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.     

Linolenic Acid Provides Multi-cellular Protective Effects After Photothrombotic Cerebral Ischemia in Rats

Alpha-linolenic acid (LIN) has been shown to provide neuroprotective effects against cerebral ischemia. LIN is a potent activator of TREK-1 channel and LIN-induced neuroprotection disappears in Trek1?/? mice, suggesting that this channel is directly related to the LIN-induced resistance of brain against ischemia. However, the cellular mechanism underlying LIN induced neuroprotective effects after ischemia remains unclear. In this study, using a rat photochemical brain ischemia model, we investigated the effects of LIN on the protein abundance of astrocytic glutamate transporter and AQP4, microglia activation, cell apoptosis and behavioral recovery following ischemia. Administration of LIN rescued the protein abundance of astrocytic glutamate transporter GLT-1, decreased the protein abundance of AQP4 and brain edema, inhibited microglia activation, attenuated cell apoptosis and improved behavioral function recovery. Meanwhile, TREK-1 was widely distributed in the cortex and hippocampus, primarily localized in astrocytes and neurons. LIN could potentiate the TREK-1 mediated astrocytic passive conductance and hyperpolarize the membrane potential. Our results suggest that LIN provides multiple cellular neuroprotective effects in cerebral ischemia. TREK-1 may serve as a promising multi-mechanism therapeutic target for the treatment of stroke.     

A human TREK-1/HEK cell line: a highly efficient screening tool for drug development in neurological diseases

TREK-1 potassium channels are involved in a number of physiopathological processes such as neuroprotection, pain and depression. Molecules able to open or to block these channels can be clinically important. Having a cell model for screening such molecules is of particular interest. Here, we describe the development of the first available cell line that constituvely expresses the TREK-1 channel. The TREK-1 channel expressed by the h-TREK-1/HEK cell line has conserved all its modulation properties. It is opened by stretch, pH, polyunsaturated fatty acids and by the neuroprotective molecule, riluzole and it is blocked by spadin or fluoxetine. We also demonstrate that the h-TREK-1/HEK cell line is protected against ischemia by using the oxygen-glucose deprivation model.     

TREK-1, a K+ channel involved in neuroprotection and general anesthesia

TREK-1 is a two-pore-domain background potassium channel expressed throughout the central nervous system. It is opened by polyunsaturated fatty acids and lysophospholipids. It is inhibited by neurotransmitters that produce an increase in intracellular cAMP and by those that activate the Gq protein pathway. TREK-1 is also activated by volatile anesthetics and has been suggested to be an important target in the action of these drugs. Using mice with a disrupted TREK-1 gene, we now show that TREK-1 has an important role in neuroprotection against epilepsy and brain and spinal chord ischemia. Trek1-/- mice display an increased sensitivity to ischemia and epilepsy. Neuroprotection by polyunsaturated fatty acids, which is impressive in Trek1+/+ mice, disappears in Trek1-/- mice indicating a central role of TREK-1 in this process. Trek1-/- mice are also resistant to anesthesia by volatile anesthetics. TREK-1 emerges as a potential innovative target for developing new therapeutic agents for neurology and anesthesiology.     

External Ba2+ block of the two-pore domain potassium channel TREK-1 defines conformational transition in its selectivity filter

TREK-1 is a member of the two-pore domain potassium channel family that is known as a leak channel and plays a key role in many physiological and pathological processes. The conformational transition of the selectivity filter is considered as an effective strategy for potassium channels to control the course of potassium efflux. It is well known that TREK-1 is regulated by a large volume of extracellular and intracellular signals. However, until now, little was known about the selectivity filter gating mechanism of the channel. In this research, it was found that Ba(2+) blocked the TREK-1 channel in a concentration- and time-dependent manner. A mutagenesis analysis showed that overlapped binding of Ba(2+) at the assumed K(+) binding site 4 (S4) within the selectivity filter was responsible for the inhibitory effects on TREK-1. Then, Ba(2+) was used as a probe to explore the conformational transition in the selectivity filter of the channel. It was confirmed that collapsed conformations were induced by extracellular K(+)-free and acidification at the selectivity filters, leading to nonconductive to permeable ions. Further detailed characterization demonstrated that the two conformations presented different properties. Additionally, the N-terminal truncated isoform (ΔN41), a product derived from alternative translation initiation, was identified as a constitutively nonconductive variant. Together, these results illustrate the important role of selectivity filter gating in the regulation of TREK-1 by the extracellular K(+) and proton.     

