Pantoprazole and riluzole target H+/K+-ATPases and pH-sensitive K+ channels in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) remains the most lethal cancer type. PDAC is characterized by fibrotic, hypoxic, and presumably acidic tumor microenvironment (TME). Acidic TME is an important player in tumor development, progression, aggressiveness, and chemoresistance. The dysregulation of ductal ion transporters/channels might contribute to extracellular pH (pHe) acidification and PDAC progression. Our aim was to test whether H+/K+-ATPases and pH-sensitive K+ channels contribute to these processes and could be targeted by clinically approved drugs. We used human pancreatic cancer cells adapted to various pHe conditions and grown in monolayers and spheroids. First, we created cells expressing pHoran4 at the outer plasma membrane and showed that pantoprazole, the H+/K+-ATPase inhibitor, alkalinized pHe. Second, we used FluoVolt to monitor the membrane voltage (Vm) and showed that riluzole hyperpolarized Vm, most likely by opening of pH-sensitive K+ channels such as TREK-1. Third, we show that pantoprazole and riluzole inhibited cell proliferation and viability of monolayers and spheroids of cancer cells adapted to various pHe conditions. Most importantly, combination of the two drugs had significantly larger inhibitory effects on PDAC cell survival. We propose that co-targeting H+/K+-ATPases and pH-sensitive K+ channels by re-purposing of pantoprazole and riluzole could provide novel acidosis-targeted therapies of PDAC.

TREK-1 channel as a therapeutic target for dexmedetomidine-mediated neuroprotection in cerebral ischemia.

Our objective is to explore the protective effect of Dexmedetomidine on brain apoptosis and its mechanism through TREK-1 pathway. Forty male Sprague-Dawley rats were allocated into four groups: Sham, Cerebral Ischemia/Reperfusion Injury (CIRI), 50 µg/kg Dex, and 100 µg/kg Dex. A rat model of middle cerebral artery occlusion (MCAO) was employed to simulate cerebral embolism. Primary cortical neurons were exposed to Dex for 48 h, with some receiving additional treatment with 100 µM yohimbine hydrochloride (YOH) or TREK-1 small interfering RNA (siRNA). Neuronal damage was assessed using hematoxylin and eosin (HE) staining. Cell viability and apoptosis were measured by Cell Counting Kit-8 (CCK8) and flow cytometry, respectively. Protein and gene expression levels of Bcl-2, Bax, and TREK-1 were determined by Western blot and real-time polymerase chain reaction (PCR). Histopathological changes revealed that Dex treatment at both 50 µg/kg and 100 µg/kg significantly mitigated neuronal damage compared to the CIRI group. YOH treatment and Trek1 siRNA significantly reduced cell viability (p < 0.05). The mRNA expression and protein levels of TREK-1 and Bax were remarkably increased, while mRNA expression and protein levels of Bcl-2 was seriously decreased after CIRI modeling. In contrast, Dex treatment at both concentrations led to decreased TREK-1 and Bax expression and increased Bcl-2 expression in primary cortical neurons. Addition of 100 µM YOH and Trek1 siRNA reversed the effects of Dex on apoptosis-related genes (p < 0.05). Dex exerts neuroprotective effects through the TREK-1 pathway in vivo and in vitro.

Development of covalent chemogenetic K2P channel activators.

K2P potassium channels regulate excitability by affecting cellular resting membrane potential in the brain, cardiovascular system, immune cells, and sensory organs. Despite their important roles in anesthesia, arrhythmia, pain, hypertension, sleep, and migraine, the ability to control K2P function remains limited. Here, we describe a chemogenetic strategy termed CATKLAMP (covalent activation of TREK family K+ channels to clamp membrane potential) that leverages the discovery of a K2P modulator pocket site that reacts with electrophile-bearing derivatives of a TREK subfamily small-molecule activator, ML335, to activate the channel irreversibly. We show that CATKLAMP can be used to probe fundamental aspects of K2P function, as a switch to silence neuronal firing, and is applicable to all TREK subfamily members. Together, our findings exemplify a means to alter K2P channel activity that should facilitate molecular and systems level studies of K2P function and enable the search for new K2P modulators.

Clostridium butyricum regulates intestinal barrier function via trek1 to improve behavioral abnormalities in mice with autism spectrum disorder.

