PKC- and PKA-dependent phosphorylation modulates TREK-1 function in naïve and neuropathic rats.

PKC and PKA phosphorylation inhibit TREK-1 channels downstream of Gs protein-coupled receptor activation in vitro. However, the role of phosphorylation of TREK-1 in neuropathic pain is unknown. The purpose of this study was to investigate whether altered TREK-1 channel function by PKA and PKC modulators contributes to antiallodynia in neuropathic rats. Furthermore, we investigated if the in vitro described sites for PKC and PKA phosphorylation (S300 and S333, respectively) participate in the modulation of TREK-1 in naïve and neuropathic rats. L5/L6 spinal nerve ligation (SNL) induced tactile allodynia. Intrathecal injection of BL-1249 (TREK-1 activator) reversed nerve injury-induced tactile allodynia, whereas spadin (TREK-1 blocker) produced tactile allodynia in naïve rats and reversed the antiallodynic effect induced by BL-1249 in neuropathic rats. Intrathecal administration of rottlerin or Rp-cAMPs (PKC and PKA inhibitors, respectively) enhanced the antiallodynia observed with BL-1249 in neuropathic rats. In contrast, pretreatment with PdBu or forskolin (PKC and PKA activators, respectively) reduced the BL-1249-induced antiallodynia. Intrathecal injection of two high-activity TREK-1 recombinant channels, using a in vivo transfection method with lipofectamine, with mutations at PKC/PKA phosphosites (S300A and S333A) reversed tactile allodynia in neuropathic rats, with no effect in naïve rats. In contrast, transfection of two low-activity TREK-1 recombinant channels with phosphomimetic mutations at those sites (S300D and S333D) produced tactile allodynia in naïve rats and interfered with antiallodynic effects of rottlerin/BL-1249 or Rp-cAMPs/BL-1249. Data suggest that TREK-1 channel activity can be dynamically tuned in vivo by PKC/PKA to provoke allodynia and modulate its antiallodynic role in neuropathic pain.

A "Target Class" Screen to Identify Activators of Two-Pore Domain Potassium (K2P) Channels.

Two-pore domain potassium (K2P) channels carry background (or leak) potassium current and play a key role in regulating resting membrane potential and cellular excitability. Accumulating evidence points to a role for K2Ps in human pathophysiologies, most notably in pain and migraine, making them attractive targets for therapeutic intervention. However, there remains a lack of selective pharmacological tools. The aim of this work was to apply a "target class" approach to investigate the K2P superfamily and identify novel activators across all the described subclasses of K2P channels. Target class drug discovery allows for the leveraging of accumulated knowledge and maximizing synergies across a family of targets and serves as an additional approach to standard target-based screening. A common assay platform using baculovirus (BacMam) to transiently express K2P channels in mammalian cells and a thallium flux assay to determine channel activity was developed, allowing the simultaneous screening of multiple targets. Importantly, this system, by allowing precise titration of channel function, allows optimization to facilitate the identification of activators. A representative set of channels (THIK-1, TWIK-1, TREK-2, TASK-3, and TASK-2) were screened against a library of Food and Drug Administration (FDA)-approved compounds and the LifeArc Index Set. Activators were then analyzed in concentration-response format across all channels to assess selectivity. Using the target class approach to investigate the K2P channels has enabled us to determine which of the K2Ps are amenable to small-molecule activation, de-risk multiple channels from a technical point of view, and identify a diverse range of previously undescribed pharmacology.

Early Stimulation of TREK Channel Transcription and Activity Induced by Oxaliplatin-Dependent Cytosolic Acidification.

