PE-22-28 was designed by a French research group as an improved, longer-lasting version of spadin, a peptide discovered in 2010 that blocks a potassium channel called TREK-1 and produced fast antidepressant-like effects in mice. Every piece of evidence on PE-22-28 and its close relatives comes from mice, rats, or cells in a dish - there are no human trials, no approved medical use, and no doctor overseeing anyone who takes it. It circulates online as a 'research peptide' pitched at people wanting a faster mood boost than standard antidepressants, but that marketing runs well ahead of the actual science.
How strong is the evidence?
Every study behind PE-22-28 is a mouse or rat behavior experiment, a cell/tissue electrophysiology experiment, or basic lab biochemistry - there is no clinical trial of PE-22-28, or even of spadin (the peptide it's built from), in humans. Only one paper tests the exact PE-22-28 molecule directly; the rest study spadin, the longer and less stable peptide PE-22-28 was engineered from, or study the TREK-1 channel more broadly. One paper measured natural blood levels of a related molecule in people with depression, but that is monitoring a biomarker, not testing the drug - it doesn't tell you what happens if a person takes PE-22-28. Read everything here as 'what happened in animals and cells,' not 'what will happen in you.'
Uses
What people use it for
Explored as a rapid-onset antidepressant
Animal / labIn mouse studies, PE-22-28 and its parent peptide spadin reduced depression-like behavior in as little as 4 days, compared with the 2-4 weeks typical antidepressants take to work. Speeding up that timeline is the whole reason researchers built this peptide.
Used as a lab tool to study mood-related brain wiring
Animal / labScientists use it to understand how blocking one specific potassium channel (TREK-1) speeds up growth of new brain-cell connections in animals - useful for basic research even without a human product in sight.
Sold online as an unapproved 'research peptide'
AnecdotalBecause of its fast-antidepressant buzz, PE-22-28 is marketed on research-chemical and biohacking sites, despite having zero testing in people and no manufacturing or purity oversight.
Potential benefits
What it may help with
May lift mood quickly in animal models
Animal / labIn mice, PE-22-28 and spadin cut 'giving up' behavior in standard antidepressant tests (like the forced swim test) within about 4 days, versus the 2-4 weeks needed for drugs like fluoxetine. Each dose of PE-22-28 also lasted longer in the body - up to about 23 hours, versus roughly 7 hours for spadin.
Encourages new brain-cell connections in lab and animal studies
Animal / labIt raised BDNF, a protein that helps brain cells grow and connect, and increased markers of new synapse formation (PSD-95, synapsin) in neurons and in mouse brains after just a few days of treatment. Researchers think this rewiring is the biological reason behind the fast mood effect.
Boosts insulin release from pancreatic cells in lab and animal studies
Animal / labSpadin, the peptide PE-22-28 is built from, increased insulin release from pancreatic beta cells in dishes and raised blood insulin levels after glucose in mice. Researchers see this as a possible future lead for blood sugar problems, but it has never been tested as a diabetes treatment.
Didn't trigger the heart, pain, or seizure problems researchers worried about
Animal / labCompletely removing TREK-1 makes animals more sensitive to pain, seizures, and heart problems, so researchers specifically checked for those issues after weeks of spadin treatment. They found no changes to heart rhythm, blood pressure, seizure threshold, or baseline pain sensitivity in mice - reassuring, though not proof of human safety.
What to watch for
Side effects & risks
- Moderate
May worsen pain in inflamed or injured tissue
In rats with formalin-induced inflammation, injecting spadin locally or into the spinal fluid increased flinching and long-lasting sensitivity to touch and pressure, while turning TREK-1 on (the opposite action) reduced pain. This suggests TREK-1 blockers like PE-22-28 could make certain inflammatory pain worse, even though whole-body dosing in the mood studies didn't change baseline pain.
- Mild
Possible effect on fat cells and fat storage (mixed evidence)
A 2025 study found that blocking TREK-1 with spadin, or removing the gene, increased fat cell growth and fat storage in mice, especially on a high-fat diet. An earlier 2017 study found spadin had no measurable effect on fat cell insulin signaling. The two results don't agree, so effects on body fat aren't settled.
- Mild
Could shift insulin release and blood sugar unpredictably
The same insulin-boosting effect that looks promising for diabetes research is a double-edged sword: it means PE-22-28 could change blood sugar levels in ways that aren't predictable outside a lab, which matters most for people already on insulin or other glucose-lowering medication.
Dosing
Dosing — what studies used
No human dose has ever been established for PE-22-28 - it has not been given to a person in any published study. Everything known comes from mice and rats dosed by injection (mostly intravenous, plus some spinal or local injection in pain studies), and even those animal papers rarely report an exact milligram amount in their abstracts. Treatment courses that showed effects ran from 4 days up to about 3 weeks in mice. Anyone using a 'PE-22-28' product today is working entirely off guesswork, with no dose-finding studies or safety monitoring behind it - this is a record of what researchers did in animals, not a dosing guide for people.
Antidepressant-like behavior tests (the PE-22-28 molecule itself)
Animal studyNot reported in the published abstract
Repeated daily dosing · 4-day sub-chronic course · Intravenous injection (mice)
This is the only study that tests PE-22-28 itself rather than its parent peptide spadin.
Original antidepressant discovery and safety testing (parent peptide, spadin)
Animal studyNot reported in the published abstract
Daily dosing · 4 days for the initial antidepressant effect; up to 3 weeks tested for safety · Intravenous injection (mice)
Establishes the timeline and safety pattern PE-22-28 was designed to improve on.
Pain-signaling research, a different purpose (parent peptide, spadin)
Animal studyNot reported in the published abstract
Single pre-treatment dose · Acute effects tracked over 6 days after injury · Local peripheral or intrathecal (spinal) injection (rats)
Used to study pain pathways, not mood - this dosing worsened certain pain responses rather than helping them.
All dosing data here comes from animal research (mainly one lab in Nice, France) and is not a guide for human use. No regulatory body has reviewed or approved a human dose, and no published study has tracked what happens when a person takes it.
These figures describe what researchers used in studies. They are not a recommendation or a prescription.
Mechanism
How it works
Nerve cells have tiny pores in their outer membrane called potassium channels that let potassium flow out and calm the cell down. One of these, TREK-1, acts like a brake pedal - when it's open, it's harder for the cell to fire a signal. PE-22-28 presses down on that brake's brake: it blocks TREK-1, so affected nerve cells, especially serotonin-producing cells tied to mood, fire more easily. In animal studies, that extra firing seemed to kick-start growth of new brain-cell connections within days, which researchers think explains why mood improved so much faster than with standard antidepressants that take weeks to work. PE-22-28 itself is a shortened, more stable, lab-made version of a naturally occurring peptide called spadin, which the body makes from the breakdown of a larger protein called sortilin.