BACKGROUND: Autism Spectrum Disorder (ASD) is a complex neurodevelopmental disorder that has been found to be associated with dysregulation of gastrointestinal functions and gut microbial homeostasis (the so-called "gut-brain axis"). ASD is often accompanied by poor performances in social interaction and repetitive behaviors. Studies on the gut-brain axis provide novel insights and candidate targets for ASD therapeutics and diagnosis. Based on the ASD mice model, this work aims to reveal the mechanisms behind the interaction of intestinal barrier function and probiotics in ASD mouse models. RESULTS: We found an altered intestinal barrier in both BTBR T+ Itpr3tf/J (BTBR) and valproic acid (VPA) mice, including increased intestinal permeability, decreased expression of intestinal tight junction proteins (claudin1, claudin3, and occludin), and increased levels of IL-6, TNF-α, and IFN-γ. Based on intestinal microbial alternation, C. butyricum can drive reduced expression of histone deacetylases 1 (HDAC1) and enhanced intestinal barrier function, significantly promoting behavioral abnormalities of ASD in BTBR mice. In parallel, we confirmed that C. butyricum was involved in the regulation of intestinal function by the Trek1 channel, indicating that it is a target of C. butyricum/butyric acid to improve intestinal barrier function in ASD mice. CONCLUSIONS: Our finding provides solid evidence for the gut microbiota involved in ASD through the brain-gut axis. In addition, the probiotics C. butyricum hold promise to improve gut health and ameliorate behavioral abnormalities associated with ASD.

Effect of chronic administration of ostruthin on depression-like behavior in chronically stressed mice.

We have previously shown that a single dose of a TREK-1 channel activator, ostruthin, exhibited antidepressant and anxiolytic effects in acute behavioral test models in mice. To assess the potential clinical application, it is essential to evaluate the effects of long-term administration of ostruthin in a chronically stressed mouse model, which is considered to be similar to the clinical condition of major depression in humans. Here, we tested the effects of a single and a 7-day administration of ostruthin on mice that were subjected to chronic unpredictable mild stress (CUMS). A single administration of ostruthin showed antidepressive effects in the tail suspension and forced swim tests of CUMS-treated mice. Unexpectedly, the 7-day administration exhibited only insignificant antidepressive and anxiolytic effects. The 7-day regimen did not affect food intake or body-weight gain, suggesting the absence of apparent cytotoxicity. The mice receiving the 7-day administration had significantly lower blood concentrations of ostruthin compared to those receiving a single dose, suggesting an upregulation of drug-metabolizing activities. These findings suggest that there is a need for stable TREK-1 channel activators that are not affected by drug metabolism.

The TREK-1 potassium channel is involved in both the analgesic and anti-proliferative effects of riluzole in bone cancer pain.

BACKGROUND: The metastasis of tumors into bone tissue typically leads to intractable pain that is both very disabling and particularly difficult to manage. We investigated here whether riluzole could have beneficial effects for the treatment of prostate cancer-induced bone pain and how it could influence the development of bone metastasis. METHODS: We used a bone pain model induced by intratibial injection of human PC3 prostate cancer cells into male SCID mice treated or not with riluzole administered in drinking water. We also used riluzole in vitro to assess its possible effect on PC3 cell viability and functionality, using patch-clamp. RESULTS: Riluzole had a significant preventive effect on both evoked and spontaneous pain involving the TREK-1 potassium channel. Riluzole did not interfere with PC3-induced bone loss or bone remodeling in vivo. It also significantly decreased PC3 cell viability in vitro. The antiproliferative effect of riluzole is correlated with a TREK-1-dependent membrane hyperpolarization in these cells. CONCLUSION: The present data suggest that riluzole could be very useful to manage evoked and spontaneous hypersensitivity in cancer-induced bone pain and has no significant adverse effect on cancer progression.

The Inhibition of TREK-1 K+ Channels via Multiple Compounds Contained in the Six Kamikihito Components, Potentially Stimulating Oxytocin Neuron Pathways.