Both TASK-3 and TREK-1 two-pore loop K channels are expressed in H295R cells and modulate their membrane potential and aldosterone secretion

The rate of aldosterone synthesis by adrenal glomerulosa cells relies on the selective permeability of the glomerulosa cell to K(+) ions. In rodent and bovine adrenal glomerulosa cells, this background potassium current is provided by a two-pore loop potassium (K2P) channel: largely TASK-3 in the rat and TREK-1 in the cow. The nature of the K2P channel in the human adrenal cortex is not known, and we have addressed this issue here using the H295R human adrenal cell line. We show that these cells express mRNA and protein for both TASK-3 and TREK-1 K2P channels. Using a potentiometric dye (FMP), we also show that TASK-3 and TREK-1 channel modulators can affect the membrane potential of H295R cells. Transfecting H295R cells with TASK-3 or TREK-1 dominant-negative mutants (TASK-3 G95E or TREK-1 G144E) produced depolarization of H295R cells and altered K-stimulated aldosterone secretion. Finally, transfection of a constitutively active mutant of Galpha(q) into H295R cells (GTPase-deficient Galpha(q)-QL) depolarized them and increased basal aldosterone secretion. Taken together, our data support both TASK-3 and TREK-1 as being functionally operational in the H295R cell line. This suggests that human adrenal glomerulosa cells may utilize both of these K2P channels for their background potassium current.     

Expression of the mechanosensitive 2PK+ channel TREK-1 in human osteoblasts

TREK-1 is a mechanosensitive member of the two-pore domain potassium channel family (2PK+) that is also sensitive to lipids, free fatty acids (including arachidonic acid), temperature, intracellular pH, and a range of clinically relevant compounds including volatile anaesthetics. TREK-1 is known to be expressed at high levels in excitable tissues, such as the nervous system, the heart and smooth muscle, where it is believed to play a prominent role in controlling resting cell membrane potential and electrical excitability. In this report, we use RT-PCR, Western blotting and immunohistochemistry to confirm that human derived osteoblasts and MG63 cells express TREK-1 mRNA and protein. In addition, we show gene expression of TREK2c and TRAAK channels. Furthermore, whole cell patch clamp electrophysiology demonstrates that these cells express a spontaneously active, outwardly rectifying potassium "background leak" current that shares many similarities to TREK-1. The outward current is largely insensitive to TEA and Ba2+, and is sensitive to application of lysophosphatidylcholine (LPC). In addition, blocking TREK-1 channel activity is shown to upregulate bone cell proliferation. It is concluded that human osteoblasts functionally express TREK-1 and that these channels contribute, at least in part, to the resting membrane potential of human osteoblast cells. We hypothesise a possible role for TREK-1 in mechanotransduction, leading to bone remodelling.     

The Purified Mechanosensitive Channel TREK-1 Is Directly Sensitive to Membrane Tension

Mechanosensitive channels are detected in all cells and are speculated to play a key role in many functions including osmoregulation, growth, hearing, balance, and touch. In prokaryotic cells, a direct gating of mechanosensitive channels by membrane tension was clearly demonstrated because the purified channels could be functionally reconstituted in a lipid bilayer. No such evidence has been presented yet in the case of mechanosensitive channels from animal cells. TREK-1, a two-pore domain K⁺ channel, was the first animal mechanosensitive channel identified at the molecular level. It is the target of a large variety of agents such as volatile anesthetics, neuroprotective agents, and antidepressants. We have produced the mouse TREK-1 in yeast, purified it, and reconstituted the protein in giant liposomes amenable to patch clamp recording. The protein exhibited the expected electrophysiological properties in terms of kinetics, selectivity, and pharmacology. Negative pressure (suction) applied through the pipette had no effect on the channel, but positive pressure could completely and reversibly close the channel. Our interpretation of these data is that the intrinsic tension in the lipid bilayer is sufficient to maximally activate the channel, which can be closed upon modification of the tension. These results indicate that TREK-1 is directly sensitive to membrane tension.     

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.     

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.     

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.     

