Lamotrigine inhibits TRESK regulated by G-protein coupled receptor agonists

Dorsal root ganglion (DRG) neurons express mRNAs for numerous two-pore domain K⁺ (K_2P) channels and G-protein coupled receptors (GPCR). Recent studies have shown that TRESK is a major background K⁺ channel in DRG neurons. Here, we demonstrate the pharmacological properties of TRESK, including GPCR agonist-induced effects on DRG neurons. TRESK mRNA was highly expressed in DRG compared to brain and spinal cord. Similar to cloned TRESK, native TRESK was inhibited by acid and arachidonic acid (AA), but not zinc. Native TRESK was also activated by GPCR agonists such as acetylcholine, glutamate, and histamine. The glutamate-activated TRESK was blocked by lamotrigine in DRG neurons. In COS-7 cells transfected with mouse TRESK, 30 μM lamotrigine inhibited TRESK by ∼50%. Since TRESK is target of modulation by acid, AA, GPCR agonists, and lamotrigine, it is likely to play an active role in the regulation of excitability in DRG neurons.     

AKAP150, a switch to convert mechano-, pH- and arachidonic acid-sensitive TREK K(+) channels into open leak channels

TREK channels are unique among two-pore-domain K(+) channels. They are activated by polyunsaturated fatty acids (PUFAs) including arachidonic acid (AA), phospholipids, mechanical stretch and intracellular acidification. They are inhibited by neurotransmitters and hormones. TREK-1 knockout mice have impaired PUFA-mediated neuroprotection to ischemia, reduced sensitivity to volatile anesthetics and altered perception of pain. Here, we show that the A-kinase-anchoring protein AKAP150 is a constituent of native TREK-1 channels. Its binding to a key regulatory domain of TREK-1 transforms low-activity outwardly rectifying currents into robust leak conductances insensitive to AA, stretch and acidification. Inhibition of the TREK-1/AKAP150 complex by Gs-coupled receptors such as serotonin 5HT4sR and noradrenaline beta2AR is as extensive as for TREK-1 alone, but is faster. Inhibition of TREK-1/AKAP150 by Gq-coupled receptors such as serotonin 5HT2bR and glutamate mGluR5 is much reduced when compared to TREK-1 alone. The association of AKAP150 with TREK channels integrates them into a postsynaptic scaffold where both G-protein-coupled membrane receptors (as demonstrated here for beta2AR) and TREK-1 dock simultaneously.     

TREK-2 (K2P10.1) and TRESK (K2P18.1) are major background K+ channels in dorsal root ganglion neurons

Dorsal root ganglion (DRG) neurons express mRNAs for many two-pore domain K⁺ (K_2P) channels that behave as background K⁺ channels. To identify functional background K⁺ channels in DRG neurons, we examined the properties of single-channel openings from cell-attached and inside-out patches from the cell bodies of DRG neurons. We found seven types of K⁺ channels, with single-channel conductance ranging from 14 to 120 pS in 150 mM KCl bath solution. Four of these K⁺ channels showed biophysical and pharmacological properties similar to TRESK (14 pS), TREK-1 (112 pS), TREK-2 (50 pS), and TRAAK (73 pS), which are members of the K_2P channel family. The molecular identity of the three other K⁺ channels could not be determined, as they showed low channel activity and were observed infrequently. Of the four K_2P channels, the TRESK-like (14 pS) K⁺ channel was most active at 24°C. At 37°C, the 50-pS (TREK-2 like) channel was the most active and contributed the most (69%) to the resting K⁺ current, followed by the TRESK-like 14-pS (16%), TREK-1-like 112-pS (12%), and TRAAK-like 73-pS (3%) channels. In DRG neurons, mRNAs of all four K_2P channels, as well as those of TASK-1 and TASK-3, were expressed, as judged by RT-PCR analysis. Our results show that TREKs and TRESK together contribute >95% of the background K⁺ conductance of DRG neurons at 37°C. As TREKs and TRESK are targets of modulation by receptor agonists, they are likely to play an active role in the regulation of excitability in DRG neurons. two-pore domain K⁺ channel; conductance; excitability     

Enhanced expressions of arachidonic acid-sensitive tandem-pore domain potassium channels in rat experimental acute cerebral ischemia