Oxytocin, a significant pleiotropic neuropeptide, regulates psychological stress adaptation and social communication, as well as peripheral actions, such as uterine contraction and milk ejection. Recently, a Japanese Kampo medicine called Kamikihito (KKT) has been reported to stimulate oxytocin neurons to induce oxytocin secretion. Two-pore-domain potassium channels (K2P) regulate the resting potential of excitable cells, and their inhibition results in accelerated depolarization that elicits neuronal and endocrine cell activation. We assessed the effects of KKT and 14 of its components on a specific K2P, the potassium channel subfamily K member 2 (TREK-1), which is predominantly expressed in oxytocin neurons in the central nervous system (CNS). KKT inhibited the activity of TREK-1 induced via the channel activator ML335. Six of the 14 components of KKT inhibited TREK-1 activity. Additionally, we identified that 22 of the 41 compounds in the six components exhibited TREK-1 inhibitory effects. In summary, several compounds included in KKT partially activated oxytocin neurons by inhibiting TREK-1. The pharmacological effects of KKT, including antistress effects, may be partially mediated through the oxytocin pathway.

The TREK-1 potassium channel is a potential pharmacological target for vasorelaxation in pulmonary hypertension.

BACKGROUND AND PURPOSE: Pulmonary arterial hypertension (PAH) is a progressive disease in which chronic membrane potential (Em) depolarisation of the pulmonary arterial smooth muscle cells (PASMCs) causes calcium overload, a key pathological alteration. Under resting conditions, the negative Em is mainly set by two pore domain potassium (K2P) channels, of which the TASK-1 has been extensively investigated. EXPERIMENTAL APPROACH: Ion channel currents and membrane potential of primary cultured human(h) PASMCs were measured using the voltage- and current clamp methods. Intracellular [Ca2+] was monitored using fluorescent microscopy. Pulmonary BP and vascular tone measurements were also performed ex vivo using a rat PAH model. KEY RESULTS: TREK-1 was the most abundantly expressed K2P in hPASMCs of healthy donors and idiopathic(I) PAH patients. Background K+-current was similar in hPASMCs for both groups and significantly enhanced by the TREK activator ML-335. In donor hPASMCs, siRNA silencing or pharmacological inhibition of TREK-1 caused depolarisation, reminiscent of the electrophysiological phenotype of idiopathic PAH. ML-335 hyperpolarised donor hPASMCs and normalised the Em of IPAH hPASMCs. A close link was found between TREK-1 activity and intracellular Ca2+-signalling using a channel activator, ML-335, and an inhibitor, spadin. In the rat, ML-335 relaxed isolated pre-constricted pulmonary arteries and significantly decreased pulmonary arterial pressure in the isolated perfused lung. CONCLUSIONS AND IMPLICATIONS: These data suggest that TREK-1is a key factor in Em setting and Ca2+ homeostasis of hPASMC, and therefore, essential for maintenance of a low resting pulmonary vascular tone. Thus TREK-1 may represent a new therapeutic target for PAH.

The Variety of Mechanosensitive Ion Channels in Retinal Neurons.

Alterations in intraocular and external pressure critically involve the pathogenesis of glaucoma, traumatic retinal injury (TRI), and other retinal disorders, and retinal neurons have been reported to express multiple mechanical-sensitive channels (MSCs) in recent decades. However, the role of MSCs in visual functions and pressure-related retinal conditions has been unclear. This review will focus on the variety and functional significance of the MSCs permeable to K+, Na+, and Ca2+, primarily including the big potassium channel (BK); the two-pore domain potassium channels TRAAK and TREK; Piezo; the epithelial sodium channel (ENaC); and the transient receptor potential channels vanilloid TRPV1, TRPV2, and TRPV4 in retinal photoreceptors, bipolar cells, horizontal cells, amacrine cells, and ganglion cells. Most MSCs do not directly mediate visual signals in vertebrate retinas. On the other hand, some studies have shown that MSCs can open in physiological conditions and regulate the activities of retinal neurons. While these data reasonably predict the crossing of visual and mechanical signals, how retinal light pathways deal with endogenous and exogenous mechanical stimulation is uncertain.

Sortilin-Mediated Inhibition of TREK1/2 Channels in Primary Sensory Neurons Promotes Prediabetic Neuropathic Pain.