Who should avoid it
- Anyone with a heart rhythm condition - TREK-1 is active in heart tissue, and there is no human cardiac safety data
- People with diabetes or on blood-sugar medication - animal data show effects on insulin release that could cause unpredictable blood sugar swings
- Pregnant or breastfeeding people - no safety data exists at all
- Anyone with chronic or inflammatory pain conditions - animal data suggest TREK-1 blockers can worsen certain pain states
- Anyone wanting a doctor-supervised depression treatment - this is not an approved medicine anywhere and has never been tested in a person with depression
Interactions to know
- May add to the effect of SSRIs or SNRIs, since both act on the same serotonin-TREK-1 pathway - never studied together in people
- Could affect blood-sugar-lowering drugs like insulin or sulfonylureas, since animal studies show it changes insulin release
- No interaction data exists for any other drug, supplement, or peptide - every combination is untested
The papers that matter most
Key studies
The paper that created PE-22-28 itself. It showed PE-22-28 binds TREK-1 far more tightly than spadin, lasts about 3x longer in the body, and produced fast antidepressant-like and neuron-growth effects in mice.
Shortened Spadin Analogs Display Better TREK-1 Inhibition, In Vivo Stability and Antidepressant Activity
The original discovery of spadin as a fast-acting antidepressant peptide in mice, and the scientific foundation PE-22-28 was later built on.
Spadin, a sortilin-derived peptide, targeting rodent TREK-1 channels: a new concept in the antidepressant drug design
Specifically tested for the heart, pain, and seizure problems you'd expect from blocking TREK-1, and found none in mice after weeks of treatment - reassuring, but still animal-only data.
Spadin as a new antidepressant: absence of TREK-1-related side effects
Shows the likely mechanism: spadin raises BDNF and builds new brain-cell connections, which may explain the fast mood effect.
In vitro and in vivo regulation of synaptogenesis by the novel antidepressant spadin
A newer study showing that blocking TREK-1 (as PE-22-28 does) increased fat cell growth and fat storage in mice - a possible downside worth watching.
Novel function of TREK-1 in regulating adipocyte differentiation and lipid accumulation
Blocking TREK-1 locally made certain inflammatory pain responses worse in rats, a caution for anyone with pain conditions.
TREK-1 potassium channels participate in acute and long-lasting nociceptive hypersensitivity induced by formalin in rats
Bottom line
PE-22-28 has a genuinely interesting fast-antidepressant story in mice, but that's where the story stops today - it's an unapproved peptide with zero human testing, and using it means accepting real unknowns around your heart, blood sugar, and pain response.
Research papers
Studies we have on file for PE-22-28. Tap a title to open it on PubMed. Labels like “animal” or “human trial” are rough guides.
40 papers
Fighting against depression with TREK-1 blockers: Past and future. A focus on spadin.
Depression is a devastating mood disorder and a leading cause of disability worldwide. Depression affects approximately one in five individuals in the world and represents heavy economic and social burdens. The neurobiological mechanisms of depression are not fully understood, but evidence highlights the role of monoamine neurotransmitter balance. Several antidepressants (ADs) are marketed to treat depression and related mood disorders. However, despite their efficacy, they remain nonspecific and unsafe because they trigger serious adverse effects. Therefore, developing new molecules for new targets in depression has become a real necessity. Eight years ago, spadin was described as a natural peptide with AD properties. This 17-amino acid peptide blocks TREK-1 channels, an original target in depression. Compared to the classical AD drugs such as fluoxetine, which requires 3-4 weeks for the AD effect to manifest, spadin acts rapidly within only 4 days of treatment. The AD properties are associated with increased neurogenesis and synaptogenesis in the brain. Despite the advantages of this fast-acting AD, the in vivo stability is weak and does not last for >7 h. The present review summarizes different strategies such as retro-inverso strategy, cyclization, and shortening the spadin sequence that has led to the development and optimization of spadin as an AD. Shortened spadin analogs present increased inhibition potency for TREK-1, an improved AD activity, and prolonged in vivo bioavailability. Finally, we also discuss about other inhibitors of TREK-1 channels with a proven efficacy in treating depression in the clinic, such as fluoxetine.
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.
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.
Spadin Modulates Astrocytic Passive Conductance via Inhibition of TWIK-1/TREK-1 Heterodimeric Channels.
Astrocytes, the most abundant cell type in the brain, are non-excitable cells and play critical roles in brain function. Mature astrocytes typically exhibit a linear current-voltage relationship termed passive conductance, which is believed to enable astrocytes to maintain potassium homeostasis in the brain. We previously demonstrated that TWIK-1/TREK-1 heterodimeric channels mainly contribute to astrocytic passive conductance. However, the molecular identity of astrocytic passive conductance is still controversial and needs to be elucidated. Here, we report that spadin, an inhibitor of TREK-1, can dramatically reduce astrocytic passive conductance in brain slices. A series of gene silencing experiments demonstrated that spadin-sensitive currents are mediated by TWIK-1/TREK-1 heterodimeric channels in cultured astrocytes and hippocampal astrocytes from brain slices. Our study clearly showed that TWIK-1/TREK-1-heterodimeric channels can act as the main molecular machinery of astrocytic passive conductance, and suggested that spadin can be used as a specific inhibitor to control astrocytic passive conductance.
Identifying fast-onset antidepressants using rodent models.
Depression is a leading cause of disability worldwide and a major contributor to the burden of suicide. A major limitation of classical antidepressants is that 2-4 weeks of continuous treatment is required to elicit therapeutic effects, prolonging the period of depression, disability and suicide risk. Therefore, the development of fast-onset antidepressants is crucial. Preclinical identification of fast-onset antidepressants requires animal models that can accurately predict the delay to therapeutic onset. Although several well-validated assay models exist that predict antidepressant potential, few thoroughly tested animal models exist that can detect therapeutic onset. In this review, we discuss and assess the validity of seven rodent models currently used to assess antidepressant onset: olfactory bulbectomy, chronic mild stress, chronic forced swim test, novelty-induced hypophagia (NIH), novelty-suppressed feeding (NSF), social defeat stress, and learned helplessness. We review the effects of classical antidepressants in these models, as well as six treatments that possess fast-onset antidepressant effects in the clinic: electroconvulsive shock therapy, sleep deprivation, ketamine, scopolamine, GLYX-13 and pindolol used in conjunction with classical antidepressants. We also discuss the effects of several compounds that have yet to be tested in humans but have fast-onset antidepressant-like effects in one or more of these antidepressant onset sensitive models. These compounds include selective serotonin (5-HT)2C receptor antagonists, a 5-HT4 receptor agonist, a 5-HT7 receptor antagonist, NMDA receptor antagonists, a TREK-1 receptor antagonist, mGluR antagonists and (2R,6R)-HNK. Finally, we provide recommendations for identifying fast-onset antidepressants using rodent behavioral models and molecular approaches.
Novel function of TREK-1 in regulating adipocyte differentiation and lipid accumulation.
