pour le traitement des douleurs osseuses du CaP dont le mécanisme d’action implique certainement le canal potassique fond TREK-1
Prostate (PCa), breast and lung cancer are often difficult to diagnose due to their initial asymptomatic nature. Rarely painful in the primary stages, these cancers have a high propensity to metastasize to the bone microenvironment. Clinically, at this stage, this translates into pain that is both very disabling and resistant to standard analgesic treatments, including morphine. The difficulty comes from the multifactorial nature of this pain, which combines nociceptive and neuropathic components. Among the factors attributed to cancer pain, an essential role is attributed to the disruption of the nervous system (peripheral and central) and to tumor growth, both of which affect the bone microenvironment.Glutamate, a major excitatory neurotransmitter of the nervous system, has recently emerged as a potential target in the management of solid cancers, including PCa. Riluzole, an anti glutamatergic molecule, which is authorized for the treatment of ALS, has demonstrated an analgesic effect in several inflammatory and neuropathic pain models and an antiproliferative effect in vitro. Therefore, we investigated whether this molecule could have beneficial effects in the treatment of PCa-induced bone pain, and how it could influence the development of bone metastatic PCa. We used a bone pain model by intratibial injection of human PCa cells, PC3-luc cells, and administered riluzole in the drinking water. In this model, riluzole demonstrated a significant analgesic effect involving the TREK-1 channel, a selective channel for potassium ions. In addition, riluzole significantly decreased cell viability in vitro and slowed tumor growth in vivo without affecting bone remodeling. The antiproliferative effect of riluzole would imply an increase in the expression of TREK-1 channels in PC3 cells participating in their hyperpolarization.In conclusion, this work highlights the importance of riluzole as a molecule of interest for the treatment of PCa bone pain whose mechanism of action certainly involves the TREK-1 potassium channel

La morphine demeure l'antalgique de référence pour le traitement de la douleur (nociception), mais elle est également responsable d‘effets secondaires importants. Des études ont montré que les animaux privés de canaux potassiques TREK-1 (TWIK-related K+channels) étaient plus sensibles à la douleur. Plus récemment, il a été démontré que le canal potassique TREK-1 joue un rôle crucial dans l'analgésie induite par la morphine chez les souris, alors qu'il n'est pas impliqué dans les effets secondaires (constipation, dépression respiratoire et dépendance). Ces résultats suggèrent que les canaux TREK-1 constituent des cibles d‘intérêt pour la conception de nouveaux antalgiques sans effets indésirables liés aux opioïdes. Des études antérieures au sein de notre laboratoire ont permis l'identification de quatre structures chefs de file, activatrices des canaux TREK-1, présentant une activité antalgique in vivo. La structure 3D du canal TREK-1 n‘étant pas élucidée au moment de nos travaux, nous avons décidé d'effectuer une optimisation basée sur une étude de relation structure-activité (RSA). Trente-six analogues ont été synthétisés par condensation de Knoevenagel et évalués pour leur effet antalgique (test de l‘acide acétique, test de la plaque chaude) et leur capacité à activer le canal TREK-1 (électrophysiologie). La capacité des substituants du noyau aromatique à établir des interactions de type liaison hydrogène ainsi que le volume de ces substituants ont une influence déterminante sur l'activité. Des résultats prometteurs ont émergé de cette étude RSA: 5 molécules présentent une très bonne activité antalgique (> 50% d'inhibition de la douleur, test de la plaque chaude) ainsi que d'une bonne activation de TREK-1 canaux (R ≥ 2 à 10 μM ou R ≥ 4 au-dessus de 20 μM)
Morphine remains the analgesic of reference for the treatment of pain (nociception), but it is also responsible for serious adverse effects. Research studies have shown that animals deprived of potassium channels TREK-1 (TWIK-related K+ channels) were over-sensitive to pain. More recently, it has been demonstrated that the TREK-1 potassium channel is a crucial contributor of morphine-induced analgesia in mice, while it is not involved in morphine-induced constipation, respiratory depression and dependence. These results suggest that the TREK-1 channels constitute targets of interest for the design of novel analgesics without opioid-like adverse effects. Previous studies within our consortium led to the identification of four lead structures as TREK-1 activators exhibiting analgesic activity in vivo.Since the 3D structure of TREK-1 was not available at the time, we decided to perform hit optimization by conventional structure-activity relationship (SAR) studies. Thirty six analogs were synthesized via Knoevenagel condensation and evaluated for their analgesic effect (writhing test, hot plate assay) and their ability to activate TREK-1 channel (electrophysiology). It turned out that the possibility to form hydrogen bonding interaction (aryl moiety) and the volume of substituents of the amide or ester has a crucial influence on activity. Promising results emerged from this SAR study: 5 molecules display a very good analgesic activity (> 50% inhibition of pain, hot plate assay) as well as a good activation of TREK-1 channels (R ≥ 2 at 10μM or R ≥ 4 above 20μM)

Aberrant expression of potassium (K+) channels contributes to cancer cell proliferation and in certain circumstances channel blockade has been shown to inhibit cell proliferation. Two pore potassium (K2P) channels are the most recently identified group of K+ channels. K2P channels have been found to play a role in several cancers including prostate and breast cancer. We investigated the K2P channels TREK-1, TREK-2 and TASK-3, in ovarian cancer and normal ovaries and described the effect of channel blockade on cell proliferation, the cell cycle and apoptosis. Immunofluorescence confirmed expression in the cell lines (n=3) normal ovaries (n=4) and ovarian cancer (n=4). Western blotting quantified channel expression in normal ovaries (n=6) and cancer (n=22). There appeared to be a significant increase in expression of TREK-1 (P=O.0019) and TASK-3 (P=O.0047) in cancer when compared to normal ovaries. Immunohistochemistry further established expression in ovarian cancer (TREK-1 and -2, n=69) and normal ovaries (n=9) and in the TMA (TASK-3 n=230). Increased TASK-3 immunostaining conferred a significant survival advantage (P=O.001). There was a significant (P