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.     

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.     

Human TREK2, a 2P domain mechano-sensitive K+ channel with multiple regulations by polyunsaturated fatty acids, lysophospholipids, and Gs, Gi, and Gq protein-coupled receptors

Mechano-sensitive and fatty acid-activated K(+) belong to the structural class of K(+) channel with two pore domains. Here, we report the isolation and the characterization of a novel member of this family. This channel, called TREK2, is closely related to TREK1 (78% of homology). Its gene is located on chromosome 14q31. TREK2 is abundantly expressed in pancreas and kidney and to a lower level in brain, testis, colon, and small intestine. In the central nervous system, TREK2 has a widespread distribution with the highest levels of expression in cerebellum, occipital lobe, putamen, and thalamus. In transfected cells, TREK2 produces rapidly activating and non-inactivating outward rectifier K(+) currents. The single-channel conductance is 100 picosiemens at +40 mV in 150 mm K(+). The currents can be strongly stimulated by polyunsaturated fatty acid such as arachidonic, docosahexaenoic, and linoleic acids and by lysophosphatidylcholine. The channel is also activated by acidification of the intracellular medium. TREK2 is blocked by application of intracellular cAMP. As with TREK1, TREK2 is activated by the volatile general anesthetics chloroform, halothane, and isoflurane and by the neuroprotective agent riluzole. TREK2 can be positively or negatively regulated by a variety of neurotransmitter receptors. Stimulation of the G(s)-coupled receptor 5HT4sR or the G(q)-coupled receptor mGluR1 inhibits channel activity, whereas activation of the G(i)-coupled receptor mGluR2 increases TREK2 currents. These multiple types of regulations suggest that TREK2 plays an important role as a target of neurotransmitter action.     

The mechano‐activated K+ channels TRAAK and TREK‐1 control both warm and cold perception

The sensation of cold or heat depends on the activation of specific nerve endings in the skin. This involves heat‐ and cold‐sensitive excitatory transient receptor potential (TRP) channels. However, we show here that the mechano‐gated and highly temperature‐sensitive potassium channels of the TREK/TRAAK family, which normally work as silencers of the excitatory channels, are also implicated. They are important for the definition of temperature thresholds and temperature ranges in which excitation of nociceptor takes place and for the intensity of excitation when it occurs. They are expressed with thermo‐TRP channels in sensory neurons. TRAAK and TREK‐1 channels control pain produced by mechanical stimulation and both heat and cold pain perception in mice. Expression of TRAAK alone or in association with TREK‐1 controls heat responses of both capsaicin‐sensitive and capsaicin‐insensitive sensory neurons. Together TREK‐1 and TRAAK channels are important regulators of nociceptor activation by cold, particularly in the nociceptor population that is not activated by menthol.     

Mechanisms underlying excitatory effects of group I metabotropic glutamate receptors via inhibition of 2P domain K+ channels

Group I metabotropic glutamate receptors (mGluRs) are implicated in diverse processes such as learning, memory, epilepsy, pain and neuronal death. By inhibiting background K(+) channels, group I mGluRs mediate slow and long-lasting excitation. The main neuronal representatives of this K(+) channel family (K(2P) or KCNK) are TASK and TREK. Here, we show that in cerebellar granule cells and in heterologous expression systems, activation of group I mGluRs inhibits TASK and TREK channels. D-myo-inositol-1,4,5-triphosphate and phosphatidyl-4,5-inositol-biphosphate depletion are involved in TASK channel inhibition, whereas diacylglycerols and phosphatidic acids directly inhibit TREK channels. Mechanisms described here with group I mGluRs will also probably stand for many other receptors of hormones and neurotransmitters.     