K2P (two-pore domain potassium) channels, a diversified class of K+-selective ion channels, have been found to affect a wide range of physiological processes in the body. Despite their established significance in regulating proliferation and differentiation in multiple cell types, K2P channels' specific role in adipogenic differentiation (adipogenesis) remains poorly understood. In this study, we investigated the engagement of K2P channels, specifically KCNK2 (also known as TREK-1), in adipogenesis using primary cultured adipocytes and TREK-1 knockout (KO) mice. Our findings showed that TREK-1 expression in adipocytes decreases substantially during adipogenesis. This typically causes an increased Ca2+ influx and alters the electrical potential of the cell membrane in 3T3-L1 cell lines. Furthermore, we observed an increase in differentiation and lipid accumulation in both 3T3-L1 cell lines and primary cultured adipocytes when the TREK-1 activity was blocked with Spadin, the specific inhibitors, and TREK-1 shRNA. Finally, our findings revealed that mice lacking TREK-1 gained more fat mass and had worse glucose tolerance when fed a high-fat diet (HFD) compared to the wild-type controls. The findings demonstrate that increase of the membrane potential at adipocytes through the downregulation of TREK-1 can influence the progression of adipogenesis.
TREK-1 channel blockade mediates the antidepressant-like effects of hydroxynorketamine.
The ketamine metabolite (2R,6R)-hydroxynorketamine [(2R,6R)-HNK] exhibits rapid antidepressant effects without the psychotomimetic or addictive side effects associated with ketamine, making it a promising therapeutic candidate. However, the precise molecular targets and mechanisms underlying its antidepressant actions remain controversial. In this study, we identified TREK-1, a two-pore domain potassium channel, as a key target of (2R,6R)-HNK. Electrophysiological experiments revealed that (2R,6R)-HNK selectively inhibits TREK-1 currents, with an IC50 close to its effective antidepressant concentration. TREK-1 is widely expressed in the brain, and its activity in the mPFC has been implicated in regulating depressive-like behaviors. Early-life stress increases TREK-1 expression in the mPFC, impairing synaptic plasticity and neural circuit formation, which contributes to emotional disorders. Further investigations demonstrated that (2R,6R)-HNK enhances the firing frequency of mPFC pyramidal neurons and upregulates synaptic plasticity-related proteins, including CREB and PSD95. Crucially, the antidepressant effects of (2R,6R)-HNK were abolished in mice with pyramidal neuron-specific knockdown of TREK-1 in the mPFC, confirming the essential role of TREK-1 inhibition in mediating (2R,6R)-HNK's actions. Our findings establish TREK-1 as a critical target for (2R,6R)-HNK's rapid antidepressant effects, providing new insights into the mechanisms of depression treatment and the development of novel therapeutics.
Shortened Spadin Analogs Display Better TREK-1 Inhibition, In Vivo Stability and Antidepressant Activity.
Depression is a devastating mental disorder that affects 20% of the population worldwide. Despite their proven efficacy, antidepressants present a delayed onset of action and serious adverse effects. Seven years ago, we described spadin (PE 12-28) as a promising endogenous peptide with antidepressant activity. Spadin specifically blocks the TREK-1 channel. Previously, we showed in vivo that, spadin activity disappeared beyond 7 h after administration. In order to improve in vivo spadin stability and bioavailability, we screened spadin analogs and derivatives. From the study of spadin blood degradation products, we designed a 7 amino-acid peptide, PE 22-28. In vitro studies on hTREK-1/HEK cells by using patch-clamp technique, showed that PE 22-28 displayed a better specificity and affinity for TREK-1 channel compared to spadin, IC50 of 0.12 nM vs. 40-60 nM for spadin. In the same conditions, we also pointed out that different modifications of its N or C-terminal ends maintained or abolished TREK-1 channel activity without affecting PE 22-28 affinity. In vivo, the antidepressant properties of PE 22-28 and its derivatives were demonstrated in behavioral models of depression, such as the forced swimming test. Mice treated with spadin-analogs showed a significant reduction of the immobility time. Moreover, in the novelty suppressed feeding test after a 4-day sub-chronic treatment PE 22-28 reduced significantly the latency to eat the food pellet. PE 22-28 and its analogs were able to induce neurogenesis after only a 4-day treatment with a prominent effect of the G/A-PE 22-28. On mouse cortical neurons, PE 22-28 and its derivatives enhanced synaptogenesis measured by the increase of PSD-95 expression level. Finally, the action duration of PE 22-28 and its analogs was largely improved in comparison with that of spadin, up to 23 h instead of 7 h. Taken together, our results demonstrated that PE 22-28 and its derivatives represent other promising molecules that could be an alternative to spadin in the treatment of depression.
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.
Production of K2P2.1 (TREK-1) for structural studies.
K2P (KCNK) potassium channels form 'background' or 'leak' currents that are important for controlling cell excitability in the brain, cardiovascular system, and somatosensory neurons. K2P2.1 (TREK-1) is one of the founding members of this family and one of the first well-characterized polymodal ion channels capable of responding to a variety of physical and chemical gating cues. Of the six K2P subfamilies, the thermo-and mechano-sensitive TREK subfamily comprising K2P2.1 (TREK-1), K2P4.1 (TRAAK), and K2P10.1 (TREK-2) is the first to have structures determined for each subfamily member. These structural studies have revealed key architectural features that provide a framework for understanding how gating cues sensed by different channel elements converge on the K2P selectivity filter C-type gate. TREK family structural studies have also revealed numerous sites where small molecules or lipids bind and affect channel function. This rich structural landscape provides the framework for probing K2P function and for the development of new K2P-directed agents. Such molecules may be useful for affecting processes where TREK channels are important such as anesthesia, pain, arrythmia, ischemia, migraine, intraocular pressure, and lung injury. Production of high quality protein samples is key to addressing new questions about K2P function and pharmacology. Here, we present methods for producing pure K2P2.1 (TREK-1) suitable for advancing towards these goals through structural and biochemical studies.
Spadin as a new antidepressant: absence of TREK-1-related side effects.
Despite several decades of research, current antidepressant (AD) treatments remain of a limited efficacy justifying the need to find new drugs. These drugs have to be more efficacious, more rapid and display lesser side effects. Using rodent models, we recently identified spadin as a new antidepressant molecule that acts more quickly than classical ADs, working within 4 days to get same effects obtained with other ADs after 21 days. Spadin blocks TREK-1 K(2P) potassium channels that are considered as new targets for ADs. Deletion of the TREK-1 channel is known to increase sensitivity to pain, seizures and ischemia. Thus blocking these channels could result in deleterious side effects. In this study we showed that spadin did not interfere with other TREK-1 controlled functions such as pain, epilepsy and ischemia. We also demonstrated that spadin was unable to inhibit currents generated by TREK-2, TRAAK, TASK and TRESK four other K2P channels. More importantly, spadin did not induce cardiac dysfunctions, did not block I(Kr) and I(Ks) and did not modify the systolic pressure or cardiac pulses. After a three week treatment spadin remained an efficacious AD and did not modify the infarct size in brain following focal ischemia. Finally, we showed that kainate induced seizures and glycemia were not modified by spadin treatments. These data, together with those previously published reinforce the idea that spadin represents a good candidate for a new generation of ADs. This article is part of a Special Issue entitled 'Anxiety and Depression'.
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.