Neuropathic pain can occur during the prediabetic stage, even in the absence of hyperglycemia. The presence of prediabetic neuropathic pain (PDNP) poses challenges to the management of individuals with prediabetes. However, the mechanisms underlying this pain remain unclear. This study aims to investigate the underlying mechanism and identify potential therapeutic targets of PDNP. A prediabetic animal model induced by a high-energy diet exhibits both mechanical allodynia and thermal hyperalgesia. Furthermore, hyperexcitability and decreased potassium currents are observed in the dorsal root ganglion (DRG) neurons of these rats. TREK1 and TREK2 channels, which belong to the two-pore-domain K+ channel (K2P) family and play an important role in controlling cellular excitability, are downregulated in DRG neurons. Moreover, this alteration is modulated by Sortilin, a molecular partner that modulates the expression of TREK1. The overexpression of Sortilin negatively affects the expression of TREK1 and TREK2, leading to increased neuronal excitability in the DRG and enhanced peripheral pain sensitivity in rats. Moreover, the downregulation of Sortilin or activation of TREK1 and TREK2 channels by genetic or pharmacological approaches can alleviate PDNP. Therefore, targeting the Sortilin-mediated TREK1/2 pathway may provide a therapeutic approach for ameliorating PDNP.

Endovascular balloon usage in endoscopic third ventriculostomy for hydrocephalus during a national shortage: case series and technical note.

Endoscopic third ventriculostomy (ETV) is a well-established surgical technique for treating hydrocephalus. Many providers have transitioned to utilizing the specialized Neuroballoon for the stoma dilation in ETV; however, these devices are intermittently unavailable during supply chain shortages. We present the experience of employing cardiac angioplasty and neurovascular balloons as substitutes for the Neuroballoon in 3 patients. The scepter balloon (Microvention), priced at $1800 compared to the standard $300 Neuroballoon (Integra), proved effective, but its pliability presented technical challenges. The substantial cost differential compared to a Neuroballoon ($300) raises economic considerations. The Cardiac TREK balloon (Abbott) was similarly effective, while also being easier to manage endoscopically and cheaper at $158. These experiences support the viability of non-neuroendoscopic specialized balloons as alternatives for ETV dilation of the floor of tuber cinereum.

Myometrium infection decreases TREK1 through NHE1 and increases contraction in pregnant mice.

Intrauterine infection during pregnancy can enhance uterine contractions. A two-pore K+ channel TREK1 is crucial for maintaining uterine quiescence and reducing contractility, with its properties regulated by pH changes in cell microenvironment. Meanwhile, the sodium hydrogen exchanger 1 (NHE1) plays a pivotal role in modulating cellular pH homeostasis, and its activation increases smooth muscle tension. By establishing an infected mouse model of Escherichia coli (E. coli) and lipopolysaccharide (LPS), we used Western blotting, real-time quantitative polymerase chain reaction, and immunofluorescence to detect changes of TREK1 and NHE1 expression in the myometrium, and isometric recording measured the uterus contraction. The NHE1 inhibitor cariporide was used to explore the effect of NHE1 on TREK1. Finally, cell contraction assay and siRNA transfection were performed to clarify the relationship between NHE1 and TREK1 in vitro. We found that the uterine contraction was notably enhanced in infected mice with E. coli and LPS administration. Meanwhile, TREK1 expression was reduced, whereas NHE1 expression was upregulated in infected mice. Cariporide alleviated the increased uterine contraction and promoted myometrium TREK1 expression in LPS-injected mice. Furthermore, suppression of NHE1 with siRNA transfection inhibited the contractility of uterine smooth muscle cells and activated the TREK1. Altogether, our findings indicate that infection increases the uterine contraction by downregulating myometrium TREK1 in mice, and the inhibition of TREK1 is attributed to the activation of NHE1.NEW & NOTEWORTHY Present work found that infection during pregnancy will increase myometrium contraction. Infection downregulated NHE1 and followed TREK1 expression and activation decrease in myometrium, resulting in increased myometrium contraction.

Mechanical activation of TWIK-related potassium channel by nanoscopic movement and rapid second messenger signaling.

