TREK-1 Channels Research Articles

TREK Channel Family Activator for Relief of Pain and Neurogenic Inflammation

TREK Channel Family Activator for Relief of Pain and Neurogenic Inflammation

TMIE Defines Pore and Gating Properties of the Mechanotransduction Channel of Mammalian Cochlear Hair Cells

TMC1 and TMC2 (TMC1/2) have been proposed to form the pore of the mechanotransduction channel of cochlear hair cells. Here, we show that TMC1/2 cannot form mechanotransduction channels in cochlear hair cells without TMIE. TMIE binds to TMC1/2, and a TMIE mutation that perturbs TMC1/2 binding abolishes mechanotransduction. N-terminal TMIE deletions affect the response of the mechanotransduction channel to mechanical force. Similar to mechanically gated TREK channels, the C-terminal cytoplasmic TMIE domain contains charged amino acids that mediate binding to phospholipids, including PIP2. TMIE point mutations in the C terminus that are linked to deafness disrupt phospholipid binding, sensitize the channel to PIP2 depletion from hair cells, and alter the channel's unitary conductance and ion selectivity. We conclude that TMIE is a subunit of the cochlear mechanotransduction channel and that channel function is regulated by a phospholipid-sensing domain in TMIE with similarity to those in other mechanically gated ion channels.

Spadin Selectively Antagonizes Arachidonic Acid Activation of TREK-1 Channels

TREK-1 channel activity is a critical regulator of neuronal, cardiac, and smooth muscle physiology and pathology. The antidepressant peptide, spadin, has been proposed to be a TREK-1-specific blocker. Here we sought to examine the mechanism of action underlying spadin inhibition of TREK-1 channels. Heterologous expression in Xenopus laevis oocytes and electrophysiological analysis using two-electrode voltage clamp in standard bath solutions was used to characterize the pharmacological profile of wild-type and mutant murine TREK-1 and TREK-2 channels using previously established human K2P activators; arachidonic acid (AA), cis-4,7,10,13,16,19-docosahexaenoic acid (DHA), BL-1249, and cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC) and inhibitors; spadin and barium (Ba2+). Mouse TREK-1 and TREK-2 channel currents were both significantly increased by AA, BL-1249, and CDC, similar to their human homologs. Under basal conditions, both TREK-1 and TREK-2 currents were insensitive to application of spadin, but could be blocked by Ba2+. Spadin did not significantly inhibit either TREK-1 or TREK-2 currents either chemically activated by AA, BL-1249, or CDC, or structurally activated via a gating mutation. However, pre-exposure to spadin significantly perturbed the subsequent activation of TREK-1 currents by AA, but not TREK-2. Furthermore, spadin was unable to prevent activation of TREK-1 by BL-1249, CDC, or the related bioactive lipid, DHA. Spadin specifically antagonizes the activation of TREK-1 channels by AA, likely via an allosteric mechanism. Lack of intrinsic activity may explain the absence of clinical side effects during antidepressant therapy.

TREK Channel Family Activator with a Well-Defined Structure-Activation Relationship for Pain and Neurogenic Inflammation.

TWIK-related K+ (TREK) channels are potential analgesic targets. However, selective activators for TREK with both defined action mechanism and analgesic ability for chronic pain have been lacking. Here, we report (1S,3R)-3-((4-(6-methylbenzo[d]thiazol-2-yl)phenyl)carbamoyl)cyclopentane-1-carboxylic acid (C3001a), a selective activator for TREK, against other two-pore domain K+ (K2P) channels. C3001a binds to the cryptic binding site formed by P1 and TM4 in TREK-1, as suggested by computational modeling and experimental analysis. Furthermore, we identify the carboxyl group of C3001a as a structural determinant for binding to TREK-1/2 and the key residue that defines the subtype selectivity of C3001a. C3001a targets TREK channels in the peripheral nervous system to reduce the excitability of nociceptive neurons. In neuropathic pain, C3001a alleviated spontaneous pain and cold hyperalgesia. In a mouse model of acute pancreatitis, C3001a alleviated mechanical allodynia and inflammation. Together, C3001a represents a lead compound which could advance the rational design of peripherally acting analgesics targeting K2P channels without opioid-like adverse effects.

