TREK-1 Channels Research Articles

Two-pore potassium channel TREK-1 (K2P2.1) regulates NLRP3 inflammasome activity in macrophages.

Because of the importance of potassium efflux in inflammasome activation, we investigated the role of the two-pore potassium (K2P) channel TREK-1 in macrophage inflammasome activity. Using primary alveolar macrophages (AMs) and bone marrow-derived macrophages (BMDMs) from wild-type (wt) and TREK-1-/- mice, we measured responses to inflammasome priming [using lipopolysaccharide (LPS)] and activation (LPS + ATP). We measured IL-1β, caspase-1, and NLRP3 via ELISA and Western blot. A membrane-permeable potassium indicator was used to measure potassium efflux during ATP exposure, and a fluorescence-based assay was used to assess changes in membrane potential. Inflammasome activation induced by LPS + ATP increased IL-1β secretion in wt AMs, whereas activation was significantly reduced in TREK-1-/- AMs. Priming of BMDMs using LPS was not affected by either genetic deficiency or pharmacological inhibition of TREK-1 with Spadin. Cleavage of caspase-1 following LPS + ATP treatment was significantly reduced in TREK-1-/- BMDMs. The intracellular potassium concentration in LPS-primed wt BMDMs was significantly lower compared with TREK-1-/- BMDMs or wt BMDMs treated with Spadin. Conversely, activation of TREK-1 with BL1249 caused a decrease in intracellular potassium in wt BMDMs. Treatment of LPS-primed BMDMs with ATP caused a rapid reduction in intracellular potassium levels, with the largest change observed in TREK-1-/- BMDMs. Intracellular K+ changes were associated with changes in the plasma membrane potential (Em), as evidenced by a more depolarized Em in TREK-1-/- BMDMs compared with wt, and Em hyperpolarization upon TREK-1 channel opening with BL1249. These results suggest that TREK-1 is an important regulator of NLRP3 inflammasome activation in macrophages.NEW & NOTEWORTHY Because of the importance of potassium efflux in inflammasome activation, we investigated the role of the two-pore potassium (K2P) channel TREK-1 in macrophage inflammasome activity. Using primary alveolar macrophages and bone marrow-derived macrophages from wild-type and TREK-1-/- mice, we measured responses to inflammasome priming (using LPS) and activation (LPS + ATP). Our results suggest that TREK-1 is an important regulator of NLRP3 inflammasome activation in macrophages.

Prolonged exposure to lactate causes TREK1 channel clustering in rat hippocampal astrocytes

Increased concentrations of lactate (15–30 mM) are associated with and found to be neuroprotective in various brain pathophysiology. In our earlier studies we showed that high levels of lactate can increase TREK1 channel activity and expression within 1 h. TREK1 channels are two pore domain leak potassium ion channels that are upregulated during cerebral ischemia, epilepsy and other brain pathologies. They play a prominent neuroprotective role against excitotoxicity. Although it has been previously shown that chronic application of lactate (6 h) causes increased gene transcription and protein expression, we observe clustering of TREK1 channels that is dependent on time of exposure (3–6 h) and concentration of lactate (15–30 mM). Using immunofluorescence techniques and image analysis, we show that the clustering of TREK1 channels is dependent on the actin cytoskeletal network of the astrocytes. Clustering of TREK1 channels can augment astrocytic functions during pathophysiological conditions and have significant implications in lactate mediated neuroprotection.