Oxaliplatin-induced peripheral neuropathy is characterized by an acute hyperexcitability syndrome triggered/exacerbated by cold. The mechanisms underlying oxaliplatin-induced peripheral neuropathy are unclear, but the alteration of ion channel expression and activity plays a well-recognized central role. Recently, we found that oxaliplatin leads to cytosolic acidification in dorsal root ganglion (DRG) neurons. Here, we investigated the early impact of oxaliplatin on the proton-sensitive TREK potassium channels. Following a 6-h oxaliplatin treatment, both channels underwent a transcription upregulation that returned to control levels after 42 h. The overexpression of TREK channels was also observed after in vivo treatment in DRG cells from mice exposed to acute treatment with oxaliplatin. Moreover, both intracellular pH and TREK channel transcription were similarly regulated after incubation with amiloride, an inhibitor of the Na+/H+ exchanger. In addition, we studied the role of oxaliplatin-induced acidification on channel behavior, and, as expected, we observed a robust positive modulation of TREK channel activity. Finally, we focused on the impact of this complex modulation on capsaicin-evoked neuronal activity finding a transient decrease in the average firing rate following 6 h of oxaliplatin treatment. In conclusion, the early activation of TREK genes may represent a mechanism of protection against the oxaliplatin-related perturbation of neuronal excitability.

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.

Spinal TASK-1 and TASK-3 modulate inflammatory and neuropathic pain.

This study assessed the participation of spinal TWIK-related acid-sensitive K+ channels 1 and 3 (TASK-1 and TASK-3) in inflammatory (formalin test) and neuropathic (spinal nerve ligation, SNL) pain in rats. Intrathecal pre-treatment (-10 min) with the TASK-1 blocker ML365 or TASK-3 blocker PK-THPP, but not vehicle, enhanced in a dose-dependent manner 1% formalin-induced acute and long-lasting secondary mechanical allodynia and mechanical hyperalgesia in rats. In contrast, intrathecal pre-treatment with terbinafine, an activator of TASK-3, reduced formalin-induced flinching and allodynia/hyperalgesia. Both blockers and terbinafine had similar effects on female and male rats. In addition, intrathecal injection of ML365 or PK-THPP blocked the terbinafine-induced antiallodynic effect in neuropathic rats, but they did not modify baseline withdrawal threshold in naïve or sham-operated rats. TASK-1 and TASK-3 mRNA and protein were expressed in L4 and L5 dorsal root ganglia (DRG) and dorsal and ventral spinal cord of naïve animals. Interestingly, formalin injection increased TASK-1 expression in ipsilateral L5 DRG, but not in the spinal cord. Moreover, formalin injection transiently enhanced TASK-3 expression in ipsilateral L5 DRG and dorsal spinal cord. In contrast, SNL down-regulated TASK-3 expression in the ipsilateral L4 and L5 DRG but not in dorsal or ventral spinal cord, while SNL did not modify TASK-1 expression at any tissue. The pharmacological and molecular results suggest that TASK-1 and TASK-3 have a relevant antinociceptive role in inflammatory and neuropathic pain.

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.

Arachidonic acid hyperpolarizes mesenchymal stromal cells from the human adipose tissue by stimulating TREK1 K+ channels.

The current knowledge of electrogenesis in mesenchymal stromal cells (MSCs) remains scarce. Earlier, we demonstrated that in MSCs from the human adipose tissue, transduction of certain agonists involved the phosphoinositide cascade. Its pivotal effector PLC generates DAG that can regulate ion channels directly or via its derivatives, including arachidonic acid (AA). Here we showed that AA strongly hyperpolarized MSCs by stimulating instantly activating, outwardly rectifying TEA-insensitive K+ channels. Among AA-regulated K+ channels, K2P channels from the TREK subfamily appeared to be an appropriate target. The expression of K2P channels in MSCs was verified by RT-PCR, which revealed TWIK-1, TREK-1, and TASK-5 transcripts. The TREK-1 inhibitor spadin antagonized the electrogenic action of AA, which was simulated by the channel activator BL 1249. This functional evidence suggested that TREK-1 channels mediated AA-dependent hyperpolarization of MSCs. Being mostly silent at rest, TREK-1 negligibly contributed to the "background" K+ current. The dramatic stimulation of TREK-1 channels by AA indicates their involvement in AA-dependent signaling in MSCs.

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.

GI-530159, a novel, selective, mechanosensitive two-pore-domain potassium (K2P ) channel opener, reduces rat dorsal root ganglion neuron excitability.