PIP2 Mediated Inhibition of TREK Potassium Currents by Bradykinin in Mouse Sympathetic Neurons.

Bradykinin (BK), a hormone inducing pain and inflammation, is known to inhibit potassium M-currents (IM) and to increase the excitability of the superior cervical ganglion (SCG) neurons by activating the Ca2+-calmodulin pathway. M-current is also reduced by muscarinic agonists through the depletion of membrane phosphatidylinositol 4,5-biphosphate (PIP2). Similarly, the activation of muscarinic receptors inhibits the current through two-pore domain potassium channels (K2P) of the “Tandem of pore-domains in a Weakly Inward rectifying K+ channel (TWIK)-related channels” (TREK) subfamily by reducing PIP2 in mouse SCG neurons (mSCG). The aim of this work was to test and characterize the modulation of TREK channels by bradykinin. We used the perforated-patch technique to investigate riluzole (RIL) activated currents in voltage- and current-clamp experiments. RIL is a drug used in the palliative treatment of amyotrophic lateral sclerosis and, in addition to blocking voltage-dependent sodium channels, it also selectively activates the K2P channels of the TREK subfamily. A cell-attached patch-clamp was also used to investigate TREK-2 single channel currents. We report here that BK reduces spike frequency adaptation (SFA), inhibits the riluzole-activated current (IRIL), which flows mainly through TREK-2 channels, by about 45%, and reduces the open probability of identified single TREK-2 channels in cultured mSCG cells. The effect of BK on IRIL was precluded by the bradykinin receptor (B2R) antagonist HOE-140 (d-Arg-[Hyp3, Thi5, d-Tic7, Oic8]BK) but also by diC8PIP2 which prevents PIP2 depletion when phospholipase C (PLC) is activated. On the contrary, antagonizing inositol triphosphate receptors (IP3R) using 2-aminoethoxydiphenylborane (2-APB) or inhibiting protein kinase C (PKC) with bisindolylmaleimide did not affect the inhibition of IRIL by BK. In conclusion, bradykinin inhibits TREK-2 channels through the activation of B2Rs resulting in PIP2 depletion, much like we have demonstrated for muscarinic agonists. This mechanism implies that TREK channels must be relevant for the capture of information about pain and visceral inflammation.

TREK for High-Speed and High-Frequency Conduction through the Axon

In this issue of Neuron, Kanda etal. (2019) find that the two-pore domain potassium channels TRAAK and TREK1 drive axonal action potential repolarization for high-speed and high-frequency nervous impulses in mammalian myelinated nerves.

Astrocytic AEG-1 regulates expression of TREK-1 under acute hypoxia.

TREK-1 (TWIK-related K+ channel), a member of the two-pore domain K+ (K2P) channel family, is highly expressed in astrocytes, where it plays a key role in glutamate release and passive conductance. In addition, TREK-1 is induced to protect neurons under pathological conditions such as hypoxia. However, the upstream regulation of TREK-1 remains poorly understood. In this study, we found that AEG-1 (astrocyte elevated gene-1) regulates the expression of astrocytic TREK-1 under hypoxic conditions. Upregulation of AEG-1 increased expression of TREK-1 in astrocytes, and knockdown of AEG-1 dramatically decreased the mRNA and protein levels of TREK-1, which were restored by expression of shRNA-insensitive AEG-1. In addition, expression of TREK-1 was not regulated in the absence of AEG-1, even when HIF1α was present. Together, these results suggest that AEG-1 acts as a major upstream regulator of TREK-1 channels in astrocytes under hypoxia. SIGNIFICANCE OF THE STUDY: Previous studies have reported that hypoxia increases the expression of astrocytic TREK-1 and that increased TREK-1 expression protects neuronal cells from apoptosis. However, its cellular mechanism is not clear. In this study we first showed that AEG-1 is a major mediator of hypoxic-regulated TREK-1 expression in normal astrocytes independently of HIF-1α.

N-Glycosylation of TREK-1/hK2P2.1 Two-Pore-Domain Potassium (K2P) Channels.