TREK-1 inhibition promotes synaptic plasticity in the prelimbic cortex

Synaptic plasticity is one of the putative mechanisms involved in the maturation of the prefrontal cortex (PFC) during postnatal development. Early life stress (ELS) affects the shaping of cortical circuitries through impairment of synaptic plasticity supporting the onset of mood disorders. Growing evidence suggests that dysfunctional postnatal maturation of the prelimbic division (PL) of the PFC might be related to the emergence of depression. The potassium channel TREK-1 has attracted particular interest among many factors that modulate plasticity, concerning synaptic modifications that could underlie mood disorders. Studies have found that ablation of TREK-1 increases the resilience to depression, while rats exposed to ELS exhibit higher TREK-1 levels in the PL. TREK-1 is regulated by multiple intracellular transduction pathways including the ones activated by metabotropic receptors. In the hippocampal neurons, TREK-1 interacts with the serotonergic system, one of the main factors involved in the action of antidepressants. To investigate possible mechanisms related to the antidepressant role of TREK-1, we used brain slice electrophysiology to evaluate the effects of TREK-1 pharmacological blockade on synaptic plasticity at PL circuitry. We extended this investigation to animals subjected to ELS. Our findings suggest that in non-stressed animals, TREK-1 activity is required for the reduction of synaptic responses mediated by the 5HT1A receptor activation. Furthermore, we demonstrate that TREK-1 blockade promotes activity-dependent long-term depression (LTD) when acting in synergy with 5HT1A receptor stimulation. On the other hand, in ELS animals, TREK-1 blockade reduces synaptic transmission and facilitates LTD expression. These results indicate that TREK-1 inhibition stimulates synaptic plasticity in the PL and this effect is more pronounced in animals subjected to ELS during postnatal development.

Mechanical activation of vagal afferents involves opposing cation and TREK1 currents and NO regulation.

Vagal afferents convey signals of mechanical stimulation in the gut to the brain, which is essential for the regulation of food intake. However, ion channels sensing mechanical stimuli are not fully understood. This study aimed to examine the ionic currents activated by mechanical stimulation and a possible neuro-modulatory role of nitric oxide on vagal afferents. Nodose neuronal currents and potentials, and intestinal afferent firing by mechanical stimulation were measured by whole-cell patch clamp, and in vitro afferent recording, respectively. Osmotically activated cation and two-pore domain K+ currents were identified in nodose neurons. The membrane potential displayed a biphasic change under hypotonic stimulation. Cation channel-mediated depolarization was followed by a hyperpolarization mediated by K+ channels. The latter was inhibited by l-methionine (TREK1 channel inhibitor) and l-NNA (nitric oxide synthase inhibitor). Correspondingly, mechanical stimulation activated opposing cation and TREK1 currents. NOS inhibition decreased TREK1 currents and potentiated jejunal afferent nerve firing induced by mechanical stimuli. This study suggested a novel activation mechanism of ion channels underlying adaptation under mechanical distension in vagal afferent neurons. The guts' ability to perceive mechanical stimuli is vital in determining how it responds to food intake. The mechanosensation through ion channels could initiate and control gut function.

TREK channels in Mechanotransduction: a Focus on the Cardiovascular System.

Mechano-electric feedback is one of the most important subsystems operating in the cardiovascular system, but the underlying molecular mechanism remains rather unknown. Several proteins have been proposed to explain the molecular mechanism of mechano-transduction. Transient receptor potential (TRP) and Piezo channels appear to be the most important candidates to constitute the molecular mechanism behind of the inward current in response to a mechanical stimulus. However, the inhibitory/regulatory processes involving potassium channels that operate on the cardiac system are less well known. TWIK-Related potassium (TREK) channels have emerged as strong candidates due to their capacity for the regulation of the flow of potassium in response to mechanical stimuli. Current data strongly suggest that TREK channels play a role as mechano-transducers in different components of the cardiovascular system, not only at central (heart) but also at peripheral (vascular) level. In this context, this review summarizes and highlights the main existing evidence connecting this important subfamily of potassium channels with the cardiac mechano-transduction process, discussing molecular and biophysical aspects of such a connection.

Prolonged stretching of rat uteri causes hypoxia and inhibits contractility via potassium channel TREK1.