To further explore the pathophysiological significance of arachidonic acid-sensitive potassium channels, RT-PCR and Western blot analysis were used to investigate the expression changes of TREK channels in cortex and hippocampus in rat experimental acute cerebral ischemia in this study. Results showed that TREK-1 and TRAAK mRNA in cortex, TREK-1 and TREK-2 mRNA in hippocampus showed significant increases 2 h after middle cerebral artery occlusion (MCAO). While the mRNA expression levels of the all three channel subtypes increased significantly 24 h after MCAO in cortex and hippocampus. At the same time, the protein expressions of all the three channel proteins showed significant increase 24 h after MCAO in cortex and hippocampus, but only TREK-1 showed increased expression 2 h after MCAO in cortex and hippocampus. Immunohistochemical experiments verified that all the three channel proteins had higher expression levels in cortical and hippocampal neurons 24 h after MCAO. These results suggested a strong correlation between TREK channels and acute cerebral ischemia. TREK channels might provide a neuroprotective mechanism in the pathological process.     

Lentiviral gene transfer into the dorsal root ganglion of adult rats

Background

Neuronal hyperexcitability is a crucial phenomenon underlying spontaneous and evoked pain. In invertebrate nociceptors, the S-type leak K⁺ channel (analogous to TREK-1 in mammals) plays a critical role of in determining neuronal excitability following nerve injury. Few data are available on the role of leak K_2P channels after peripheral axotomy in mammals.

Results

Here we describe that rat sciatic nerve axotomy induces hyperexcitability of L4-L5 DRG sensory neurons and decreases TRESK (K2P18.1) expression, a channel with a major contribution to total leak current in DRGs. While the expression of other channels from the same family did not significantly change, injury markers ATF3 and Cacna2d1 were highly upregulated. Similarly, acute sensory neuron dissociation (in vitro axotomy) produced marked hyperexcitability and similar total background currents compared with neurons injured in vivo. In addition, the sanshool derivative IBA, which blocked TRESK currents in transfected HEK293 cells and DRGs, increased intracellular calcium in 49% of DRG neurons in culture. Most IBA-responding neurons (71%) also responded to the TRPV1 agonist capsaicin, indicating that they were nociceptors. Additional evidence of a biological role of TRESK channels was provided by behavioral evidence of pain (flinching and licking), in vivo electrophysiological evidence of C-nociceptor activation following IBA injection in the rat hindpaw, and increased sensitivity to painful pressure after TRESK knockdown in vivo.

Conclusions

In summary, our results clearly support an important role of TRESK channels in determining neuronal excitability in specific DRG neurons subpopulations, and show that axonal injury down-regulates TRESK channels, therefore contributing to neuronal hyperexcitability.     

Involvement of intracellular transport in TREK-1c current run-up in 293T cells

The TREK-1 channel, the TWIK-1-related potassium (K⁺) channel, is a member of a family of 2-pore-domain K⁺ (K2P) channels, through which background or leak K⁺ currents occur. An interesting feature of the TREK-1 channel is the run-up of current: i.e. the current through TREK-1 channels spontaneously increases within several minutes of the formation of the whole-cell configuration. To investigate whether intracellular transport is involved in the run-up, we established 293T cell lines stably expressing the TREK-1c channel (K2P2.1) and examined the effects of inhibitors of membrane protein transport, N-methylmaleimide (NEM), brefeldin-A, and an endocytosis inhibitor, pitstop2, on the run-up. The results showing that NEM and brefeldin-A inhibited and pitstop2 facilitated the run-up suggest the involvement of intracellular protein transport. Correspondingly, in cells stably expressing the mCherry-TREK-1 fusion protein, NEM decreased and pitstop2 increased the cell surface localization of the fusion protein. Furthermore, the run-up was inhibited by the intracellular application of a peptide of the C-terminal fragment TREK335–360, corresponding to the interaction site with microtubule-associated protein 2 (Mtap2). This peptide also inhibited the co-immunoprecipitation of Mtap2 with anti-mCherry antibody. The extracellular application of an ezrin inhibitor (NSC668394) also suppressed the run-up and surface localization of the fusion protein. The co-application of these inhibitors abolished the TREK-1c current, suggesting that the additive effects of ezrin and Mtap2 enhance the surface expression of TREK-1c channels and the run-up. These findings clearly showed the involvement of intracellular transport in TREK-1c current run-up and its mechanism.     