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.

Spadin and shortened spadin analogs as efficient new antidepressants in mouse models of post-stroke depression

Stroke is a major disease associated with high mortality and serious long-term disability. Usual treatments fail to improve long-term recovery and thrombolysis is only efficient on 10% of patients. Consequently, developing new treatments is necessary. The TREK-1 channel was shown to be protective against stroke. We have identified a peptide (spadin or PE 12-28) and its shortened analogs that are potent antidepressant and are able to inhibit TREK-1. Our aim was to demonstrate that these peptides have protective effects against post-stroke depression. For mimicking stroke on mice, we used the in vivo model of MCAO. Thanks to electrophysiology studies, we developed a protocol consisting in a two phase treatment, a low dose (0.03 mg/kg) for one week followed by a high dose (3 mg/kg) treatment for several weeks. At different time points, behavioral tests were performed in order measure both motor and cognitive performance of the animals. Treated mice showed a significant reduction of the immobility time in the Forced Swimming Test and in the latency to eat in the Novelty Suppressed Feeding test. Both tests demonstrate that the depressive state is improved. The learning capacity was increased in the Morris Water Maze and the motor coordination was improved in both rotarod and pole tests. Additionally, the increase in neurogenesis, measured by BrdU incorporation was still present even at 10 weeks post trauma. Taken together our results suggest that spadin and its analogs are very potent candidates for the development of new treatments improving stroke recovery, mainly by preventing the depression.

Polymodal Mechanism for TWIK-Related K+ Channel Inhibition by Local Anesthetic.

Local anesthetics cause reversible block of pain and robustly inhibit TWIK-related K channel (TREK-1) currents. Before local anesthesia onset, injection of local anesthetics can cause unwanted transient pain. TREK-1 is an anesthetic-sensitive potassium channel that when inhibited produces pain. A disordered C-terminal loop of TREK-1 is thought to contribute to anesthetic sensitivity, but the molecular basis for TREK-1 inhibition by local anesthetics is unknown. Phospholipase D2 (PLD2) is an enzyme that produces phosphatidic acid (PA) required for TREK-1 activation and also binds to the channel's C terminus. Here, we use biophysical and cellular techniques to characterize direct and indirect lipid-mediated mechanism for TREK-1 inhibition (respectively). We characterized direct binding of local anesthetic to TREK-1 by reconstituting the purified channel into artificial membranes and measuring ion flux. We characterized indirect PA-mediated inhibition of TREK-1 by monitoring lipid production in live whole cells using a fluorescent PLD2 product release assay and ion channel current using live whole-cell patch-clamp electrophysiology. We monitored anesthetic-induced nanoscale translocation of PLD2 to TREK-1 channels with super-resolution direct stochastic reconstruction microscopy (dSTORM). We find local anesthetics tetracaine, lidocaine, and bupivacaine directly bind to and inhibit PLD2 enzymatic activity. The lack of PLD2 activity indirectly inhibited TREK-1 currents. Select local anesthetics also partially blocked the open pore of TREK-1 through direct binding. The amount of pore block was variable with tetracaine greater than bupivacaine and lidocaine exhibiting a minor effect. Local anesthetics also disrupt lipid rafts, a mechanism that would normally activate PLD2 were it not for their direct inhibition of enzyme catalysis. We propose a mechanism of TREK-1 inhibition comprised of (1) primarily indirect PLD2-dependent inhibition of lipid catalysis and (2) limited direct inhibition for select local anesthetics through partial open pore block. The inhibition through PLD2 explains how the C terminus can regulate the channel despite being devoid of structure and putative binding sites for local anesthetics.