During pregnancy, uterine kept quiescence along with uterine overdistention before labor. Prolonged stretching induced uterus myometrial hypoxia, increased TREK1 expression, and relaxed the myometrium, which may contribute to uterine quiescence and atony during pregnancy. The mechanisms underlying pre-labor uterine quiescence and uterine atony during overdistention are unclear. TREK1 (a two-pore domain potassium channel) and hypoxia-inducible factor-1α (HIF-1α) are activated by mechanical stretch, and their expression is upregulated by decreased uterine contractility. HIF-1α is a nuclear factor which regulates numerous target proteins, but whether it regulates TREK1 during the uterine stretch to cause uterine quiescence and/or atony is unclear. We investigated uterine contractility at different gestational stages in rats, as well as in non-pregnant uteri, which were induced by prolonged stretching and hypoxia. We also assessed the effects of incubating the uteri with or without echinomycin or l-methionine. Moreover, we analyzed HIF-1α and TREK1 expression levels in each group, as well as at various gestational stages of pregnant human uteri. We found that contractility was significantly decreased in pregnant uteri when compared with non-pregnant uteri, and this decrease was associated with increases in HIF-1α and TREK1 expression levels. HIF-1α and TREK1 expression levels in human uteri increased with the gestational length. Decreased uterine contractility and increased HIF-1α and TREK1 expression levels were also observed in non-pregnant rat uteri under 8 g of stretching tension or hypoxia. Inhibition of hypoxia with echinomycin restored normal uterine contractility, while HIF-1α and TREK1 protein expression remained reduced. TREK1 inhibition with l-methionine also restored uterine contractility under tension or hypoxia. In conclusion, we demonstrated that prolonged stretching induces myometrial hypoxia, increases TREK1 expression, and relaxes the myometrium, which may contribute to uterine quiescence and atony.

PO-01-241 ROLE OF THE TWO-PORE K+CHANNEL TREK1 IN REGULATING HEART FAILURE-INDUCED VENTRICULAR ARRHYTHMIA

Despite therapeutic advances, heart failure (HF) remains a leading cause of death in the United States and is growing in prevalence. Although disease-induced defects in ion channel excitability and expression put patients at higher risk for life-threatening ventricular arrhythmias, the exact mechanisms by which HF leads to proarrhythmic ion channel dysfunction are not completely understood. Notably, several recent studies have identified important roles for the two-pore background K+ channel TREK1 in regulating sinus rhythm, atrial arrhythmia, and ventricular excitability. In the present study, we examined a putative role for TREK1 dysfunction in promoting heart failure-induced ventricular arrhythmia. We subjected wild type (WT) mice and mice with cardiac-specific TREK1 deficiency (TREK1cKO) to transaortic constriction (TAC) surgery as a model of heart failure or to a sham procedure. Over the course of 6-12 weeks following surgery, we examined structural and electrical remodeling via echocardiography and subsurface ECGs. At the end of the time course, ventricular cells were isolated and used for patch clamp, Ca2+/contractility imaging, and immunostaining. A subset of hearts were isolated for optical voltage mapping. We observed that TREK1cKO mice experienced greater reductions in cardiac function earlier in the development of pressure overload compared to WT mice, which was maintained throughout the time course. Additionally, TREK1cKO mice exhibited proarrhythmic changes to ECG parameters over 6-12 weeks of pressure overload including action potential duration (APD) prolongation, QTc prolongation, and QRS widening. However, the TREK1cKOs also experienced faster conduction, lower APD heterogeneity, and lower ventricular arrhythmia inducibility at 6 weeks post-surgery. Overall, these results demonstrate a potentially complex role of TREK1 in regulating ventricular excitability, ion dynamics, and arrhythmia susceptibility in the context of pressure overload-induced heart failure.

TREK-1 and TREK-2 Knockout Mice Are Not Resistant to Halothane or Isoflurane.