A TREK inhibitor takes multiple tracks

Single-channel recordings reveal that norfluoxetine inhibits the two-pore domain K⁺ channel TREK-2 by a complex array of mechanisms.

The TREK subfamily of two-pore domain K⁺ channels are expressed throughout the central and peripheral nervous systems and are involved in a diverse range of processes such as mechanosensation, thermosensation, and nociception. Accordingly, channel gating—which is thought to involve changes in the selectivity filter of TREKs—can be regulated by a wide variety of factors, including pressure, temperature, and multiple endogenous ligands (1). In this issue of JGP, Proks et al. reveal that this regulatory complexity is reflected in the fact that the TREK inhibitor norfluoxetine impairs channel activity via several different mechanisms (2).Peter Proks (left), Stephen J. Tucker (center), and colleagues use single-channel recordings to investigate how norfluoxetine inhibits the two-pore domain K⁺ channel TREK-2. Norfluoxetine binds exclusively to the “down” conformation of TREK-2 (right) and prevents the channel’s transmembrane domains from transitioning to the “up” configuration. But Proks et al. find that TREK-2 can be fully active in the down conformation and that norfluoxetine works via multiple mechanisms to inhibit both the open and closed states of the channel.Norfluoxetine is a metabolite of fluoxetine (Prozac), and both compounds are among the few known inhibitors of TREK activity (3). “TREK channels are not the principal targets of fluoxetine, which is mainly a selective serotonin reuptake inhibitor,” explains Stephen J. Tucker from the University of Oxford. “But fluoxetine and norfluoxetine are useful tools to study the mechanisms of TREK channel gating.”Tucker and colleagues previously helped solve the crystal structures of TREK-2 in the presence and absence of norfluoxetine (4). The channel can adopt two distinct conformations, named “up” or “down” depending on the orientation of its transmembrane helices, and norfluoxetine was found to bind within the inner cavity of TREK-2 in a gap that is only formed when the transmembrane helices are in the down configuration. Norfluoxetine can therefore block the transition from the down to up conformation, and it was originally suggested that this might inhibit channel activity by locking the selectivity filter in its closed state. But the mechanism of filter gating appears to be more complex. Tucker’s group, for example, has previously shown using macroscopic recordings that TREK-2 can adopt several open states, some of which may occur in the down conformation (5).To learn more about the mechanisms underlying filter gating and norfluoxetine inhibition, Tucker and colleagues, including first author Peter Proks, turned to single-channel recordings of purified TREK-2 channels embedded in lipid bilayers (2). “We found that norfluoxetine affects both the open and closed states of the channel and is therefore a state-independent inhibitor of TREK-2,” Tucker says. “That information is lost in macroscopic recordings.”Moreover, the fact that highly active channels are sensitive to norfluoxetine inhibition confirms that TREK channels can be fully open in the down conformation. It also indicates that, in addition to blocking changes in transmembrane conformation, norfluoxetine must inhibit TREK channels by other mechanisms as well.“We found that there are several mechanisms involved, all of which converge on the selectivity filter gate,” Tucker says. The researchers also observed a mild voltage dependence of norfluoxetine inhibition, suggesting that it can influence voltage-dependent gating as well.“The complexity with which the drug works reflects the many different ways in which the selectivity filter can gate the channel,” Tucker says. “This, in turn, reflects the polymodal regulation of TREK channels and their ability to integrate a wide variety of signals.”     

Antipsychotics inhibit TREK but not TRAAK channels

Schizophrenia is a chronic mental illness affecting 0.4% of the population. Existing antipsychotic drugs are mainly used to treat positive symptoms such as hallucinations but have only poor effects on negative symptoms such as cognitive deficits or depression. TREK and TRAAK channels are two P domain background potassium channels activated by polyunsaturated fatty acids and mechanical stress. TREK but not TRAAK channels are regulated by Gs- and Gq-coupled pathways. The inactivation of the TREK-1 but not the TRAAK channel in mice results in a depression-resistant phenotype. In addition, it has been shown that antidepressants such as fluoxetine or paroxetine directly inhibit TREK channel activity. Here we show that different antipsychotic drugs directly inhibit TREK currents with IC(50) values of approximately 1 to approximately 20 microM. No effect is seen on TRAAK channel activity. We conclude that TREK channels might be involved in the therapeutic action of antipsychotics or in their secondary effects. Furthermore, TREK channels could play a role in the pathophysiology of psychiatric disorders such as depression and schizophrenia.     