The Involvement of Sortilin/NTSR3 in Depression as the Progenitor of Spadin and Its Role in the Membrane Expression of TREK-1.
The molecular identification of sortilin, also called neurotensin receptor-3, from three different biochemical approaches already predicted the involvement of the protein in numerous biological and cellular functions. The first important observation was that sortilin is synthesized as a precursor that is converted to a mature protein after cleavage by the protein convertase furin in late Golgi compartments. This maturation leads to the formation of a 44 amino acid peptide, the propeptide (PE). The release of this peptide when matured sortilin reached the plasma membrane remained to be demonstrated. Sortilin has been also shown to be shedded by matrix metalloproteases releasing a large extracellular fragment identified as soluble sortilin. Therefore, sortilin has been shown to interact with several proteins and receptors confirming its role in the sorting of cellular components to the plasma membrane and/or to the lysosomal pathway. Interestingly, sortilin physically interacts with the two pore domain potassium channel TREK-1 and the PE as well as its synthetic analog spadin is able to block the activation of TREK-1 highlighting their role in the depression pathology. The present review describes the advance of research that led to these results and how both the soluble form of sortilin and the sortilin-derived PE have been detected in human serum and whose levels are affected in patients with major depressive disorder (MDD). The use of spadin as an antidepressant and the further role of soluble sortilin and of sortilin-derived PE as potential biomarkers during depression statement and/or remission of the pathology are considered and discussed in this review.
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.
Tandem pore TWIK-related potassium channels and neuroprotection.
TWIK-related potassium channels (TREK) belong to a subfamily of the two-pore domain potassium channels family with three members, TREK1, TREK2 and TWIK-related arachidonic acid-activated potassium channels. The two-pore domain potassium channels is the last big family of channels being discovered, therefore it is not surprising that most of the information we know about TREK channels predominantly comes from the study of heterologously expressed channels. Notwithstanding, in this review we pay special attention to the limited amount of information available on native TREK-like channels and real neurons in relation to neuroprotection. Mainly we focus on the role of free fatty acids, lysophospholipids and other neuroprotective agents like riluzole in the modulation of TREK channels, emphasizing on how important this modulation may be for the development of new therapies against neuropathic pain, depression, schizophrenia, epilepsy, ischemia and cardiac complications.
Retroinverso analogs of spadin display increased antidepressant effects.
Although depression is the most common mood disorder, only one third of patients are treated with success. Finding new targets, new drugs, and also new drug intake way are the main challenges in the depression field. Several years ago, we identified a new target with the TWIK-related potassium channel-1 (TREK-1) potassium channel, and more recently, we have discovered a peptide of 17 amino acids with antidepressant properties. This peptide, that we called spadin, can be considered as a new concept in antidepressant drug design. Spadin derives from a larger peptide resulting to a posttranslational maturation of sortilin; consequently, spadin can be considered as a natural molecule. Moreover, spadin acts more rapidly than classical antidepressants and does not induce side effects. In this work, we sought analogs of spadin displaying a better affinity on TREK-1 channels and an increased action duration. Analogs were characterized by electrophysiology measurements, by behavioral tests, and by their ability to induce neurogenesis. We identified two retro-inverso peptides that have kept the antidepressant properties of spadin; particularly, they increased the hippocampal neurogenesis after a 4-day treatment. As spadin, these analogs did not induce side effects on either pain, epilepsy processes, or at the cardiac level. Together, our results indicated that spadin retro-inverso peptides could represent new potent antidepressant drugs. As exemplified by spadin in the field of depression, retro-inverso strategies could represent a useful technique for developing new classes of drugs in a number of pathologies.
Sortilin-derived peptides promote pancreatic beta-cell survival through CREB signaling pathway.
Deterioration of insulin secretion and pancreatic beta-cell mass by inflammatory attacks is one of the main pathophysiological features of type 2 diabetes (T2D). Therefore, preserving beta-cell mass and stimulating insulin secretion only in response to glucose for avoiding the hypoglycemia risks, are the most state-of-the-art option for the treatment of T2D. In this study we tested two correlated hypothesis that 1/ the endogenous peptide released from sortilin, known as PE, that stimulates insulin secretion only in response to glucose, protects beta-cells against death induced by cytokines, and 2/ Spadin and Mini-Spadin, two synthetic peptides derived from PE, that mimic the effects of PE in insulin secretion, also provide beneficial effect on beta-cells survival. We show that PE and its derivatives by inducing a rise of intracellular calcium concentration by depolarizing the membrane protect beta-cells against death induced by Interleukin-1β. Using biochemical, confocal imaging and cell biology techniques, we reveal that the protective effects of PE and its derivatives rely on the activation of the CaM-Kinase pathway, and on the phosphorylation and activation of the transcription factor CREB. In addition, Mini-Spadin promotes beta-cell proliferation, suggesting its possible regenerative effect. This study highlights new possible roles of PE in pancreatic beta-cell survival and its derivatives as pharmacological tools against diabetes.
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.
Genetic and pharmacological inhibition of two-pore domain potassium channel TREK-1 alters depression-related behaviors and neuronal plasticity in the hippocampus in mice.
The two-pore domain potassium channel TREK-1 is a member of background K+ channels that are thought to provide baseline regulation of membrane excitability. Recent studies have highlighted the putative role of TREK-1 in the action of antidepressants, and its antagonists might be potentially effective antidepressants. However, the mechanisms underlying the actions of TREK-1 are not yet fully understood. The expression of TREK-1 was examined in a mouse model of chronic unpredictable mild stress (CUMS) using immunoblotting. Neuron-specific genetic manipulation of TREK-1 was performed through adeno-associated virus. Behavioral tests were performed to evaluate depression-related behaviors. Electrophysiological recordings were used to evaluate synaptic plasticity. Golgi staining was used to examine neuroplasticity. TREK-1 expression was increased in the mouse hippocampus after CUMS. Knockdown of TREK-1 in hippocampal neurons significantly attenuated depressive-like behaviors and prevented the decrease of CUMS-induced synaptic proteins in mice. Further examination indicated that neuron-specific knockdown of TREK-1 in the hippocampus prevented stress-induced impairment of glutamatergic synaptic transmission in the CA1 region. Moreover, chronic TREK-1 inhibition protected against CUMS-induced depressive-like behaviors and impairment of synaptogenesis in the hippocampus. Our results indicate a role for TREK-1 in the modulation of synaptic plasticity in a mouse model of depression. These findings will provide insight into the pathological mechanism of depression and further evidence for a novel target for antidepressant treatment.
The peptidic antidepressant spadin interacts with prefrontal 5-HT(4) and mGluR(2) receptors in the control of serotonergic function.