Expression of the two pore domain potassium channel TREK-1 in human intervertebral disc cells

Potassium channels play a major role in intracellular homeostasis and regulation of cell volume. Intervertebral disc cells respond to mechanical loading in a complex manner. Mechanical loading may play a role in disc degeneration. Lumbar intervertebral disc samples from 5 patients (average age: 47 years, range: 25-64 years) were used for this study, investigating cells from the nucleus pulposus and the annulus fibrosus duplicate samples to determine RNA expression and protein expression. Analysis of mRNA expression by RT-PCR demonstrated that TREK 1 was expressed by nucleus pulposus (n=5) and annulus fibrosus (n=5) cells. Currently, TREK-1 is the only potassium channel known to be activated by intracellular acidosis, and responds to mechanical and chemical stimuli. Whilst the precise role of potassium channels in cellular homeostasis remains to be determined, TREK-1 may be important to protect disc cells against ischaemic damage, and subsequent disc degeneration, and may also play a role in effecting mechanotransduction. Further research is required to fully elucidate the role of the TREK-1 ion channel in intervertebral disc cells.     

匹罗卡品癫痫模型中海马区TREK-2钾离子通道表达变化及意义

目的:研究匹罗卡品癫痫模型中海马区TREK-2双孔钾离子通道的表达变化,初步探讨TREK-2在癫痫发病过程中的机制及意义。方法:选用成年雄性SD大鼠腹腔注射氯化锂-匹罗卡品(lithium-pilocarpine)构建癫痫模型,分别在癫痫持续状态(status epilepticus,SE)后不同时间点(6 h、1 d、3 d、1 w、2 w、4 w、8 w)提取海马组织,利用western-blot检测海马区TREK-2随时间表达变化。并用TREK-2 si RNA下调海马区TREK-2表达,进一步观察对大鼠癫痫状态的影响。结果:与对照组相比,TREK-2在诱导癫痫持续状态发作后的3d开始降低(P0.05),1 w,2 w,4 w明显降低(P0.01),8 w时仍维持在很低水平(P0.001)。在TREK-2表达下调后,大鼠癫痫潜伏时间(latent period)明显缩短,癫痫持续状态1 h 5级以上发作频率(seizure frequency)明显增加。结论:TREK-2在氯化锂-匹罗卡品致痫大鼠海马组织中表达的降低,且其下调加重癫痫状态的事实提示TREK-2参与了癫痫的发生发展过程。     

Sequential phosphorylation mediates receptor- and kinase-induced inhibition of TREK-1 background potassium channels

Background potassium channels determine membrane potential and input resistance and serve as prominent effectors for modulatory regulation of cellular excitability. TREK-1 is a two-pore domain background K+ channel (KCNK2, K2P2.1) that is sensitive to a variety of physicochemical and humoral factors. In this work, we used a recombinant expression system to show that activation of G alpha(q)-coupled receptors leads to inhibition of TREK-1 channels via protein kinase C (PKC), and we identified a critical phosphorylation site in a key regulatory domain that mediates inhibition of the channel. In HEK 293 cells co-expressing TREK-1 and either the thyrotropin-releasing hormone receptor (TRHR1) or the Orexin receptor (Orx1R), agonist stimulation induced robust channel inhibition that was suppressed by a bisindolylmaleimide PKC inhibitor but not by a protein kinase A blocker ((R(p))-cAMP-S). Channel inhibition by agonists or by direct activators of PKC (phorbol dibutyrate) and PKA (forskolin) was disrupted not only by alanine or aspartate mutations at an identified PKA site (Ser-333) in the C terminus, but also at a more proximal regulatory site in the cytoplasmic C terminus (Ser-300); S333A and S300A mutations enhanced basal TREK-1 current, whereas S333D and S300D substitutions mimicked phosphorylation and strongly diminished currents. When studied in combination, TREK-1 current density was enhanced in S300A/S333D but reduced in S300D/S333A mutant channels. Channel mutants were expressed and appropriately targeted to cell membranes. Together, these data support a sequential phosphorylation model in which receptor-induced kinase activation drives modification at Ser-333 that enables subsequent phosphorylation at Ser-300 to inhibit TREK-1 channel activity.     