BACKGROUND AND PURPOSE: TREK two-pore-domain potassium (K2P ) channels play a critical role in regulating the excitability of somatosensory nociceptive neurons and are important mediators of pain perception. An understanding of the roles of TREK channels in pain perception and, indeed, in other pathophysiological conditions, has been severely hampered by the lack of potent and/or selective activators and inhibitors. In this study, we describe a new, selective opener of TREK channels, GI-530159. EXPERIMENTAL APPROACH: The effect of GI-530159 on TREK channels was demonstrated using 86 Rb efflux assays, whole-cell and single-channel patch-clamp recordings from recombinant TREK channels. The expression of K2P 2.1 (TREK1), K2P 10.1 (TREK2) and K2P 4.1 (TRAAK) channels was determined using transcriptome analysis from single dorsal root ganglion (DRG) cells. Current-clamp recordings from cultured rat DRG neurons were used to measure the effect of GI-530159 on neuronal excitability. KEY RESULTS: For recombinant human TREK1 channels, GI-530159 had similar low EC50 values in Rb efflux experiments and electrophysiological recordings. It activated TREK2 channels, but it had no detectable action on TRAAK channels nor any significant effect on other K channels tested. Current-clamp recordings from cultured rat DRG neurones showed that application of GI-530159 at 1 µM resulted in a significant reduction in firing frequency and a small hyperpolarization of resting membrane potential. CONCLUSIONS AND IMPLICATIONS: This study provides pharmacological evidence for the presence of mechanosensitive TREK K2P channels in sensory neurones and suggests that development of selective K2P channel openers like GI-530159 could aid in the development of novel analgesic agents. LINKED ARTICLES: This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.

Heterodimerization of two pore domain K+ channel TASK1 and TALK2 in living heterologous expression systems.

Two-pore-domain K+ (K2P) channels sense a wide variety of stimuli such as mechanical stress, inhalational anesthetics, and changes in extracellular pH or temperature. The K2P channel activity forms a background K+ current and, thereby, contributes to resting membrane potentials. Six subfamilies including fifteen subtypes of K2P channels have been identified. Each K2P channel molecule with two pores consists of a homodimer of each subtype. In addition, a few heterodimers mainly within the same subfamilies have been found recently. In the present study, the possibility of heterodimerization between TASK1 (TWIK-Related Acid-Sensitive K+ channel) and TALK2 (TWIK-Related Alkaline pH-Activated K+ channel) was examined. These channels belong to separate subfamilies and show extremely different channel properties. Surprisingly, single molecular imaging analyses in this study using a total internal reflection microscope suggested the heterodimerization of TASK1 and TALK2 in a pancreatic cell line, QGP-1. This heterodimer was also detected using a bimolecular fluorescence complementation assay in a HEK293 heterologous expression system. Fluorescence resonance energy transfer analyses showed that the affinity between TASK1 and TALK2 appeared to be close to those of homodimers. Whole-cell patch-clamp recordings revealed that TASK1 currents in HEK293 cells were significantly attenuated by co-expression of a dominant-negative form of TALK2 in comparison with that of wild-type TALK2. The sensitivities of TASK1-TALK2 tandem constructs to extracellular pH and halothane were characterized as a unique hybrid of TASK1 and TALK2. These results suggested that heterodimerization of TASK1 and TALK2 provides cells with the ability to make multiple responses to a variety of physiological and pharmacological stimuli.

Terbinafine is a novel and selective activator of the two-pore domain potassium channel TASK3.