The neuronal background K2P channels: focus on TREK1

Two-pore-domain K(+) (K(2P)) channel subunits are made up of four transmembrane segments and two pore-forming domains that are arranged in tandem and function as either homo- or heterodimeric channels. This structural motif is associated with unusual gating properties, including background channel activity and sensitivity to membrane stretch. Moreover, K(2P) channels are modulated by a variety of cellular lipids and pharmacological agents, including polyunsaturated fatty acids and volatile general anaesthetics. Recent in vivo studies have demonstrated that TREK1, the most thoroughly studied K(2P) channel, has a key role in the cellular mechanisms of neuroprotection, anaesthesia, pain and depression.     

Lysophospholipids open the two-pore domain mechano-gated K(+) channels TREK-1 and TRAAK

The two-pore (2P) domain K(+) channels TREK-1 and TRAAK are opened by membrane stretch as well as arachidonic acid (AA) (Patel, A. J., Honoré, E., Maingret, F., Lesage, F., Fink, M., Duprat, F., and Lazdunski, M. (1998) EMBO J. 17, 4283-4290; Maingret, F., Patel, A. J., Lesage, F., Lazdunski, M., and Honoré, E. (1999) J. Biol. Chem. 274, 26691-26696; Maingret, F., Fosset, M., Lesage, F., Lazdunski, M. , and Honoré, E. (1999) J. Biol. Chem. 274, 1381-1387. We demonstrate that lysophospholipids (LPs) and platelet-activating factor also produce large specific and reversible activations of TREK-1 and TRAAK. LPs activation is a function of the size of the polar head and length of the acyl chain but is independent of the charge of the molecule. Bath application of lysophosphatidylcholine (LPC) immediately opens TREK-1 and TRAAK in the cell-attached patch configuration. In excised patches, LPC activation is lost, whereas AA still produces maximal opening. The carboxyl-terminal region of TREK-1, but not the amino terminus and the extracellular loop M1P1, is critically required for LPC activation. LPC activation is indirect and may possibly involve a cytosolic factor, whereas AA directly interacts with either the channel proteins or the bilayer and mimics stretch. Opening of TREK-1 and TRAAK by fatty acids and LPs may be an important switch in the regulation of synaptic function and may also play a protective role during ischemia and inflammation.     

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.     

The Influence of Cold Temperature on Cellular Excitability of Hippocampal Networks

The hippocampus plays an important role in short term memory, learning and spatial navigation. A characteristic feature of the hippocampal region is its expression of different electrical population rhythms and activities during different brain states. Physiological fluctuations in brain temperature affect the activity patterns in hippocampus, but the underlying cellular mechanisms are poorly understood. In this work, we investigated the thermal modulation of hippocampal activity at the cellular network level. Primary cell cultures of mouse E17 hippocampus displayed robust network activation upon light cooling of the extracellular solution from baseline physiological temperatures. The activity generated was dependent on action potential firing and excitatory glutamatergic synaptic transmission. Involvement of thermosensitive channels from the transient receptor potential (TRP) family in network activation by temperature changes was ruled out, whereas pharmacological and immunochemical experiments strongly pointed towards the involvement of temperature-sensitive two-pore-domain potassium channels (K_2P), TREK/TRAAK family. In hippocampal slices we could show an increase in evoked and spontaneous synaptic activity produced by mild cooling in the physiological range that was prevented by chloroform, a K_2P channel opener. We propose that cold-induced closure of background TREK/TRAAK family channels increases the excitability of some hippocampal neurons, acting as a temperature-sensitive gate of network activation. Our findings in the hippocampus open the possibility that small temperature variations in the brain in vivo, associated with metabolism or blood flow oscillations, act as a switch mechanism of neuronal activity and determination of firing patterns through regulation of thermosensitive background potassium channel activity.     

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.     

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.     