This study investigates the mechanism of action of spadin, a putative fast-acting peptidic antidepressant (AD) and a functional blocker of the K(+) TREK-1 channel, in relation with the medial prefrontal cortex (mPFC)-dorsal raphé (DRN) serotonergic (5-HT) neurons connectivity. Spadin increased 5-HT neuron firing rate by 113%, an augmentation abolished after electrolytic lesion of the mPFC. Among the few receptor subtypes known to modulate TREK-1, the stimulation of 5-HT4 receptors and the blockade of mGluR2/3 ones both activated 5-HT impulse flow, effects also suppressed by mPFC lesion. The combination of spadin with the 5-HT4 agonist RS 67333 paradoxically reduced 5-HT firing, an effect reversed by acutely administering the 5-HT1A agonist flesinoxan. It also had a robust synergetic effect on the expression of Zif268 within the DRN. Together, these results strongly suggest that 5-HT neurons underwent a state of depolarization block, and that the mechanisms underlying the influences exerted by spadin and RS 67333 are additive and independent from each other. In contrast, the mGluR2/3 antagonist LY 341495 occluded the effect of spadin, showing that it likely depends on mPFC TREK-1 channels coupled to mGluR2/3 receptors. These in vivo electrophysiological data were confirmed by in vitro Ca(2+) cell imaging performed in cultured cortical neurons. Altogether, our results indicate that spadin, as a natural compound, constitutes a very good candidate to explore the "glutamatergic path" of fast-acting AD research. In addition, they provide the first evidence of 5-HT depolarization block, showing that the combination of 5-HT activators for strategies of AD augmentation should be performed with extreme caution.
Esketamine reduces postoperative depression in breast cancer through TREK-1 channel inhibition and neurotransmitter modulation.
Postoperative depression significantly affects the quality of life of breast cancer patients. This study explores the potential therapeutic effects of esketamine on postoperative depression through modulation of the TREK-1 two-pore domain potassium channel. We analyzed data from 54 female breast cancer patients who underwent surgery at our hospital between 2019 and 2023, dividing them into experimental and control groups based on esketamine treatment. Transcriptomic sequencing of hippocampal neurons from rats identified potassium ion-related pathways and key regulatory genes, including TREK-1, influenced by esketamine. In vitro studies showed that esketamine primarily alleviates depressive symptoms by inhibiting TREK-1 protein expression, enhancing GABA neurotransmitter release, and improving neuronal activity, while overexpression of TREK-1 reversed these effects. Esketamine's inhibition of TREK-1 channels and promotion of hippocampal neuron activity effectively alleviate postoperative depression in breast cancer patients, suggesting a novel therapeutic strategy.
Two-pore domain potassium channel TREK-1 contributes to arachidonic acid-induced Ca2+ signaling in human fibroblast-like synovial cells.
Human fibroblast-like synovial cells (hFLSs) are essential in maintaining the structural integrity of the articular cartilage and promoting joint inflammation. These cells are highly responsive to various physical and chemical stimuli, many of which influence cellular processes through intracellular Ca2+ signaling and membrane ion channel activity. In this study, we investigated the role of the TREK-1 two-pore domain potassium (K2P) channel as a molecular sensor of arachidonic acid (AA) in FLSs. Patch-clamp recordings revealed an outwardly rectifying K+ conductance resistant to conventional K+ channel blockers (4-AP and TEA) but sensitive to inhibition by quinidine, a broad-spectrum K2P blocker. Activation of the TREK-1 channel with 4-(2-Butyl-6,7-dichloro-2-cyclopentyl-indan-1-on-5-yl) oxobutyric acid (DCPIB) and ML402 increased this current, and immunocytochemical staining demonstrated TREK-1 expression in hFLSs. AA exposure potentiated the K+ current in a concentration-dependent manner and caused hyperpolarization of the resting membrane potential, effects fully antagonized by pretreatment of the cells with spadin, a TREK-1 selective blocker. Fluorescent Ca2+ measurements showed that AA-induced variable increase in the intracellular Ca2+ concentration ([Ca2+]i) in different FLSs, and spadin attenuated these responses, reducing the number of cells exhibiting oscillatory and sustained [Ca2+]i elevations. In a nominally Ca2+-free medium, spadin had no effect, suggesting that TREK-1 channels regulate plasma membrane Ca2+ influx. Our findings provide the first electrophysiological and pharmacological evidence for the involvement of TREK-1 channels in AA-induced Ca2+ signaling in hFLSs.
Migraine is a dysfunction of neuronal potassium ion channels.
Migraine is a primary headache disorder characterized by unilateral pain usually with aura, that affects approximately one in six individuals in India. The underlying biomechanical processes of migraine are still poorly understood, and new research is constantly being published. One of the major factors in migraine pathogenesis is the dysfunction of ion channels in the trigeminal nuclei and sensory cortices. Potassium channels are modulators and regulators of neuronal signaling and conductance, playing an important role in maintenance of the membrane potential and neuronal conduction. Therefore, potassium channel dysfunctions are potential factors in migraine pathogenesis, and thus targets for specific antimigraine prophylaxis. This review reveals that potassium channels play a significant role in pathogenesis and management of migraine. Dysfunctions in KATP channels, K2P channels including TRESK and TREK-1, small and large conductance calcium-sensitive potassium channels (SKCa and BKCa), and voltage-gated potassium channels (KV) are known to affect the incidence and progression of migraine in the general populace. KATP openers can induce migraine like phenotype, but KATP blockers have so far not been effective in reducing the intensity of migraine headache. Potassium channels are a potential druggable target for migraine prophylaxis with several compounds currently in preclinical trials.
Spadin, a sortilin-derived peptide, targeting rodent TREK-1 channels: a new concept in the antidepressant drug design.
Current antidepressant treatments are inadequate for many individuals, and when they are effective, they require several weeks of administration before a therapeutic effect can be observed. Improving the treatment of depression is challenging. Recently, the two-pore domain potassium channel TREK-1 has been identified as a new target in depression, and its antagonists might become effective antidepressants. In mice, deletion of the TREK-1 gene results in a depression-resistant phenotype that mimics antidepressant treatments. Here, we validate in mice the antidepressant effects of spadin, a secreted peptide derived from the propeptide generated by the maturation of the neurotensin receptor 3 (NTSR3/Sortilin) and acting through TREK-1 inhibition. NTSR3/Sortilin interacted with the TREK-1 channel, as shown by immunoprecipitation of TREK-1 and NTSR3/Sortilin from COS-7 cells and cortical neurons co-expressing both proteins. TREK-1 and NTSR3/Sortilin were colocalized in mouse cortical neurons. Spadin bound specifically to TREK-1 with an affinity of 10 nM. Electrophysiological studies showed that spadin efficiently blocked the TREK-1 activity in COS-7 cells, cultured hippocampal pyramidal neurons, and CA3 hippocampal neurons in brain slices. Spadin also induced in vivo an increase of the 5-HT neuron firing rate in the Dorsal Raphe Nucleus. In five behavioral tests predicting an antidepressant response, spadin-treated mice showed a resistance to depression as found in TREK-1 deficient mice. More importantly, an intravenous 4-d treatment with spadin not only induced a strong antidepressant effect but also enhanced hippocampal phosphorylation of CREB protein and neurogenesis, considered to be key markers of antidepressant action after chronic treatment with selective serotonin reuptake inhibitors. This work also shows the development of a reliable method for dosing the propeptide in serum of mice by using AlphaScreen technology. These findings point out spadin as a putative antidepressant of new generation with a rapid onset of action. Spadin can be regarded as the first natural antidepressant peptide identified. It corresponds to a new concept to address the treatment of depression.