Pranlukast is a novel small molecule activator of the two-pore domain potassium channel TREK2

TREK2 (KCNK10, K2P10.1) is a two-pore domain potassium (K2P) channel and a potential target for the treatment of pain. Like the majority of the K2P superfamily, there is currently a lack of useful pharmacological tools to study TREK2. Here we present a strategy for identifying novel TREK2 activators. A cell-based thallium flux assay was developed and used to screen a library of drug-like molecules, from which we identified the CysLT1 antagonist Pranlukast as a novel activator of TREK2. This compound was selective for TREK2 versus TREK1 and showed no activity at TRAAK. Pranlukast was also screened against other members of the K2P superfamily. Several close analogues of Pranlukast and other CysLT1 antagonists were also tested for their ability to activate K2P channels. Consistent with previous work, structure activity relationships showed that subtle structural changes to these analogues completely attenuated the activation of TREK2, whereas for TREK1, analogues moved from activators to inhibitors. Pranlukast’s activity was also confirmed using whole-cell patch clamp electrophysiology. Studies using mutant forms of TREK2 suggest Pranlukast does not bind in the K2P modulator pocket or the BL-1249 binding site. Pranlukast therefore represents a novel tool by which to study the mechanism of TREK2 activation.

TREK1 channel in DGGCs ameliorates depression-like behaviour and increases adult hippocampal neurogenesis in mice

TREK1 channel in DGGCs ameliorates depression-like behaviour and increases adult hippocampal neurogenesis in mice

Pharmacologically reversible, loss of function mutations in the TM2 and TM4 inner pore helices of TREK-1 K2P channels

A better understanding of the gating of TREK two pore domain potassium (K2P) channels and their activation by compounds such as the negatively charged activator, flufenamic acid (FFA) is critical in the search for more potent and selective activators of these channels. Currents through wild-type and mutated human K2P channels expressed in tsA201 cells were measured using whole-cell patch-clamp recordings in the presence and absence of FFA. Mutation of the TM2.6 residue of TREK-1 to a phenylalanine (G171F) and a similar mutation of TM4.6 (A286F) substantially reduced current through TREK-1 channels. In complementary experiments, replacing the natural F residues at the equivalent position in TRESK channels, significantly enhanced current. Known, gain of function mutations of TREK-1 (G137I, Y284A) recovered current through these mutated channels. This reduction in current could be also be reversed pharmacologically, by FFA. However, an appropriate length MTS (MethaneThioSulfonate) cross-linking reagent (MTS14) restricted the activation of TREK-1_A286C channels by repeated application of FFA. This suggests that the cross-linker stabilises the channel in a conformation which blunts FFA activation. Pharmacologically reversible mutations of TREK channels will help to clarify the importance of these channels in pathophysiological conditions such as pain and depression.

First evidence of protective effects on stroke recovery and post-stroke depression induced by sortilin-derived peptides

Post-stroke depression (PSD) is the most common mood disorder following stroke with high relevance for outcome and survival of patients. The TREK-1 channel represents a crucial target in the pathogenesis of stroke and depression. Spadin and its short analog mini-spadin were reported to display potent antidepressant properties. We investigated the therapeutic effects of mini-spadin in a mouse model of focal ischemia and PSD. To activate TREK-1 and induce neuroprotection a single low dose of mini-spadin (0.03 μg/kg) was intraperitoneally injected 30 min after the onset of ischemia, once a day during 7 days post-ischemia. Then, to inhibit TREK-1 and induce antidepressant effect, the peptide was injected at higher concentration (3 μg/kg) once a day for 4 days/week until the sacrifice of animals. Electrophysiological studies showed that mini-spadin had a biphasic action on TREK-1. At low doses, the channel activity was increased whereas at higher doses it was inhibited. Mini-spadin prevented the loss of body weight and the delayed dopaminergic degeneration in substantia nigra and improved the motor and cognitive ischemia-induced deficits. Moreover, mini-spadin prevented PSD analyzed in the Forced Swim (FST) and Novelty Suppressed Feeding (NSF) tests. Finally, enhanced neurogenesis and synaptogenesis contributed to the beneficial effects of mini-spadin against stroke and PSD. This work reveals the first evidence that the modulation of TREK-1 channels in the early and chronic phases of stroke as well as the stimulation of brain plasticity by mini-spadin could play a key role in its brain protective effects against stroke and its deleterious consequences such as PSD.

Trabecular Meshwork TREK-1 Channels Function as Polymodal Integrators of Pressure and pH.