Altered acetylcholine, bradykinin and cutaneous pressure-induced vasodilation in mice lacking the TREK1 potassium channel: the endothelial link

The TWIK related K+ channel TREK1 is an important member of the class of two-pore-domain K+ channels. It is a background K+ channel and is regulated by hormones, neurotransmitters, intracellular pH and mechanical stretch. This work shows that TREK1 is present both in mesenteric resistance arteries and in skin microvessels. It is particularly well expressed in endothelial cells. Deletion of TREK1 in mice leads to an important alteration in vasodilation of mesenteric arteries induced by acetylcholine and bradykinin. Iontophoretic delivery of acetylcholine and bradykinin in the skin of TREK1+/+ and TREK1-/- mice also shows the important role of TREK1 in cutaneous endothelium-dependent vasodilation. The vasodilator response to local pressure application is also markedly decreased in TREK1-/- mice, mimicking the decreased response to pressure observed in diabetes. Deletion of TREK1 is associated with a marked alteration in the efficacy of the G-protein-coupled receptor-associated cascade producing NO that leads to major endothelial dysfunction.     

A phospholipid sensor controls mechanogating of the K+ channel TREK-1

TREK-1 (KCNK2 or K(2P)2.1) is a mechanosensitive K(2P) channel that is opened by membrane stretch as well as cell swelling. Here, we demonstrate that membrane phospholipids, including PIP(2), control channel gating and transform TREK-1 into a leak K(+) conductance. A carboxy-terminal positively charged cluster is the phospholipid-sensing domain that interacts with the plasma membrane. This region also encompasses the proton sensor E306 that is required for activation of TREK-1 by cytosolic acidosis. Protonation of E306 drastically tightens channel-phospholipid interaction and leads to TREK-1 opening at atmospheric pressure. The TREK-1-phospholipid interaction is critical for channel mechano-, pH(i)- and voltage-dependent gating.     

A neuronal two P domain K+ channel stimulated by arachidonic acid and polyunsaturated fatty acids.

TWIK-1, TREK-1 and TASK K+ channels comprise a class of pore-forming subunits with four membrane-spanning segments and two P domains. Here we report the cloning of TRAAK, a 398 amino acid protein which is a new member of this mammalian class of K+ channels. Unlike TWIK-1, TREK-1 and TASK which are widely distributed in many different mouse tissues, TRAAK is present exclusively in brain, spinal cord and retina. Expression of TRAAK in Xenopus oocytes and COS cells induces instantaneous and non-inactivating currents that are not gated by voltage. These currents are only partially inhibited by Ba2+ at high concentrations and are insensitive to the other classical K+ channel blockers tetraethylammonium, 4-aminopyridine and Cs+. A particularly salient feature of TRAAK is that they can be stimulated by arachidonic acid (AA) and other unsaturated fatty acids but not by saturated fatty acids. These channels probably correspond to the functional class of fatty acid-stimulated K+ currents that recently were identified in native neuronal cells but have not yet been cloned. These TRAAK channels might be essential in normal physiological processes in which AA is known to play an important role, such as synaptic transmission, and also in pathophysiological processes such as brain ischemia. TRAAK channels are stimulated by the neuroprotective drug riluzole.     

Enhancement of TREK1 channel surface expression by protein-protein interaction with β-COP

TREK1 belongs to a family of two-pore-domain K⁺ (K_2P) channels and produce background currents that regulate cell excitability. In the present study, we identified a vesicle transport protein, β-COP, as an interacting partner by yeast two-hybrid screening of a human brain cDNA library with N-terminal region of TREK1 (TREK1-N) as bait. Several in vitro and in vivo binding assays confirmed the protein-protein interaction between TREK1 and β-COP. We also found that β-COP was associated with TREK1 in native condition at the PC3 cells. When RFP-β-COP was co-transfected with GFP-TREK1 into COS-7 cells, both proteins were found localized to the plasma membrane. In addition, the channel activity and surface expression of GFP-TREK1 increased dramatically by co-transfection with RFP-β-COP. Surface expression of the TREK1 channel was also clearly reduced with the addition of β-COP-specific shRNA. Collectively, these data suggest that β-COP plays a critical role in the forward transport of TREK1 channel to the plasma membrane.