In vitro and in vivo regulation of synaptogenesis by the novel antidepressant spadin.
We have described a novel antidepressant peptide, spadin, that acts by blocking the TWIK-related-potassium channel, type 1 (TREK-1). Here, we examined possible mechanisms of action of spadin at both molecular and cellular levels. Effects of spadin were measured in primary cultures of neurons or tissues from mice injected i.v. with spadin. Western blots, qPCR, histochemical and electrophysiological techniques were used. In vitro, spadin increased neuronal membrane potential and activated both the MAPK and PI3K signalling pathways, in a time- and concentration-dependent manner. The latter pathway was involved in the protective effect of spadin against staurosporine-induced apoptosis. Also, spadin enhanced both mRNA expression and protein of two markers of synaptogenesis, the post-synaptic density protein of 95 kDalton (PSD-95) and synapsin. We confirmed these effects on synaptogenesis by the observation that spadin treatment significantly increased the proportion of mature spines in cortical neurons. Finally, in vivo injections of spadin led to a rapid increase in both mRNA expression and protein level of brain-derived neurotrophic factor (BDNF) in the hippocampus, confirming the antidepressant action of the peptide. We argue for a new role of spadin in synaptogenesis as both PSD-95 and synapsin mRNA expression and protein levels were further enhanced in the hippocampus, following treatment in vivo with the peptide. These findings provide new mechanisms of action for the rapidly acting antidepressant peptide spadin by stimulating expression of BDNF and synaptic proteins, both in vitro and in vivo.
Potentiation of Calcium Influx and Insulin Secretion in Pancreatic Beta Cell by the Specific TREK-1 Blocker Spadin.
Inhibition of the potassium channels TREK-1 by spadin (SPA) is currently thought to be a promising therapeutic target for the treatment of depression. Since these channels are expressed in pancreatic β-cells, we investigated their role in the control of insulin secretion and glucose homeostasis. In this study, we confirmed the expression of TREK-1 channels in the insulin secreting MIN6-B1 β-cell line and in mouse islets. We found that their blockade by SPA potentiated insulin secretion induced by potassium chloride dependent membrane depolarization. Inhibition of TREK-1 by SPA induced a decrease of the resting membrane potential (ΔVm ~ 12 mV) and increased the cytosolic calcium concentration. In mice, administration of SPA enhanced the plasma insulin level stimulated by glucose, confirming its secretagogue effect observed in vitro. Taken together, this work identifies SPA as a novel potential pharmacological agent able to control insulin secretion and glucose homeostasis.
Fluoxetine Protection in Decompression Sickness in Mice is Enhanced by Blocking TREK-1 Potassium Channel with the "spadin" Antidepressant.
In mice, disseminated coagulation, inflammation, and ischemia induce neurological damage that can lead to death. These symptoms result from circulating bubbles generated by a pathogenic decompression. Acute fluoxetine treatment or the presence of the TREK-1 potassium channel increases the survival rate when mice are subjected to an experimental dive/decompression protocol. This is a paradox because fluoxetine is a blocker of TREK-1 channels. First, we studied the effects of an acute dose of fluoxetine (50 mg/kg) in wild-type (WT) and TREK-1 deficient mice (knockout homozygous KO and heterozygous HET). Then, we combined the same fluoxetine treatment with a 5-day treatment protocol with spadin, in order to specifically block TREK-1 activity (KO-like mice). KO and KO-like mice were regarded as antidepressed models. In total, 167 mice (45 WTcont 46 WTflux 30 HETflux and 46 KOflux) constituting the flux-pool and 113 supplementary mice (27 KO-like 24 WTflux2 24 KO-likeflux 21 WTcont2 17 WTno dive) constituting the spad-pool were included in this study. Only 7% of KO-TREK-1 treated with fluoxetine (KOflux) and 4% of mice treated with both spadin and fluoxetine (KO-likeflux) died from decompression sickness (DCS) symptoms. These values are much lower than those of WT control (62%) or KO-like mice (41%). After the decompression protocol, mice showed significant consumption of their circulating platelets and leukocytes. Spadin antidepressed mice were more likely to exhibit DCS. Nevertheless, mice which had both blocked TREK-1 channels and fluoxetine treatment were better protected against DCS. We conclude that the protective effect of such an acute dose of fluoxetine is enhanced when TREK-1 is inhibited. We confirmed that antidepressed models may have worse DCS outcomes, but concomitant fluoxetine treatment not only decreased DCS severity but increased the survival rate.
Molecular regulations governing TREK and TRAAK channel functions.
K+ channels with two-pore domain (K2p) form a large family of hyperpolarizing channels. They produce background currents that oppose membrane depolarization and cell excitability. They are involved in cellular mechanisms of apoptosis, vasodilatation, anesthesia, pain, neuroprotection and depression. This review focuses on TREK-1, TREK-2 and TRAAK channels subfamily and on the mechanisms that contribute to their molecular heterogeneity and functional regulations. Their molecular diversity is determined not only by the number of genes but also by alternative splicing and alternative initiation of translation. These channels are sensitive to a wide array of biophysical parameters that affect their activity such as unsaturated fatty acids, intra- and extracellular pH, membrane stretch, temperature, and intracellular signaling pathways. They interact with partner proteins that influence their activity and their plasma membrane expression. Molecular heterogeneity, regulatory mechanisms and protein partners are all expected to contribute to cell specific functions of TREK currents in many tissues.
Improved AAV vector system for cell-type-specific RNA interference.
RNA interference (RNAi) is a powerful technique to effectively silence or knock down gene function in mammalian cells. For better cell-type RNAi experiments in vivo, AAV vector-based RNA interference systems need to be improved. New method: In this study, we developed an AAV vector (CREon shRNA) that expressed CRE-dependent short hairpin RNA (shRNA) and fluorescent proteins simultaneously. We verified the Cre-dependent knockdown efficiency of the newly developed CREon shRNA vector in both HEK293T cells overexpressing TREK-1 and PC3 cells with endogenous TREK-1. Next, we packaged this TREK-1 CREon vector with AAV and injected it into the hippocampus of the brain together with a synapsin or GFAP promoter-driven CRE virus, confirming that it works well cell-selectively even in vivo. Finally, this viral vector was applied to an animal model of LPS-induced depression to determine whether behavioral changes occurred. Comparison with existing methods: With the existing pSico or pAAV-Sico-Red vectors, expression of fluorescent protein disappears when shRNA is conditionally activated by CRE recombinase, but our Creon shRNA vector showed simultaneous expression of both shRNA and fluorescent protein. Thus, it offers the advantage of allowing easy visual distinction of knocked-down cells. The newly improved CREon shRNA vector can be used as a novel research tool for conditional shRNA, and may be useful for various in vivo studies such as cancer and neurobiology.
Sortilin derived propeptide regulation during adipocyte differentiation and inflammation.