Mechanism of inhibition of TREK-2 (K2P10.1) by the Gq-coupled M3 muscarinic receptor

TREK-2 is a member of the two-pore domain K⁺ channel family and provides part of the background K⁺ current in many types of cells. Neurotransmitters that act on receptors coupled to G_q strongly inhibit TREK-2 and thus enhance cell excitability. The molecular basis for the inhibition of TREK-2 was studied. In COS-7 cells expressing TREK-2 and M₃ receptor, acetylcholine (ACh) applied to the bath solution strongly inhibited the whole cell current, and this was markedly reduced in the presence of U-73122, an inhibitor of PLC. The inhibition was also observed in cell-attached patches when ACh was applied to the bath solution. In inside-out patches, direct application of guanosine 5'-O-(3-thiotriphosphate) (10 µM), Ca²⁺ (5 µM), or diacylglycerol (DAG; 10 µM) produced no inhibition of TREK-2 in >75% of patches tested. Phosphatidic acid, a product of DAG kinase, had no effect on TREK-2. Pretreatment of cells with 20 µM wortmannin, an inhibitor of phosphatidylinositol kinases, did not affect the inhibition or the recovery from inhibition of TREK-2, suggesting that phosphatidylinositol 4,5-bisphosphate depletion did not mediate the inhibition. Pretreatment of cells with a protein kinase C inhibitor (bisindolylmaleimide, 10 µM) markedly inhibited ACh-induced inhibition of TREK-2. Mutation of two putative PKC sites (S326A, S359C) abolished inhibition by ACh. Mutation of these amino acids to aspartate to mimic the phosphorylated state resulted in diminished TREK-2 current and no inhibition by ACh. These results suggest that the agonist-induced inhibition of TREK-2 via M₃ receptor occurs primarily via PKC-mediated phosphorylation. two-pore domain potassium channel; G_q protein; background potassium conductance; protein kinase C; phosphatidylinositol 4,5-bisphosphate     

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.     

Expression of Stretch-Activated Two-Pore Potassium Channels in Human Myometrium in Pregnancy and Labor

Background

We tested the hypothesis that the stretch-activated, four-transmembrane domain, two pore potassium channels (K2P), TREK-1 and TRAAK are gestationally-regulated in human myometrium and contribute to uterine relaxation during pregnancy until labor.

Methodology

We determined the gene and protein expression of K2P channels in non-pregnant, pregnant term and preterm laboring myometrium. We employed both molecular biological and functional studies of K2P channels in myometrial samples taken from women undergoing cesarean delivery of a fetus.

Principal Findings

TREK-1, but not TREK-2, channels are expressed in human myometrium and significantly up-regulated during pregnancy. Down-regulation of TREK-1 message was seen by Q-PCR in laboring tissues consistent with a role for TREK-1 in maintaining uterine quiescence prior to labor. The TRAAK channel was unregulated in the same women. Blockade of stretch-activated channels with a channel non-specific tarantula toxin (GsMTx-4) or the more specific TREK-1 antagonist L-methionine ethyl ester altered contractile frequency in a dose-dependent manner in pregnant myometrium. Arachidonic acid treatment lowered contractile tension an effect blocked by fluphenazine. Functional studies are consistent with a role for TREK-1 in uterine quiescence.

Conclusions

We provide evidence supporting a role for TREK-1 in contributing to uterine quiescence during gestation and hypothesize that dysregulation of this mechanism may underlie certain cases of spontaneous pre-term birth.     

Metabolic and thermal stimuli control K(2P)2.1 (TREK-1) through modular sensory and gating domains

K(2P)2.1 (TREK-1) is a polymodal two-pore domain leak potassium channel that responds to external pH, GPCR-mediated phosphorylation signals, and temperature through the action of distinct sensors within the channel. How the various intracellular and extracellular sensory elements control channel function remains unresolved. Here, we show that the K(2P)2.1 (TREK-1) intracellular C-terminal tail (Ct), a major sensory element of the channel, perceives metabolic and thermal commands and relays them to the extracellular C-type gate through transmembrane helix M4 and pore helix 1. By decoupling Ct from the pore-forming core, we further demonstrate that Ct is the primary heat-sensing element of the channel, whereas, in contrast, the pore domain lacks robust temperature sensitivity. Together, our findings outline a mechanism for signal transduction within K(2P)2.1 (TREK-1) in which there is a clear crosstalk between the C-type gate and intracellular Ct domain. In addition, our findings support the general notion of the existence of modular temperature-sensing domains in temperature-sensitive ion channels. This marked distinction between gating and sensory elements suggests a general design principle that may underlie the function of a variety of temperature-sensitive channels.     

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