Two-pore domain potassium channels (K2Ps) are characterized by their four transmembrane domain and two-pore topology. They carry background (or leak) potassium current in a variety of cell types. Despite a number of important roles there is currently a lack of pharmacological tools with which to further probe K2P function. We have developed a cell-based thallium flux assay, using baculovirus delivered TASK3 (TWIK-related acid-sensitive K+ channel 3, KCNK9, K2P9.1) with the aim of identifying novel, selective TASK3 activators. After screening a library of 1000 compounds, including drug-like and FDA approved molecules, we identified Terbinafine as an activator of TASK3. In a thallium flux assay a pEC50 of 6.2 ( ±0.12) was observed. When Terbinafine was screened against TASK2, TREK2, THIK1, TWIK1 and TRESK no activation was observed in thallium flux assays. Several analogues of Terbinafine were also purchased and structure activity relationships examined. To confirm Terbinafine's activation of TASK3 whole cell patch clamp electrophysiology was carried out and clear potentiation observed in both the wild type channel and the pathophysiological, Birk-Barel syndrome associated, G236R TASK3 mutant. No activity at TASK1 was observed in electrophysiology studies. In conclusion, we have identified the first selective activator of the two-pore domain potassium channel TASK3.

The Impact of Heterozygous KCNK3 Mutations Associated With Pulmonary Arterial Hypertension on Channel Function and Pharmacological Recovery.

BACKGROUND: Heterozygous loss of function mutations in the KCNK3 gene cause hereditary pulmonary arterial hypertension (PAH). KCNK3 encodes an acid-sensitive potassium channel, which contributes to the resting potential of human pulmonary artery smooth muscle cells. KCNK3 is widely expressed in the body, and dimerizes with other KCNK3 subunits, or the closely related, acid-sensitive KCNK9 channel. METHODS AND RESULTS: We engineered homomeric and heterodimeric mutant and nonmutant KCNK3 channels associated with PAH. Using whole-cell patch-clamp electrophysiology in human pulmonary artery smooth muscle and COS7 cell lines, we determined that homomeric and heterodimeric mutant channels in heterozygous KCNK3 conditions lead to mutation-specific severity of channel dysfunction. Both wildtype and mutant KCNK3 channels were activated by ONO-RS-082 (10 µmol/L), causing cell hyperpolarization. We observed robust gene expression of KCNK3 in healthy and familial PAH patient lungs, but no quantifiable expression of KCNK9, and demonstrated in functional studies that KCNK9 minimizes the impact of select KCNK3 mutations when the 2 channel subunits co-assemble. CONCLUSIONS: Heterozygous KCNK3 mutations in PAH lead to variable loss of channel function via distinct mechanisms. Homomeric and heterodimeric mutant KCNK3 channels represent novel therapeutic substrates in PAH. Pharmacological and pH-dependent activation of wildtype and mutant KCNK3 channels in pulmonary artery smooth muscle cells leads to membrane hyperpolarization. Co-assembly of KCNK3 with KCNK9 subunits may provide protection against KCNK3 loss of function in tissues where both KCNK9 and KCNK3 are expressed, contributing to the lung-specific phenotype observed clinically in patients with PAH because of KCNK3 mutations.

K2P2.1 (TREK-1)-activator complexes reveal a cryptic selectivity filter binding site.

Polymodal thermo- and mechanosensitive two-pore domain potassium (K2P) channels of the TREK subfamily generate 'leak' currents that regulate neuronal excitability, respond to lipids, temperature and mechanical stretch, and influence pain, temperature perception and anaesthetic responses. These dimeric voltage-gated ion channel (VGIC) superfamily members have a unique topology comprising two pore-forming regions per subunit. In contrast to other potassium channels, K2P channels use a selectivity filter 'C-type' gate as the principal gating site. Despite recent advances, poor pharmacological profiles of K2P channels limit mechanistic and biological studies. Here we describe a class of small-molecule TREK activators that directly stimulate the C-type gate by acting as molecular wedges that restrict interdomain interface movement behind the selectivity filter. Structures of K2P2.1 (also known as TREK-1) alone and with two selective K2P2.1 (TREK-1) and K2P10.1 (TREK-2) activators-an N-aryl-sulfonamide, ML335, and a thiophene-carboxamide, ML402-define a cryptic binding pocket unlike other ion channel small-molecule binding sites and, together with functional studies, identify a cation-π interaction that controls selectivity. Together, our data reveal a druggable K2P site that stabilizes the C-type gate 'leak mode' and provide direct evidence for K2P selectivity filter gating.