A variety of molecular targets for volatile anesthetics have been suggested, including the anesthetic-sensitive potassium leak channel, TREK-1. Knockout of TREK-1 is reported to render mice resistant to volatile anesthetics, making TREK-1 channels compelling targets for anesthetic action. Spinal cord slices from mice, either wild type or an anesthetic- hypersensitive mutant, Ndufs4, display an isoflurane-induced outward potassium leak that correlates with their minimum alveolar concentrations and is blocked by norfluoxetine. The hypothesis was that TREK-1 channels conveyed this current and contribute to the anesthetic hypersensitivity of Ndufs4. The results led to evaluation of a second TREK channel, TREK-2, in control of anesthetic sensitivity. The anesthetic sensitivities of mice carrying knockout alleles of Trek-1 and Trek-2, the double knockout Trek-1;Trek-2, and Ndufs4;Trek-1 were measured. Neurons from spinal cord slices from each mutant were patch clamped to characterize isoflurane-sensitive currents. Norfluoxetine was used to identify TREK-dependent currents. The mean values for minimum alveolar concentrations (± SD) between wild type and two Trek-1 knockout alleles in mice (P values, Trek-1 compared to wild type) were compared. For wild type, minimum alveolar concentration of halothane was 1.30% (0.10), and minimum alveolar concentration of isoflurane was 1.40% (0.11); for Trek-1tm1Lex, minimum alveolar concentration of halothane was 1.27% (0.11; P = 0.387), and minimum alveolar concentration of isoflurane was 1.38% (0.09; P = 0.268); and for Trek-1tm1Lzd, minimum alveolar concentration of halothane was 1.27% (0.11; P = 0.482), and minimum alveolar concentration of isoflurane was 1.41% (0.12; P = 0.188). Neither allele was resistant for loss of righting reflex. The EC50 values of Ndufs4;Trek-1tm1Lex did not differ from Ndufs4 (for Ndufs4, EC50 of halothane, 0.65% [0.05]; EC50 of isoflurane, 0.63% [0.05]; and for Ndufs4;Trek-1tm1Lex, EC50 of halothane, 0.58% [0.07; P = 0.004]; and EC50 of isoflurane, 0.61% [0.06; P = 0.442]). Loss of TREK-2 did not alter anesthetic sensitivity in a wild-type or Trek-1 genetic background. Loss of TREK-1, TREK-2, or both did not alter the isoflurane-induced currents in wild-type cells but did cause them to be norfluoxetine insensitive. Loss of TREK channels did not alter anesthetic sensitivity in mice, nor did it eliminate isoflurane-induced transmembrane currents. However, the isoflurane-induced currents are norfluoxetine-resistant in Trek mutants, indicating that other channels may function in this role when TREK channels are deleted.

MP03-07 THE LACK OF MECHANOSENSITIVE TREK-1 CHANNEL MIMICS THE DEVELOPMENT OF AGING-RELATED BLADDER PHENOTYPES IN MICE

You have accessJournal of UrologyCME1 Apr 2023MP03-07 THE LACK OF MECHANOSENSITIVE TREK-1 CHANNEL MIMICS THE DEVELOPMENT OF AGING-RELATED BLADDER PHENOTYPES IN MICE Alison Xiaoqiao Xie, Nao Iguchi, and Anna Malykhina Alison Xiaoqiao XieAlison Xiaoqiao Xie More articles by this author , Nao IguchiNao Iguchi More articles by this author , and Anna MalykhinaAnna Malykhina More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003214.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Previous animal and human studies have confirmed that the potassium channel subfamily K member 2 (TREK-1), a member of two-pore domain mechanosensitive K+ channels (K2P), acts as a mechanosensor in the urinary bladder. We reported that TREK-1 is a predominantly expressed K2P channel in the human detrusor, and its expression and function are altered in patients with idiopathic detrusor overactivity (DO). TREK-1 expression in the peripheral organs and in the nervous system also declines during aging. This study aimed to investigate the role of TREK-1-related mechanosensitivity in the detrusor in aging mice. METHODS: Bladder histology, detrusor contractility, and voiding function were investigated in adult (3, 6, 9 and 12 months old) TREK-1 KO mice of both sexes and age-matched wildtype controls. Bladder weight, detrusor smooth muscle (DSM) thickness, and collagen content were compared between the groups using paraffin-embedded bladder samples. Bladder strip assay was used to evaluate detrusor muscle contractility and nerve-induced bladder contractions. Voiding spot assay (VSA) and awake cystometry recordings were performed to assess micturition patterns in vivo. RESULTS: Increased bladder weight and bladder wall thickness were observed in TREK-1 KO mice of both sexes, starting as early as at 6-month-old. and threshold volume in cystometry recordings. In comparison to age- and sex-matched controls, bladder strips isolated from TREK-1 KO mice showed significantly decreased DSM contractility including contractile responses to electric field stimulation and cholinergic receptor activation. Interestingly, while the peak forces of evoked DSM contractions decreased with aging in control mice, TREK-1 KO animals at 6-12 month-old displayed DSM contractility that was lower than in 12-month-old control animals. Increased numbers of non-voiding contractions were observed in both sexes of TREK-1 KO mice, whereas the bladder capacity, intermicturition interval, and micturition threshold volume were significantly increased in comparison to age-matched controls. CONCLUSIONS: In summary, the lack of TREK-1 expression in adult mice significantly increased a number of functional changes typically observed in underactive bladders with detrusor overactivity, including increases in bladder size, weight, capacity, and a significant decrease in DSM contractility. Our findings suggest that decreased TREK-1 expression may mimic the development of age-related voiding phenotypes in mice. Source of Funding: DK095817 (Malykhina), DK116648 (Malykhina) and Department of Surgery Academic Enrichment Fund (Xie) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e23 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Alison Xiaoqiao Xie More articles by this author Nao Iguchi More articles by this author Anna Malykhina More articles by this author Expand All Advertisement PDF downloadLoading ...