In this work, we aimed to correlate the expression of sortilin with the production of sortilin-derived propeptide (PE) during adipocyte differentiation, insulin resistance and inflammation. We also investigated the effect of spadin, a shorter analogue of PE that exerts a potent antidepressant in mice, on adipocyte functions. During adipogenesis, insulin resistance and inflammation, we measured the mRNA and protein expression of sortilin, by quantitative PCR and Western-blot, and quantified the expression of PE by a specific dosing method. We observed that the production of PE was correlated with the sortilin expression during adipogenesis. Immunostaining experiments allowed to visualize the co-localization of sortilin, PE and VAMP2 in 3T3-L1 adipocytes. TNFα treatment induced insulin resistance and a decrease of sortilin expression (mRNA and protein), correlated with the decrease of the PE production. By contrast, treatment with dexamethasone, which also induced insulin resistance, was without effect on sortilin expression and PE production. As a putative bioactive peptide, we have evaluated its autocrine effect by the use of spadin on 3T3-L1 adipocytes by performing glucose uptake and signalling experiments. Any effect was measured on adipocytes indicating that the use of spadin as an antidepressant would have no side effects on adipocyte physiology.
Corticosteroids elevate intraocular pressure through suppression of TREK-1 signaling.
Clinicians are often forced into the dilemma of whether to battle ocular inflammation or preserve vision imperiled by elevated intraocular pressure (IOP). Anti-inflammatory treatments utilizing glucocorticosteroid regimens may induce glaucoma by chronically elevating IOP via increased trabecular meshwork (TM) resistance to the flow of aqueous humor, but it is not known whether pressure transduction itself is impacted by steroids and how changes in TM mechanosignaling affect conventional outflow resistance and IOP. To address this, we investigated the role of TREK-1 (TWIK-related potassium channel-1), a mechanosensitive K + channel, in regulation of outflow facility, transmembrane signaling and dexamethasone (DEX)-induced ocular hypertension (OHT). The expression of tandem-pore potassium channels in mouse TM cells was dominated by Trek-1 (Kcnk2 ) mRNA, with residual expression of Traak, Tresk2 and Twik3 and vanishingly low levels of Task1 and Trek2 . DEX suppressed Trek1 transcription by ∼80% but did not affect expression of Trpv4 and Piezo1 genes. Chronic DEX administration depolarized the membrane potential of TM cells and elevated IOP in mice whereas the selective TREK-1 agonist ML-402 lowered IOP in rodent OHT models. ML-402 doubled the outflow facility in perfused mouse eyes at all applied pressures and hyperpolarized DEX-treated TM cells. These in vitro, ex vivo and in vivo results implicate TREK-1 channels in homeostatic regulation of TM mechanosignaling, conventional outflow regulation and IOP homeostasis. Suppression of TREK-1 signaling by corticosteroids underlies OHT and could contribute to steroid glaucoma but this can be obviated by pharmacological stimulation of the channel with cornea-permeant ML-402 eye drops.
TREK-1 potassium channels participate in acute and long-lasting nociceptive hypersensitivity induced by formalin in rats.
TREK-1 channels are expressed in small nociceptive dorsal root ganglion (DRG) neurons where they participate in acute inflammatory and neuropathic pain. However, the role of TREK-1 in persistent pain is not well understood. The aim of this study was to investigate the local peripheral and spinal participation of TREK-1 in formalin-induced acute and long-lasting nociceptive hypersensitivity. Local peripheral or intrathecal pre-treatment with spadin, selective blocker of TREK-1, increased acute flinching behavior and secondary mechanical allodynia and hyperalgesia behavior observed 6 days after formalin injection. Local peripheral or intrathecal pre-treatment with BL-1249, selective opener of TREK-1, decreased long-lasting secondary mechanical allodynia and hyperalgesia induced by formalin. Pre-treatment with BL-1249 prevented the pro-nociceptive effect of spadin on acute nociception and long-lasting mechanical allodynia and hyperalgesia in rats. Pre-treatment with two recombinant channels that produce a high TREK-1 current, S300A and S333A (non-phosphorylated state of TREK-1), reduced formalin-induced acute pain and long-lasting mechanical allodynia and hyperalgesia. Besides, post-treatment with S300A, S333A or BL-1249 reversed long-lasting mechanical allodynia and hyperalgesia induced by formalin. Formalin increased TREK-1 expression at 1 and 6 days in DRG and dorsal spinal cord in rats, whereas that it increased c-fos expression at the DRG. Intrathecal repeated transfection of rats with S300A and S333A or injection with BL-1249 reduced formalin-induced enhanced c-fos expression. Data suggest that TREK-1 activity at peripheral and spinal sites reduces neuronal excitability in the process of acute and long-lasting nociception induced by formalin in rats.
Bilayer-Mediated Structural Transitions Control Mechanosensitivity of the TREK-2 K2P Channel.
The mechanosensitive two-pore domain (K2P) K+ channels (TREK-1, TREK-2, and TRAAK) are important for mechanical and thermal nociception. However, the mechanisms underlying their gating by membrane stretch remain controversial. Here we use molecular dynamics simulations to examine their behavior in a lipid bilayer. We show that TREK-2 moves from the "down" to "up" conformation in direct response to membrane stretch, and examine the role of the transmembrane pressure profile in this process. Furthermore, we show how state-dependent interactions with lipids affect the movement of TREK-2, and how stretch influences both the inner pore and selectivity filter. Finally, we present functional studies that demonstrate why direct pore block by lipid tails does not represent the principal mechanism of mechanogating. Overall, this study provides a dynamic structural insight into K2P channel mechanosensitivity and illustrates how the structure of a eukaryotic mechanosensitive ion channel responds to changes in forces within the bilayer.
Role for TREK-1 as a polymodal sensor and regulator of cell activity.
TREK-1 (KCNK2) is a polymodal two-pore domain potassium (K2P) channel that functions as a background K+ conductance and integrator of mechanical, chemical, and thermal stimuli across diverse cell types. Its unique structure enables sensitivity to membrane stretch, lipid composition, pH, temperature, pharmacologic agents, and intracellular signaling pathways. Beyond shaping resting membrane potential and excitability, TREK-1 engages in noncanonical signaling roles involving protein-protein interactions, trafficking, and modulation of intracellular signaling cascades such as MAPK and calcineurin pathways. TREK-1 is widely expressed in the nervous system, where it regulates neuronal firing, pain sensitivity, mood, and neuroprotection. In the heart, TREK-1 influences action potential duration, mechano-electric feedback, sinoatrial node function, and stress-induced remodeling, with mutations linked to arrhythmogenesis. In fibroblasts and fibroblast-like cells, TREK-1 acts as a mechanotransducer driving differentiation and fibrosis through MAPK signaling. TREK-1 also modulates immune activation, inflammasome signaling, adipogenesis, epithelial injury responses, vascular tone, and cancer cell proliferation. Across tissues, dysregulation of TREK-1 contributes to pathological excitability, fibrosis, inflammation, and degeneration. Given its multimodal regulation and broad impact on cellular function, TREK-1 represents a compelling therapeutic target, though challenges remain due to limited subtype-selective pharmacology and incomplete understanding of its nonionic signaling roles.