Towards a TREK-1/2 (TWIK-Related K+ Channel 1 and 2) dual activator tool compound: Multi-dimensional optimization of BL-1249

This letter describes a focused, multi-dimensional optimization campaign around BL-1249, a fenamate class non-steroidal anti-inflammatory and a known activator of the K_2P potassium channels TREK-1 (K_2P2.1) and TREK-2 (K_2P10.1). While BL-1249 has been widely profiled in vitro as a dual TREK-1/2 activator, poor physicochemical and DMPK properties have precluded a deeper understanding of the therapeutic potential of these key K_2P channels across a broad spectrum of peripheral and central human disease. Here, we report multi-dimensional SAR that led to a novel TREK-1/2 dual activator chemotype, exemplified by ONO-2960632/VU6011992, with improved DMPK properties, representing a new lead for further optimization towards robust in vivo tool compounds.     

Chemoselective regulation of TREK2 channel: Activation by sulfonate chalcones and inhibition by sulfonamide chalcones

Although it has not been extensively studied, a significant volume of literature suggests that TREK2 will probably turn out to be an important channel in charge of tuning neuronal transmitter and hormone levels. Thus, pharmacological tools which can manipulate this channel, such as selective agonists are essential both in drug design and to further our understanding of this system. Our investigations have shown that sulfonate (‘O’) chalcone and sulfonamide (‘N’) chalcones regulate the TREK2 channel in remarkably different ways: sulfonamide chalcone 5 behaved as an inhibitor with an IC₅₀ of 62 μM, whereas the sulfonate analogue 11 activated TREK2 with EC₅₀ value of 167 μM.     

A role for two‐pore potassium (K2P) channels in endometrial epithelial function

The human endometrial epithelium is pivotal to menstrual cycle progression, implantation and early pregnancy. Endometrial function is directly regulated by local factors that include pH, oxygen tension and ion concentrations to generate an environment conducive to fertilization. A superfamily of potassium channels characterized by two‐pore domains (K2P) and encoded by KCNK genes is implicated in the control of the cell resting membrane potential through the generation of leak currents and modulation by various physicochemical stimuli. The aims of the study were to determine the expression and function of K2P channel subtypes in proliferative and secretory phase endometrium obtained from normo‐ovulatory women and in an endometrial cancer cell line. Using immunochemical methods, real‐time qRT‐PCR proliferation assays and electrophysiology. Our results demonstrate mRNA for several K2P channel subtypes in human endometrium with molecular expression of TREK‐1 shown to be higher in proliferative than secretory phase endometrium (P < 0.001). The K2P channel blockers methanandamide, lidocaine, zinc and curcumin had antiproliferative effects (P < 0.01) in an endometrial epithelial cancer cell line indicating a role for TASK and TREK‐1 channels in proliferation. Tetraethylammonium‐ and 4‐aminopyridine‐insensitive outwards currents were inhibited at all voltages by reducing extracellular pH from 7.4 to 6.6. Higher expression of TREK‐1 expression in proliferative phase endometrium may, in part, underlie linked to increased cell division. The effects of pH and a lack of effect of non‐specific channel blockers of voltage‐gated potassium channels imply a role for K2P channels in the regulation of human endometrial function.     

Transition between conformational states of the TREK-1 K2P channel promoted by interaction with PIP2

Members of the TREK family of two-pore domain potassium channels are highly sensitive to regulation by membrane lipids, including phosphatidylinositol-4,5-bisphosphate (PIP₂). Previous studies have demonstrated that PIP₂ increases TREK-1 channel activity; however, the mechanistic understanding of the conformational transitions induced by PIP₂ remain unclear. Here, we used coarse-grained molecular dynamics and atomistic molecular dynamics simulations to model the PIP₂-binding site on both the up and down state conformations of TREK-1. We also calculated the free energy of PIP₂ binding relative to other anionic phospholipids in both conformational states using potential of mean force and free-energy-perturbation calculations. Our results identify state-dependent binding of PIP₂ to sites involving the proximal C-terminus, and we show that PIP₂ promotes a conformational transition from a down state toward an intermediate that resembles the up state. These results are consistent with functional data for PIP₂ regulation, and together provide evidence for a structural mechanism of TREK-1 channel activation by phosphoinositides.     