Development of the First Two-Pore Domain Potassium Channel TWIK-Related K+ Channel 1-Selective Agonist Possessing in Vivo Antinociceptive Activity.

The TWIK-related K+ channel, TREK-1, has recently emerged as an attractive therapeutic target for the development of a novel class of analgesic drugs, suggesting that activation of TREK-1 could result in pain inhibition. Here, we report the synthesis of a series of substituted acrylic acids (1-54) based on our previous work with caffeate esters. The analogues were evaluated for their ability to modulate TREK-1 channel by electrophysiology and for their in vivo antinociceptive activity (acetic acid-induced writhing and hot plate assays), leading to the identification of a series of novel molecules able to activate TREK-1 and displaying potent antinociceptive activity in vivo. Furyl analogue 36 is the most promising of the series.

Regulation of TWIK-related potassium channel-1 (Trek1) restitutes intestinal epithelial barrier function.

The disruption of epithelial barrier integrity is an important factor in the pathogenesis of various immune disorders. However, the restitution of the compromised barrier functions is difficult. This study investigates the regulation of TWIK-related potassium channel-1 (Trek1) in the restitution of intestinal epithelial barrier functions. The human colon epithelial cell line T84 was cultured in monolayers and used to observe epithelial barrier functions in vitro. An intestinal allergy mouse model was created. Cytokine levels were determined by enzyme-linked immunosorbent assay and western blotting. The results showed that Trek1 deficiency induced T84 monolayer barrier disruption. Allergic responses markedly suppressed the expression of Trek1 in the intestinal epithelia via activating the mitogen-activated protein kinase pathways and increasing the expression of histone deacetylase-1. The inhibition of histone deacetylase-1 by sodium butyrate or the administration of a butyrate-producing probiotic (Clostridium butyricum) restored the intestinal epithelial barrier functions and markedly enhanced the effect of antigen-specific immunotherapy. The data suggest that Trek1 is required for the maintenance of intestinal epithelial barrier integrity. Allergic responses induce an insufficiency of Trek1 expression in the intestinal epithelia. Trek1 expression facilitates the restoration of intestinal epithelial barrier functions in an allergic environment.

Perspectives on the Two-Pore Domain Potassium Channel TREK-1 (TWIK-Related K(+) Channel 1). A Novel Therapeutic Target?

Potassium (K(+)) channels are membrane proteins expressed in most living cells that selectively control the flow of K(+) ions. More than 80 genes encode the K(+) channel subunits in the human genome. The TWIK-related K(+) channel (TREK-1) belongs to the two-pore domain K(+) channels (K2P) and displays various properties including sensitivity to physical (membrane stretch, acidosis, temperature) and chemical stimuli (signaling lipids, volatile anesthetics). The distribution of TREK-1 in the central nervous system, coupled with the physiological consequences of its opening and closing, leads to the emergence of this channel as an attractive therapeutic target. We review the TREK-1 channel, its structural and functional properties, and the pharmacological agents (agonists and antagonists) able to modulate its gating.

Influence of the N terminus on the biophysical properties and pharmacology of TREK1 potassium channels.

TWIK-related K(+) 1 (TREK1) potassium channels are members of the two-pore domain potassium channel family and contribute to background potassium conductances in many cell types, where their activity can be regulated by a variety of physiologic and pharmacologic mediators. Fenamates such as FFA (flufenamic acid; 2-{[3-(trifluoromethyl)phenyl]amino}benzoic acid), MFA [mefenamic acid; 2-(2,3-dimethylphenyl)aminobenzoic acid], NFA [niflumic acid; 2-{[3-(trifluoromethyl)phenyl]amino}nicotinic acid], and diclofenac [2-(2-(2,6-dichlorophenylamino)phenyl)acetic acid] and the related experimental drug BL-1249 [(5,6,7,8-tetrahydro-naphthalen-1-yl)-[2-(1H-tetrazol-5-yl)-phenyl]-amine] enhance the activity of TREK1 currents, and we show that BL-1249 is the most potent of these compounds. Alternative translation initiation produces a shorter, N terminus truncated form of TREK1 with a much reduced open probability and a proposed increased permeability to sodium compared with the longer form. We show that both forms of TREK1 can be activated by fenamates and that a number of mutations that affect TREK1 channel gating occlude the action of fenamates but only in the longer form of TREK1. Furthermore, fenamates produce a marked enhancement of current through the shorter, truncated form of TREK1 and reveal a K(+)-selective channel, like the long form. These results provide insight into the mechanism of TREK1 channel activation by fenamates, and, given the role of TREK1 channels in pain, they suggest a novel analgesic mechanism for these compounds.