A photoswitchable inhibitor of TREK channels controls pain in wild-type intact freely moving animals

By endowing light control of neuronal activity, optogenetics and photopharmacology are powerful methods notably used to probe the transmission of pain signals. However, costs, animal handling and ethical issues have reduced their dissemination and routine use. Here we report LAKI (Light Activated K+ channel Inhibitor), a specific photoswitchable inhibitor of the pain-related two-pore-domain potassium TREK and TRESK channels. In the dark or ambient light, LAKI is inactive. However, alternating transdermal illumination at 365 nm and 480 nm reversibly blocks and unblocks TREK/TRESK current in nociceptors, enabling rapid control of pain and nociception in intact and freely moving mice and nematode. These results demonstrate, in vivo, the subcellular localization of TREK/TRESK at the nociceptor free nerve endings in which their acute inhibition is sufficient to induce pain, showing LAKI potential as a valuable tool for TREK/TRESK channel studies. More importantly, LAKI gives the ability to reversibly remote-control pain in a non-invasive and physiological manner in naive animals, which has utility in basic and translational pain research but also in in vivo analgesic drug screening and validation, without the need of genetic manipulations or viral infection.

Membrane phospholipids control gating of the mechanosensitive potassium leak channel TREK1

Tandem pore domain (K2P) potassium channels modulate resting membrane potentials and shape cellular excitability. For the mechanosensitive subfamily of K2Ps, the composition of phospholipids within the bilayer strongly influences channel activity. To examine the molecular details of K2P lipid modulation, we solved cryo-EM structures of the TREK1 K2P channel bound to either the anionic lipid phosphatidic acid (PA) or the zwitterionic lipid phosphatidylethanolamine (PE). At the extracellular face of TREK1, a PA lipid inserts its hydrocarbon tail into a pocket behind the selectivity filter, causing a structural rearrangement that recapitulates mutations and pharmacology known to activate TREK1. At the cytoplasmic face, PA and PE lipids compete to modulate the conformation of the TREK1 TM4 gating helix. Our findings demonstrate two distinct pathways by which anionic lipids enhance TREK1 activity and provide a framework for a model that integrates lipid gating with the effects of other mechanosensitive K2P modulators.

The energetics of rapid cellular mechanotransduction

Cells throughout the human body detect mechanical forces. While it is known that the rapid (millisecond) detection of mechanical forces is mediated by force-gated ion channels, a detailed quantitative understanding of cells as sensors of mechanical energy is still lacking. Here, we combine atomic force microscopy with patch-clamp electrophysiology to determine the physical limits of cells expressing the force-gated ion channels (FGICs) Piezo1, Piezo2, TREK1, and TRAAK. We find that, depending on the ion channel expressed, cells can function either as proportional or nonlinear transducers of mechanical energy and detect mechanical energies as little as ~100 fJ, with a resolution of up to ~1 fJ. These specific energetic values depend on cell size, channel density, and cytoskeletal architecture. We also make the surprising discovery that cells can transduce forces either nearly instantaneously (<1 ms) or with a substantial time delay (~10 ms). Using a chimeric experimental approach and simulations, we show how such delays can emerge from channel-intrinsic properties and the slow diffusion of tension in the membrane. Overall, our experiments reveal the capabilities and limits of cellular mechanosensing and provide insights into molecular mechanisms that different cell types may employ to specialize for their distinct physiological roles.

The energetics of rapid cellular mechanotransduction.