PurposeThe concentration of protons in the aqueous humor (AH) of the vertebrate eye is maintained close to blood pH; however, pathologic conditions and surgery may shift it by orders of magnitude. We investigated whether and how changes in extra- and intracellular pH affect the physiology and function of trabecular meshwork (TM) cells that regulate AH outflow.MethodsElectrophysiology, in conjunction with pharmacology, gene knockdown, and optical recording, was used to track the pH dependence of transmembrane currents and mechanotransduction in primary and immortalized human TM cells.ResultsExtracellular acidification depolarized the resting membrane potential by inhibiting an outward K+-mediated current, whereas alkalinization hyperpolarized the cells and augmented the outward conductance. Intracellular acidification with sodium bicarbonate hyperpolarized TM cells, whereas removal of intracellular protons with ammonium chloride depolarized the membrane potential. The effects of extra- and intracellular acid and alkaline loading were abolished by quinine, a pan-selective inhibitor of two-pore domain potassium (K2P) channels, and suppressed by shRNA-mediated downregulation of the mechanosensitive K2P channel TREK-1. Extracellular acidosis suppressed, whereas alkalosis facilitated, the amplitude of the pressure-evoked TREK-1–mediated outward current.ConclusionsThese results demonstrate that TM mechanotransduction mediated by TREK-1 channels is profoundly sensitive to extra- and intracellular pH shifts. Intracellular acidification might modulate aqueous outflow and IOP by stimulating TREK-1 channels.

Altered detrusor contractility and voiding patterns in mice lacking the mechanosensitive TREK-1 channel

BackgroundPreviously published results from our laboratory identified a mechano-gated two-pore domain potassium channel, TREK-1, as a main mechanosensor in the smooth muscle of the human urinary bladder. One of the limitations of in vitro experiments on isolated human detrusor included inability to evaluate in vivo effects of TREK-1 on voiding function, as the channel is also expressed in the nervous system, and may modulate micturition via neural pathways. Therefore, in the present study, we aimed to assess the role of TREK-1 channel in bladder function and voiding patterns in vivo by using TREK-1 knockout (KO) mice.MethodsAdult C57BL/6 J wild-type (WT, N = 32) and TREK-1 KO (N = 33) mice were used in this study. The overall phenotype and bladder function were evaluated by gene and protein expression of TREK-1 channel, in vitro contractile experiments using detrusor strips in response to stretch and pharmacological stimuli, and cystometry in unanesthetized animals.ResultsTREK-1 KO animals had an elevated basal muscle tone and enhanced spontaneous activity in the detrusor without detectable changes in bladder morphology/histology. Stretch applied to isolated detrusor strips increased the amplitude of spontaneous contractions by 109% in the TREK-1 KO group in contrast to a 61% increase in WT mice (p ≤ 0.05 to respective baseline for each group). The detrusor strips from TREK-1 KO mice also generated more contractile force in response to electric field stimulation and high potassium concentration in comparison to WT group (p ≤ 0.05 for both tests). However, cystometric recordings from TREK-1 KO mice revealed a significant increase in the duration of the intermicturition interval, enhanced bladder capacity and increased number of non-voiding contractions in comparison to WT mice.ConclusionsOur results provide evidence that global down-regulation of TREK-1 channels has dual effects on detrusor contractility and micturition patterns in vivo. The observed differences are likely due to expression of TREK-1 channel not only in detrusor myocytes but also in afferent and efferent neural pathways involved in regulation of micturition which may underly the “mixed” voiding phenotype in TREK-1 KO mice.

Involvement of TREK-1 Channel in Cell Viability of H9c2 Rat Cardiomyoblasts Affected by Bupivacaine and Lipid Emulsion.