Electrophysiology and pharmacology of tandem domain potassium channel TREK-1 related BDNF synthesis in rat astrocytes.

In the present study, the functional properties and pharmacology of two-pore domain potassium channel (K2P) TREK-1 in primary cultured rat brain astrocytes were investigated. Western blot, patch clamping techniques, and ELISA were used to detect the distribution and function of TREK-1 as well as the expression of brain-derived neurotrophic factor (BDNF) on the primary cultured astrocytes. It was shown that TREK-1 protein expressed in astrocytes was 2.4-fold higher than it was expressed in microglia. Single channel recording via patch clamping showed that the TREK-1 outward currents in astrocytes could be activated by arachidonic acid (AA) or chloroform with the conductance of 113 ± 14 and 120 ± 13 pS, respectively. The current was also sensitive to mechanical stretch and intracellular acidification. Negative pressure (-30 cm H2O) and acidification of intracellular solution (pH 6.8 or 6.3) both enhanced TREK-1 channel open probability significantly. Further pharmacological studies showed that TREK-1 antagonist penfluridol inhibited AA-induced currents, and both penfluridol and methionine (TREK-1 blockers) significantly increased BDNF level in astrocytes by 50 %. These results indicated that TREK-1 channel current was a major component of K2P currents in astrocytes. TREK-1 channels might play important roles in regulating the function of astrocytes and might be used as a drug target for neuroprotection.

The inhibitor of volume-regulated anion channels DCPIB activates TREK potassium channels in cultured astrocytes.

BACKGROUND AND PURPOSE: The ethacrynic acid derivative, 4-(2-butyl-6,7-dichlor-2-cyclopentylindan-1-on-5-yl) oxobutyric acid (DCPIB) is considered to be a specific and potent inhibitor of volume-regulated anion channels (VRACs). In the CNS, DCPIB was shown to be neuroprotective through mechanisms principally associated to its action on VRACs. We hypothesized that DCPIB could also regulate the activity of other astroglial channels involved in cell volume homeostasis. EXPERIMENTAL APPROACH: Experiments were performed in rat cortical astrocytes in primary culture and in hippocampal astrocytes in situ. The effect of DCPIB was evaluated by patch-clamp electrophysiology and immunocytochemical techniques. Results were verified by comparative analysis with recombinant channels expressed in COS-7 cells. KEY RESULTS: In cultured astrocytes, DCPIB promoted the activation of a K(+) conductance mediated by two-pore-domain K(+) (K(2P) ) channels. The DCPIB effect occluded that of arachidonic acid, which activates K(2P) channels K(2P) 2.1 (TREK-1) and K(2P) 10.1 (TREK-2) in cultured astrocytes. Immunocytochemical analysis suggests that cultured astrocytes express K(2P) 2.1 and K(2P) 10.1 proteins. Moreover, DCPIB opened recombinant K(2P) 2.1 and K(2P) 10.1 expressed in heterologous system. In brain slices, DCPIB did not augment the large background K(+) conductance in hippocampal astrocytes, but caused an increment in basal K(+) current of neurons. CONCLUSION AND IMPLICATIONS: Our results indicate that the neuroprotective effect of DCPIB could be due, at least in part, to activation of TREK channels. DCPIB could be used as template to build new pharmacological tools able to increase background K(+) conductance in astroglia and neuronal cells.