Cells throughout the human body detect mechanical forces. While it is known that the rapid (millisecond) detection of mechanical forces is mediated by force-gated ion channels, a detailed quantitative understanding of cells as sensors of mechanical energy is still lacking. Here, we combine atomic force microscopy with patch-clamp electrophysiology to determine the physical limits of cells expressing the force-gated ion channels Piezo1, Piezo2, TREK1, and TRAAK. We find that, depending on the ion channel expressed, cells can function either as proportional or nonlinear transducers of mechanical energy, detect mechanical energies as little as ∼100 fJ, and with a resolution of up to ∼1 fJ. These specific energetic values depend on cell size, channel density, and cytoskeletal architecture. We also make the surprising discovery that cells can transduce forces either nearly instantaneously (<1ms), or with substantial time delay (∼10 ms). Using a chimeric experimental approach and simulations we show how such delays can emerge from channel-intrinsic properties and the slow diffusion of tension in the membrane. Overall, our experiments reveal the capabilities and limits of cellular mechanosensing and provide insights into molecular mechanisms that different cell types may employ to specialize for their distinct physiological roles.

Virtual screening (VS) based in the common binding site for negativelycharged activators of K+ channels: A comparison with a pharmacophore-based VS.

A master key mechanism related to the pharmacophore of negatively charged activators (NCAs) to modulate TREK, BKca and hERG potassium (K+) channels was recently described. A polypharmacological behavior of NCAs was assessed, because all NCA found could activate these three type of K+ channels. However, the role of the binding sites (BS) in the polypharmacological nature of NCAs remains unknown. In this context, the presence of structural common patterns at the BS appears to be a valid hypothesis. Residues determined experimentally as having a significant role on K+ channel-NCAs interaction were retrieved. Structural common patterns at the NCAs BS were found between TREK, BKca and hERG K+ channels using our program Geomfinder comparing by pairs. These common BS were used for a structure-based virtual screening (VS). In parallel, a pharmacophore-based VS was carried out based in the common features of the NCAs. The VS results were organized using MM-GBSA. Nineteen compounds target in silico at least two of the three types of K+ channels studied. They also accomplish two features: 1) they were found simultaneously by structure-based VS and pharmacophore-based VS; 2) all of them point their negative charge to the pore, a feature that it is mandatory for the activation of TREK, BKca and hERG K+ channelsby NCAs. These nineteen compounds will be proposed to test by means of two-electrode voltage clamp technique in the three types of K+ channels studied.

Pharmacological modulation of TREK-1 channel.

Potassium channels (PC) are a wide family of ionic channels that regulates membrane potential allowing the permeation of ions nearly at the diffusion rate limit and with high selectivity. TREK-1 is a PC of the K2P family involved in regulating the resting potential after a nerve impulse in mammals. Therefore, its pharmacological modulation is of interest. A possible gating mechanism for TREK-1 is a C-type inactivation, in which the selectivity filter (SF) is found either in a non-inactive (ordered or rigid) or inactive (disordered or collapsed) state. This mechanism was proposed in the literature based on previous findings of two modulators (namely Q6F and Q5F) that are bound behind the SF and apparently stabilize the ordered state by a cation-pi interaction with a transmembrane helix. Still, the exact mechanisms of action of those molecules and how the binding event affects the SF state allosterically, and consequently, its gating, is still elusive. Here, we describe using molecular dynamics (MD) simulations the main dynamics involved in the interaction of those modulators with the channel, including the approximated regions sampled by the MD and the crystal structures.

A nanobody toolkit for the regulation of K2P channel function.

Two-Pore Domain (K2P) K+ channels act as important regulators of the membrane potential in both excitable and non-excitable cells. Their dysfunction is implicated in a range of disorders and they represent attractive therapeutic targets. In particular, drugs that modulate TREK K2P channels may be useful for the treatment of certain forms of pain, depression and migraine. In a previous study, we demonstrated that a class of small molecule negatively-charged activators target a common gating mechanism within the selectivity filter of K2P channels to increase channel activity. Although some of these drugs are highly effective, structural conservation within the inner pore and filter gating mechanism of many related K+ channels means that target specificity is difficult to achieve. We now show that a class of biologics based on VHH domains (nanobodies), can achieve far greater selectivity as both activators and inhibitors of TREK-2 channel function and can differentiate between individual members of this family. Structures of TREK-2 in complex with these nanobodies also provides important new insights into their mechanism of action and highlight a new toolkit for the dissection of K2P channel function.