Lipid emulsion (LE) therapy has been used to reduce overdose of bupivacaine (BPV)-induced cardiotoxicity. The TWIK-related potassium channel-1 (TREK-1) is inhibited by BPV and activated by polyunsaturated fatty acids, which are the main component in LE. These pharmacological properties inspired us to investigate whether the TREK-1 channel is associated with cell viability of H9c2 cardiomyoblasts affected by BPV and LE. Consistent with previous studies, BPV-induced cell death was reduced by LE treatment. The reduction in the TREK-1 expression level by BPV was alleviated by LE. The BPV cytotoxicity highly decreased in TREK-1 overexpressed cells but was the opposite in TREK-1 knocked-down cells. TREK-1 channel activators and inhibitors increased and decreased cell viability, respectively. BPV-induced depolarization of the plasma and mitochondrial membrane potential and increase in intracellular Ca2+ level were blocked by LE treatment. BPV-induced depolarization of membrane potential was reduced in TREK-1 overexpressed cells, indicating that TREK-1 channels mediate setting the resting membrane potentials as a background K+ channel in H9c2 cells. These results show that TREK-1 activity is involved in the BPV cytotoxicity and the antagonistic effect of LE in H9c2 cells and suggest that TREK-1 could be a target for action of BPV and LE.

Role of TREK-1 in Health and Disease, Focus on the Central Nervous System.

TREK-1 is the most studied background K2P channel. Its main role is to control cell excitability and maintain the membrane potential below the threshold of depolarization. TREK-1 is multi-regulated by a variety of physical and chemical stimuli which makes it a very promising and challenging target in the treatment of several pathologies. It is mainly expressed in the brain but also in heart, smooth muscle cells, endocrine pancreas, and prostate. In the nervous system, TREK-1 is involved in many physiological and pathological processes such as depression, neuroprotection, pain, and anesthesia. These properties explain why many laboratories and pharmaceutical companies have been focusing their research on screening and developing highly efficient modulators of TREK-1 channels. In this review, we summarize the different roles of TREK-1 that have been investigated so far in attempt to characterize pharmacological tools and new molecules to modulate cellular functions controlled by TREK-1.

DCPIB, an Inhibitor of Volume-Regulated Anion Channels, Distinctly Modulates K2P Channels.

K2P potassium channels stabilize the resting membrane potential in nearly all cells and have been implicated in several neuronal, cardiovascular, and immune diseases. DCPIB, a known specific and potent inhibitor of volume-regulated anion channels (VRAC), has been reported to activate TREK1 and TREK2 in astrocytes and in vitro recently. In the present study, we demonstrated DCPIB also voltage dependently activated TRAAK besides TREK1/TREK2, showing DCPIB activated all TREK subfamily members. In contrast, the compound potently inhibited several other K2P channels with no voltage dependence, including TRESK, TASK1, and TASK3. DCPIB displayed superior selectivity toward TRESK with an IC50 of 0.14 μM, demonstrating at least 100-fold higher affinity over TREK1/TRAAK channels. Furthermore, the impaired ion selectivity filter region greatly impaired the activating effect of DCPIB on TREK1 but not the inhibitory effect of DCPIB on TRESK. This indicates distinct molecular determinants underlying the effect of DCPIB on TREK1 or TRESK channels. Our results showed that DCPIB played diverse effects on K2P channels and could be a useful tool for further investigating structure-function studies of K2P channels.

Extracellular divalent ions modulate TREK-2-like channel conductance in prefrontal pyramidal neurons in rats

Background: The aim of the study was to investigate, with the use of the patch clamp technique, the dependence of the conductance of the TREK-2-like potassium leak channel in the medial prefrontal cortex pyramidal neurons on the presence of extracellular magnesium and calcium ions. It is suspected that TREK-2 channels regulate mood and may be associated with the pathophysiology of depression. Since magnesium and calcium deficiency contribute to depressive symptoms, we investigated how TREK-2- like channel pore propertie change in the absence of divalent cations. Results: Single-channel currents were recorded in a cell-attached configuration in enzymatically dispersed pyramidal neurons of the prefrontal cortex in rats. Spontaneous TREK-2-like channel activity was recorded either in the presence or absence of magnesium and calcium ions in extracellular solution. A significant increase in the inward channel conductance was observed when divalent cations were removed from the extracellular solution. Inward rectification was also increased when the bath temperature was raised to 34-37°C. Conclusions: The study confirmed that the activity of TREK-2-like channels is affected by the presence of magnesium and calcium ions in the extracellular solution. Therefore, in vivo, the TREK-2-like channel may possibly participate in the prefrontal cortex dysfunction associated with the deficiency of divalent cations.