Rapid conversion of force into a biological signal enables living cells to respond to mechanical forces in their environment. The force is believed to initially affect the plasma membrane and then alter the behavior of membrane proteins. Phospholipase D2 (PLD2) is a mechanosensitive enzyme that is regulated by a structured membrane-lipid site comprised of cholesterol and saturated ganglioside (GM1). Here we show stretch activation of TWIK-related K+ channel (TREK-1) is mechanically evoked by PLD2 and spatial patterning involving ordered GM1 and 4,5-bisphosphate (PIP2) clusters in mammalian cells. First, mechanical force deforms the ordered lipids, which disrupts the interaction of PLD2 with the GM1 lipids and allows a complex of TREK-1 and PLD2 to associate with PIP2 clusters. The association with PIP2 activates the enzyme, which produces the second messenger phosphatidic acid (PA) that gates the channel. Co-expression of catalytically inactive PLD2 inhibits TREK-1 stretch currents in a biological membrane. Cellular uptake of cholesterol inhibits TREK-1 currents in culture and depletion of cholesterol from astrocytes releases TREK-1 from GM1 lipids in mouse brain. Depletion of the PLD2 ortholog in flies results in hypersensitivity to mechanical force. We conclude PLD2 mechanosensitivity combines with TREK-1 ion permeability to elicit a mechanically evoked response.

"Ouch!": you have just stabbed your little toe on the sharp corner of a coffee table. That painful sensation stems from nerve cells converting information about external forces into electric signals the brain can interpret. Increasingly, new evidence is suggesting that this process may be starting at fat-based structures within the membrane of these cells. The cell membrane is formed of two interconnected, flexible sheets of lipids in which embedded structures or molecules are free to move. This organisation allows the membrane to physically respond to external forces and, in turn, to set in motion chains of molecular events that help fine-tune how cells relay such information to the brain. For instance, an enzyme known as PLD2 is bound to lipid rafts ­ precisely arranged, rigid fatty 'clumps' in the membrane that are partly formed of cholesterol. PLD2 has also been shown to physically interact with and then activate the ion channel TREK-1, a membrane-based protein that helps to prevent nerve cells from relaying pain signals. However, the exact mechanism underpinning these interactions is difficult to study due to the nature and size of the molecules involved. To address this question, Petersen et al. combined a technology called super-resolution imaging with a new approach that allowed them to observe how membrane lipids respond to pressure and fluid shear. The experiments showed that mechanical forces disrupt the careful arrangement of lipid rafts, causing PLD2 and TREK-1 to be released. They can then move through the surrounding membrane where they reach a switch that turns on TREK-1. Further work revealed that the levels of cholesterol available to mouse cells directly influenced how the clumps could form and bind to PLD2, and in turn, dialled up and down the protective signal mediated by TREK-1. Overall, the study by Petersen et al. shows that the membrane of nerve cells can contain cholesterol-based 'fat sensors' that help to detect external forces and participate in pain regulation. By dissecting these processes, it may be possible to better understand and treat conditions such as diabetes and lupus, which are associated with both pain sensitivity and elevated levels of cholesterol in tissues.

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-1ß, 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-1ß 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.

Evolution of two-pore domain potassium channels and their gene expression in zebrafish embryos.

BACKGROUND: The two-pore domain potassium (K2P) channels are a major type of potassium channels that maintain the cell membrane potential by conducting passive potassium leak currents independent of voltage change. They play prominent roles in multiple physiological processes, including neuromodulation, perception of pain, breathing and mood control, and response to volatile anesthetics. Mutations in K2P channels have been linked to many human diseases, such as neuronal and cardiovascular disorders and cancers. Significant progress has been made to understand their protein structures, physiological functions, and pharmacological modifiers. However, their expression and function during embryonic development remain largely unknown. RESULTS: We employed the zebrafish model and identified 23 k2p genes using BLAST search and gene cloning. We first analyzed vertebrate K2P channel evolution by phylogenetic and syntenic analyses. Our data revealed that the six subtypes of the K2P genes have already evolved in invertebrates long before the emergence of vertebrates. Moreover, the vertebrate K2P gene number increased, most likely due to two whole-genome duplications. Furthermore, we examined zebrafish k2p gene expression during early embryogenesis by in situ hybridization. Each subgroup's genes showed similar but distinct gene expression domains with some exceptions. Most of them were expressed in neural tissues consistent with their known function of neural excitability regulation. However, a few k2p genes were expressed temporarily in specific tissues or organs, suggesting that these K2P channels may be needed for embryonic development. CONCLUSIONS: Our phylogenetic and developmental analyses of K2P channels shed light on their evolutionary history and potential roles during embryogenesis related to their physiological functions and human channelopathies.