Conformational plasticity of NaK2K and TREK2 potassium channel selectivity filters

The K+ channel selectivity filter (SF) is defined by TxGYG amino acid sequences that generate four identical K+ binding sites (S1-S4). Only two sites (S3, S4) are present in the non-selective bacterial NaK channel, but a four-site K+-selective SF is obtained by mutating the wild-type TVGDGN SF sequence to a canonical K+ channel TVGYGD sequence (NaK2K mutant). Using single molecule FRET (smFRET), we show that the SF of NaK2K, but not of non-selective NaK, is ion-dependent, with the constricted SF configuration stabilized in high K+ conditions. Patch-clamp electrophysiology and non-canonical fluorescent amino acid incorporation show that NaK2K selectivity is reduced by crosslinking to limit SF conformational movement. Finally, the eukaryotic K+ channel TREK2 SF exhibits essentially identical smFRET-reported ion-dependent conformations as in prokaryotic K+ channels. Our results establish the generality of K+-induced SF conformational stability across the K+ channel superfamily, and introduce an approach to study manipulation of channel selectivity.

ЕКСПРЕСІЯ ГЕНІВ МЕХАНОЧУТЛИВИХ РЕЦЕПТОРІВ У ДЕТРУЗОРІ СЕЧОВОГО МІХУРА ЩУРА

B a c k g r o u n d . Autonomic mechanosensitivity of the muscle layer (detrusor) of the urinary bladder is an actual problem that still remains understudied. It is known that a number of mechanosensitive channels are probably expressed, but to date, only the leading role of the mechanosensitive channel TREK-1 has been established. The aim of the study was to check the expression of mechanoreceptor genes of smooth muscle tissue (KCNK2 (TREK-1), TRPV4, Piezo1, and Piezo2) in the bladder detrusor. M e t h o d s . Unique primers were selected for the detection of the specified channel genes, as well as the reference gene GAPDH, using the BLAST software. Validation was performed using one-step PCR with reverse transcription on the CFX96 amplifier with fluorescence detection of the Fam sample. A matrix RNA sample was isolated from the muscle and urothelial layers of the urinary bladder and dorsal root ganglia (DRG) of healthy Wistar rats using a commercial nucleic acid extraction column kit (Biocorp, Ukraine). R e s u l t s . In our samples, Piezo1 and TREK-1 channels were shown to be expressed in the urothelial and muscle layers of the bladder, whereas TRPV4 was only expressed in the urothelium. Piezo2 gene expression was not detected. C o n c l u s i o n s . The selected sequences of primers for TREK-1, Piezo1, TRPV4, and the "housekeeping" gene GAPDH are effective for studying the expression of these genes in rats. The synthesized sequences correspond to the expected fragment sizes. The genes of three mechanosensitive channels are expressed in the urinary bladder: TREK-1, Piezo1, and TRPV4.

Retracted: Activation of TREK1 Channel in the Anterior Cingulate Cortex Improves Neuropathic Pain in a Rat Model.

[This retracts the article DOI: 10.1155/2022/1372823.].

TREK-1 in the heart: Potential physiological and pathophysiological roles.

The TREK-1 channel belongs to the TREK subfamily of two-pore domains channels that are activated by stretch and polyunsaturated fatty acids and inactivated by Protein Kinase A phosphorylation. The activation of this potassium channel must induce a hyperpolarization of the resting membrane potential and a shortening of the action potential duration in neurons and cardiac cells, two phenomena being beneficial for these tissues in pathological situations like ischemia-reperfusion. Surprisingly, the physiological role of TREK-1 in cardiac function has never been thoroughly investigated, very likely because of the lack of a specific inhibitor. However, possible roles have been unraveled in pathological situations such as atrial fibrillation worsened by heart failure, right ventricular outflow tract tachycardia or pulmonary arterial hypertension. The inhomogeneous distribution of TREK-1 channel within the heart reinforces the idea that this stretch-activated potassium channel might play a role in cardiac areas where the mechanical constraints are important and need a particular protection afforded by TREK-1. Consequently, the main purpose of this mini review is to discuss the possible role played by TREK -1 in physiological and pathophysiological conditions and its potential role in mechano-electrical feedback. Improved understanding of the role of TREK-1 in the heart may help the development of promising treatments for challenging cardiac diseases.