TREK-1 regulation by nitric oxide and cGMP-dependent protein kinase. An essential role in smooth muscle inhibitory neurotransmission

Potassium channels activated by membrane stretch may contribute to maintenance of relaxation of smooth muscle cells in visceral hollow organs. Previous work has identified K(+) channels in murine colon that are activated by stretch and further regulated by NO-dependent mechanisms. We have screened murine gastrointestinal, vascular, bladder, and uterine smooth muscles for the expression of TREK and TRAAK mRNA. Although TREK-1 was expressed in many of these smooth muscles, TREK-2 was expressed only in murine antrum and pulmonary artery. TRAAK was not expressed in any smooth muscle cells tested. Whole cell currents from TREK-1 expressed in mammalian COS cells were activated by stretch, and single channel recordings showed that the stretch-dependent conductance was due to 90 pS channels. Sodium nitroprusside (10(-6) or 10(-5) m) and 8-Br-cGMP (10(-4) or 10(-3) m) increased TREK-1 currents in perforated whole cell and single channel recordings. Mutation of the PKG consensus sequence at serine 351 blocked the stimulatory effects of sodium nitroprusside and 8-Br-cGMP on open probability without affecting the inhibitory effects of 8-Br-cAMP. TREK-1 encodes a component of the stretch-activated K(+) conductance in smooth muscles and may contribute to nitrergic inhibition of gastrointestinal muscles.     

The pore structure and gating mechanism of K2P channels

Two-pore domain (K2P) potassium channels are important regulators of cellular electrical excitability. However, the structure of these channels and their gating mechanism, in particular the role of the bundle-crossing gate, are not well understood. Here, we report that quaternary ammonium (QA) ions bind with high-affinity deep within the pore of TREK-1 and have free access to their binding site before channel activation by intracellular pH or pressure. This demonstrates that, unlike most other K(+) channels, the bundle-crossing gate in this K2P channel is constitutively open. Furthermore, we used QA ions to probe the pore structure of TREK-1 by systematic scanning mutagenesis and comparison of these results with different possible structural models. This revealed that the TREK-1 pore most closely resembles the open-state structure of KvAP. We also found that mutations close to the selectivity filter and the nature of the permeant ion profoundly influence TREK-1 channel gating. These results demonstrate that the primary activation mechanisms in TREK-1 reside close to, or within the selectivity filter and do not involve gating at the cytoplasmic bundle crossing.     

Differential Expression of Potassium Channels in Placentas from Normal and Pathological Pregnancies: Targeting of the K<Subscript>ir</Subscript> 2.1 Channel to Lipid Rafts

Potassium channels play important physiological roles in human syncytiotrophoblasts (hSTBs) from placenta, an epithelium responsible for maternal–fetal exchange. Basal and apical plasma membranes differ in their lipid and protein composition, and the latter contains cholesterol-enriched microdomains. In placental tissue, the specific localization of potassium channels is unknown. Previously, we described two isolated subdomains from the apical membrane (MVM and LMVM) and their respective microdomains (lipid rafts). Here, we report on the distribution of K_ir2.1, K_v2.1, TASK-1, and TREK-1 in hSTB membranes and the lipid rafts that segregate them. Immunoblotting experiments showed that these channels are present mainly in the apical membrane from healthy hSTBs. Apical expression versus basal membrane was 84 and 16% for K_ir2.1 and K_v2.1, 60 and 30% for TREK-1, and 74 and 26% for TASK-1. Interestingly, K_v2.1 showed differences between apical membrane subdomains: 26 ± 8% was located in the LMVM and 59 ± 9% in MVM. In pathological placentas, the expression distribution changed in the basal membrane: preeclampsia shifted to 50% and intrauterine growth restriction to 42% for TASK-1 and both pathologies increased to 25% for K_ir2.1 and K_v2.1, K_ir2.1 appeared to be associated with rafts that were sensitive to cholesterol depletion in healthy, but not in pathological, placentas. K_v2.1 and TREK-1 emerged in the nonraft fractions. The precise membrane localization of ion channels in hSTB membranes is necessary to understand the physiological events.     