Assessment of Research Ethics Knowledge of Pediatricians.

Objectives. To assess knowledge of ethics knowledge among child healthcare professionals. Methods. We translated the Test of Residents' Ethics Knowledge for Pediatrics (TREK-P) in Azeri and administered it to: (i) third-year medical students (n = 21), (ii) pediatrics residents (n = 24), (iii) practicing pediatricians (n = 21), and (iv) fellows (n = 9) who participated in a Fogarty International Center/National Institute of Health (Fogarty/NIH) R25 research ethics education program. The difference in the TREK-P score between the groups and the correlation between the TREK-P score and other factors were evaluated. Results. The fellows scored significantly higher than the other groups (P = .006). There was no significant difference between the other 3 groups. Within a joined group of pediatricians and fellows, previous training on ethics was the only factor that correlated with the higher TREK-P scores (P < .05). Conclusions. The higher scores in fellows support the effectiveness of postgraduate Fogarty/NIH training programs in research ethics.

K2P2.1 is a regulator of inflammatory cell responses in idiopathic inflammatory myopathies.

K2P2.1 (TREK1), a two-pore domain potassium channel, has emerged as regulator of leukocyte transmigration into the central nervous system. In the context of skeletal muscle, immune cell infiltration constitutes the pathogenic hallmark of idiopathic inflammatory myopathies (IIMs). However, the underlying mechanisms remain to be elucidated. In this study, we investigated the role of K2P2.1 in the autoimmune response of IIMs. We detected K2P2.1 expression in primary skeletal muscle and endothelial cells of murine and human origin. We observed an increased pro-inflammatory cell response, adhesion and transmigration by pharmacological blockade or genetic deletion of K2P2.1 in vitro and in in vivo myositis mouse models. Of note, our findings were not restricted to endothelial cells as skeletal muscle cells with impaired K2P2.1 function also demonstrated a strong pro-inflammatory response. Conversely, these features were abrogated by activation of K2P2.1 and improved the disease course of a myositis mouse model. In humans, K2P2.1 expression was diminished in IIM patients compared to non-diseased controls arguing for the translatability of our findings. In summary, K2P2.1 may regulate the inflammatory response of skeletal muscle. Further research is required to understand whether K2P2.1 could serve as novel therapeutic target.

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.

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.

Novel potent blockers for TWIK-1/TREK-1 heterodimers as potential antidepressants.

TREK-1 (TWIK-related potassium channel-1) is a subunit of the two-pore domain potassium (K2p) channel and is widely expressed in the brain. TREK-1 knockout mice were shown to have antidepressant-like effects, providing evidence for the channel's potential as a therapeutic target. However, currently there is no good pharmacological inhibitor specifically targeting TREK-1 containing K2p channels that also displays similar antidepressant-like effects. Here, we sought to find selective and potent inhibitors for TREK-1 related dimers both in vitro and in vivo. We synthesized and evaluated 2-hydroxy-3-phenoxypropyl piperidine derivatives yielding a library from which many TREK-1 targeting candidates emerged. Among these, hydroxyl-phenyl- (2a), piperidino- (2g), and pyrrolidino- (2h) piperidinyl substituted compounds showed high potencies to TREK-1 homodimers with significant antidepressant-like effects in forced swim test and tail suspension test. Interestingly, these compounds were found to have high potencies to TWIK-1/TREK-1 heterodimers. Contrastingly, difluoropiperidinyl-4-fluorophenoxy (3e) and 4-hydroxyphenyl-piperidinyl-4-fluorophenoxy (3j) compounds had high potencies to TREK-1 homodimer but lower potency to TWIK-1/TREK-1 heterodimers without significant antidepressant-like effects. We observed positive correlation between inhibition potency to TWIK-1/TREK-1 and immobility time, and no correlation between inhibition potency to TREK-1 homodimer and immobility time. This was consistent with molecular docking simulations of selected compounds to TREK-1 homodimeric and TWIK-1/TREK-1 heterodimeric models. Existing antidepressant fluoxetine was also found to potently inhibit TWIK-1/TREK-1 heterodimers. Our study reveals novel potent TWIK-1/TREK-1 inhibitors 2a, 2g, and 2h as potential antidepressants and suggest that the TWIK-1/TREK-1 heterodimer could be a potential novel molecular therapeutic target for antidepressants.