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.
Targeting two-pore domain K(+) channels TREK-1 and TASK-3 for the treatment of depression: a new therapeutic concept.
Depression is a disease that is particularly frequent, affecting up to 20% of the population in Western countries. The origins of this pathology involve multiple genes as well as environmental and developmental factors leading to a disorder that remains difficult to treat. Several therapies for depression have been developed and these mainly target monoamine neurotransmitters. However, these treatments are not only associated with numerous adverse effects, but they are also ineffective for more than one-third of patients. Therefore, the need to develop new concepts to treat depression is crucial. Recently, studies using knockout mouse models have provided evidence for a crucial role of two members of the two-pore domain potassium channel (K2P ) family, tandem P-domain weak inward rectifying K(+) (TWIK)-related K(+) channel 1 (TREK-1) and TWIK-related acid-sensitive K(+) channel 3 (TASK-3) in the pathophysiology of depression. It is believed that TREK-1 and TASK-3 antagonists could lead to the development of new antidepressants. Herein, we describe the discovery of spadin, a natural peptide released from the maturation of the neurotensin receptor-3 (also known as sortilin), which specifically blocks the activity of the TREK-1 channel and displays particular antidepressant properties, with a rapid onset of action and the absence of adverse effects. The development of such molecules may open a new era in the field of psychiatry.
Activation of TREK-1, but Not TREK-2, Channel by Mood Stabilizers.
Earlier studies have demonstrated that the tandem pore domain weak inward rectifying K⁺ channel (TWIK)-related K⁺ (TREK)-1 channel is inhibited by antidepressants and is associated with major depression. However, little is known about the effect of mood stabilizers that are commonly used for treatment of bipolar disorder on TREK channels, members of the two-pore domain K⁺ (K2P) channel family. This study sought to investigate the effect of mood stabilizers on TREK-1 and TREK-2 channels. HEK-293A cells were transfected with human TREK-1 or TREK-2 DNA. The effect of mood stabilizers on TREK-1 and TREK-2 was studied using the patch clamp technique. Changes in TREK protein expression by mood stabilizers were studied in the HT-22 mouse hippocampal neuronal cells using western blot analysis. Lithium chloride (LiCl, 1 mM), gabapentin (100 μM), valproate (100 μM), and carbamazepine (100 μM) increased TREK-1 currents by 31 ± 14%, 25 ± 11%, 28 ± 12%, and 72 ± 12%, respectively, whereas they had no effect on TREK-2 channel activity. In addition, western blot analysis showed LiCl and carbamazepine slightly upregulated TREK-1 expression, but not TREK-2 in the HT-22 cells. These results suggest that TREK-1 could be a potential therapeutic target for treatment of bipolar disorders as well as depression, while TREK-2 is a target well suited for treatment of major depression.
Antidepressive and anxiolytic effects of ostruthin, a TREK-1 channel activator.
We screened a library of botanical compounds purified from plants of Vietnam for modulators of the activity of a two-pore domain K+ channel, TREK-1, and we identified a hydroxycoumarin-related compound, ostruthin, as an activator of this channel. Ostruthin increased whole-cell TREK-1 channel currents in 293T cells at a low concentration (EC50 = 5.3 μM), and also activity of the TREK-2 channel (EC50 = 3.7 mM). In contrast, ostruthin inhibited other K+ channels, e.g. human ether-à-go-go-related gene (HERG1), inward-rectifier (Kir2.1), voltage-gated (Kv1.4), and two-pore domain (TASK-1) at higher concentrations, without affecting voltage-gated potassium channel (KCNQ1 and 3). We tested the effect of this compound on mouse anxiety- and depression-like behaviors and found anxiolytic activity in the open-field, elevated plus maze, and light/dark box tests. Of note, ostruthin also showed antidepressive effects in the forced swim and tail suspension tests, although previous studies reported that inhibition of TREK-1 channels resulted in an antidepressive effect. The anxiolytic and antidepressive effect was diminished by co-administration of a TREK-1 blocker, amlodipine, indicating the involvement of TREK-1 channels. Administration of ostruthin suppressed the stress-induced increase in anti-c-Fos immunoreactivity in the lateral septum, without affecting immunoreactivity in other mood disorder-related nuclei, e.g. the amygdala, paraventricular nuclei, and dorsal raphe nucleus. Ostruthin may exert its anxiolytic and antidepressive effects through a different mechanism from current drugs.
TREK1 channel activation as a new analgesic strategy devoid of opioid adverse effects.
Opioids are effective painkillers. However, their risk-benefit ratio is dampened by numerous adverse effects and opioid misuse has led to a public health crisis. Safer alternatives are required, but isolating the antinociceptive effect of opioids from their adverse effects is a pharmacological challenge because activation of the μ opioid receptor triggers both the antinociceptive and adverse effects of opioids. The TREK1 potassium channel is activated downstream of μ receptor and involved in the antinociceptive activity of morphine but not in its adverse effects. Bypassing the μ opioid receptor to directly activate TREK1 could therefore be a safer analgesic strategy. We developed a selective TREK1 activator, RNE28, with antinociceptive activity in naive rodents and in models of inflammatory and neuropathic pain. This activity was lost in TREK1 knockout mice or wild-type mice treated with the TREK1 blocker spadin, showing that TREK1 is required for the antinociceptive activity of RNE28. RNE28 did not induce respiratory depression, constipation, rewarding effects, or sedation at the analgesic doses tested. This proof-of-concept study shows that TREK1 activators could constitute a novel class of painkillers, inspired by the mechanism of action of opioids but devoid of their adverse effects.
Osmotically Sensitive TREK Channels in Rat Articular Chondrocytes: Expression and Functional Role.
Articular chondrocytes are the primary cells responsible for maintaining the integrity and functionality of articular cartilage, which is essential for smooth joint movement. A key aspect of their role involves mechanosensitive ion channels, which allow chondrocytes to detect and respond to mechanical forces encountered during joint activity; nonetheless, the variety of mechanosensitive ion channels involved in this process has not been fully resolved so far. Because some members of the two-pore domain potassium (K2P) channel family have been described as mechanosensors in other cell types, in this study, we investigate whether articular chondrocytes express such channels. RT-PCR analysis reveals the presence of TREK-1 and TREK-2 channels in these cells. Subsequent protein expression assessments, including Western blotting and immunohistochemistry, confirm the presence of TREK-1 in articular cartilage samples. Furthermore, whole-cell patch clamp assays demonstrate that freshly isolated chondrocytes exhibit currents attributable to TREK-1 channels, as evidenced by activation by arachidonic acid (AA) and ml335 and further inhibition by spadin. Additionally, exposure to hypo-osmolar shock activates currents, which can be attributed to the presence of TREK-1 channels, as indicated by their inhibition with spadin. Therefore, these findings highlight the expression of TREK channels in rat articular chondrocytes and suggest their potential involvement in regulating the integrity of cartilage extracellular matrix.
Quick links (PubMed)
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