A novel two-pore domain K+ channel,TRESK, is localized in the spinal cord

To find a novel human ion channel gene we have executed an extensive search by using a human genome draft sequencing data base. Here we report a novel two-pore domain K+ channel, TRESK (TWIK-related spinal cord K+ channel). TRESK is coded by 385 amino acids and shows low homology (19%) with previously characterized two-pore domain K+ channels. However, the most similar channel is TREK-2 (two-pore domain K+ channel), and TRESK also has two pore-forming domains and four transmembrane domains that are evolutionarily conserved in the two-pore domain K+ channel family. Moreover, we confirmed that TRESK is expressed in the spinal cord. Electrophysiological analysis demonstrated that TRESK induced outward rectification and functioned as a background K+ channel. Pharmacological analysis showed TRESK to be inhibited by previously reported K+ channel inhibitors Ba2+, propafenone, glyburide, lidocaine, quinine, quinidine, and triethanolamine. Functional analysis demonstrated TRESK to be inhibited by unsaturated free fatty acids such as arachidonic acid and docosahexaenoic acid. TRESK is also sensitive to extreme changes in extracellular and intracellular pH. These results indicate that TRESK is a novel two-pore domain K+ channel that may set the resting membrane potential of cells in the spinal cord.     

Single-channel properties and pH sensitivity of two-pore domain K+ channels of the TALK family

The two-pore K2P channel family comprises TASK, TREK, TWIK, TRESK, TALK, and THIK subfamilies, and TALK-1, TALK-2, and TASK-2 are functional members of the TALK subfamily. Here we report for the first time the single-channel properties of TALK-2 and its pHo sensitivity, and compare them to those of TALK-1 and TASK-2. In transfected COS-7 cells, the three TALK K2P channels could be identified easily by their differences in single-channel conductance and gating kinetics. The single-channel conductances of TALK-1, TALK-2, and TASK-2 in symmetrical 150 mM KCl were 21, 33, and 70 pS (-60 mV), respectively. TALK-2 was sensitive mainly to the alkaline range (pH 7-10), whereas TALK-1 and TASK-2 were sensitive to a wider pHo range (6-10). The effect of pH changes was mainly on the opening frequency. Thus, members of the TALK family expressed in native tissues may be identified based on their single-channel kinetics and pHo sensitivity.     

Lipid and mechano-gated 2P domain K(+) channels.

The two pore domain K(+) channels TREK and TRAAK are opened by membrane stretch. The activating mechanical force comes from the bilayer membrane and is independent of the cytoskeleton. Emerging work shows that mechano-gated TREK and TRAAK are opened by various lipids, including long chain polyunsaturated anionic fatty acids and neutral cone-shaped lysophospholipids. TREK-1 shares the properties of the Aplysia neuronal S channel, a presynaptic background K(+) channel involved in behavioral sensitization, a simple form of learning.     

The Thermosensitive Potassium Channel TREK-1 Contributes to Coolness-Evoked Responses of Grueneberg Ganglion Neurons

Neurons of the Grueneberg ganglion (GG) residing in the vestibule of the murine nose are activated by cool ambient temperatures. Activation of thermosensory neurons is usually mediated by thermosensitive ion channels of the transient receptor potential (TRP) family. However, there is no evidence for the expression of thermo-TRPs in the GG, suggesting that GG neurons utilize distinct mechanisms for their responsiveness to cool temperatures. In search for proteins that render GG neurons responsive to coolness, we have investigated whether TREK/TRAAK channels may play a role; in heterologous expression systems, these potassium channels have been previously found to close upon exposure to coolness, leading to a membrane depolarization. The results of the present study indicate that the thermosensitive potassium channel TREK-1 is expressed in those GG neurons that are responsive to cool temperatures. Studies analyzing TREK-deficient mice revealed that coolness-evoked responses of GG neurons were clearly attenuated in these animals compared with wild-type conspecifics. These data suggest that TREK-1 channels significantly contribute to the responsiveness of GG neurons to cool temperatures, further supporting the concept that TREK channels serve as thermoreceptors in sensory cells. Moreover, the present findings provide the first evidence of how thermosensory GG neurons are activated by given temperature stimuli in the absence of thermo-TRPs.