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Background K2P channels are tightly regulated by different stimuli including variations of external and internal pH. pH sensitivity relies on proton-sensing residues that influence channel gating and activity. Gene inactivation in the mouse is a revealing implication of K2P channels in many physiological functions ranging from hormone secretion to central respiratory adaptation. Surprisingly, only a few phenotypic traits of these mice have yet been directly related to the pH sensitivity of K2P channels.

The versatility of neuronal electrical activity is largely conditioned by the expression of different structural and functional classes of K+ channels. More than 80 genes encoding the main K+ channel alpha subunits have been identified in the human genome. Alternative splicing, heteromultimeric assembly, post-translational modification and interaction with auxiliary regulatory subunits further increase the molecular and functional diversity of K+ channels. Mammalian two-pore domain K+ channels (K(2P)) make up one class of K+ channels along with the inward rectifiers and the voltage- and/or calcium-dependent K+ channels. Each K(2P) channel subunit is made up of four transmembrane segments and two pore-forming (P) domains, which are arranged in tandem and function as either homo- or heterodimeric channels. This novel structural arrangement is associated with unusual gating properties including "background" or "leak" K+ channel activity, in which the channels show constitutive activity at rest. In this review article, we will focus on the lipid-sensitive mechano-gated K(2P) channel TREK-1 and will emphasize on the polymodal function of this "unconventional" K+ channel.

Specialized chemo- and nutrient-sensing cells share a common electrophysiological mechanism by transducing low O(2), high CO(2) and low glucose stimuli into a compensatory cellular response: the closing of background K(+) channels encoded by the K(2P) subunits. Inhibition of the TASK K(2P) channels by extracellular acidosis leads to an increased excitability of brainstem respiratory neurons. Moreover, hypoxic down-modulation of TASK channels is implicated in the activation of glomus cells in the carotid body. Stimulation of both types of cell leads to an enhanced ventilation and to cardiocirculatory adjustments. Differential modulation of TASK channels by acidosis and high glucose alters excitability of the hypothalamic orexin neurons, which influence arousal, food seeking and breathing. These recent results shed light on the role of TASK channels in sensing physiological stimuli.

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.

We have previously shown that K+-selective TASK2 channels and swelling-activated Cl−currents are involved in a regulatory volume decrease (RVD; Barriere H, Belfodil R, Rubera I, Tauc M, Lesage F, Poujeol C, Guy N, Barhanin J, Poujeol P. J Gen Physiol 122: 177–190, 2003; Belfodil R, Barriere H, Rubera I, Tauc M, Poujeol C, Bidet M, Poujeol P. Am J Physiol Renal Physiol 284: F812–F828, 2003). The aim of this study was to determine the mechanism responsible for the activation of TASK2 channels during RVD in proximal cell lines from mouse kidney. For this purpose, the patch-clamp whole-cell technique was used to test the effect of pH and the buffering capacity of external bath on Cl−and K+currents during hypotonic shock. In the presence of a high buffer concentration (30 mM HEPES), the cells did not undergo RVD and did not develop outward K+currents (TASK2). Interestingly, the hypotonic shock reduced the cytosolic pH (pHi) and increased the external pH (pHe) in wild-type but not in cftr−/−cells. The inhibitory effect of DIDS suggests that the acidification of pHiand the alkalinization of pHeinduced by hypotonicity in wild-type cells could be due to an exit of HCO3−. In conclusion, these results indicate that Cl−influx will be the driving force for HCO3−exit through the activation of the Cl−/HCO3−exchanger. This efflux of HCO3−then alkalinizes pHe, which in turn activates TASK2 channels.

TWIK-1, a member of the two-pore domain K(+) channel family, is expressed in brain, kidney, and lung. The aim of this study was to examine the effect of loss of TWIK-1 on the renal cortical collecting duct. Ducts were isolated from wild-type and TWIK-1 knockout mice by enzyme digestion and whole-cell clamp obtained via the basolateral membrane. Current- and voltage-clamp approaches were used to examine K(+) conductances. No difference was observed between intercalated cells from wild-type or knockout ducts. In contrast, knockout principal cells were hyperpolarized compared to wild-type cells and had a reduced membrane conductance. This was a consequence of a fall in a barium-insensitive, quinidine-sensitive conductance (G (Quin)). G (Quin) demonstrated outward rectification and had a relatively low K(+) to Na(+) selectivity ratio. Loss of G (Quin) would be expected to lead to the hyperpolarization observed in knockout ducts by increasing fractional K(+) conductance and Na(+) uptake by the cell. Consistent with this hypothesis, knockout ducts had an increased diameter in comparison to wild-type ducts. These data suggest that G (Quin) contributes to the resting membrane potential in the cortical collecting duct and that a fall in G (Quin) could be an adaptive response in TWIK-1 knockout ducts.

International audience; We have recently discovered a new class of potassium channels with two pore-forming domains and four membrane-spanning domains. When heterologously expressed, these channels produce time- and voltage-independent currents that classify them as background or leak channels. TWIK (for tandem of P domains in a weak inwardly rectifying K+ channel) was the first member of this family to be cloned. Here, we describe the genomic organization of TWIK in the mouse. The coding sequence as well as the untranslated sequences are contained in three exons. The twik gene (or KCNK1) has been mapped to chromosome 8, consistent with its localization to 1q42-43 in human. The twik gene is expressed in virtually all mouse tissues. It is most abundantly expressed in brain and moderately in other organs such as kidney. The level of expression is increased in brain and kidney from neonate to adult animals, but the TWIK message is also detected during embryogenesis, as early as day 7 post conception.

International audience; Despite several decades of research, current antidepressant (AD) treatments remain of a limited efficacy justifying the need to find new drugs. These drugs have to be more efficacious, more rapid and display lesser side effects. Using rodent models, we recently identified spadin as a new antidepressant molecule that acts more quickly than classical ADs, working within 4 days to get same effects obtained with other ADs after 21 days. Spadin blocks TREK-1 K(2P) potassium channels that are considered as new targets for ADs. Deletion of the TREK-1 channel is known to increase sensitivity to pain, seizures and ischemia. Thus blocking these channels could result in deleterious side effects. In this study we showed that spadin did not interfere with other TREK-1 controlled functions such as pain, epilepsy and ischemia. We also demonstrated that spadin was unable to inhibit currents generated by TREK-2, TRAAK, TASK and TRESK four other K2P channels. More importantly, spadin did not induce cardiac dysfunctions, did not block I(Kr) and I(Ks) and did not modify the systolic pressure or cardiac pulses. After a three week treatment spadin remained an efficacious AD and did not modify the infarct size in brain following focal ischemia. Finally, we showed that kainate induced seizures and glycemia were not modified by spadin treatments. These data, together with those previously published reinforce the idea that spadin represents a good candidate for a new generation of ADs. This article is part of a Special Issue entitled 'Anxiety and Depression'.

Modulation of the standing outward current (I (SO)) by muscarinic acetylcholine (ACh) receptor (MAChR) stimulation is fundamental for the state-dependent change in activity mode of thalamocortical relay (TC) neurons. Here, we probe the contribution of MAChR subtypes, G proteins, phospholipase C (PLC), and two pore domain K(+) (K(2P)) channels to this signaling cascade. By the use of spadin and A293 as specific blockers, we identify TWIK-related K(+) (TREK)-1 channel as new targets and confirm TWIK-related acid-sensitve K(+) (TASK)-1 channels as known effectors of muscarinic signaling in TC neurons. These findings were confirmed using a high affinity blocker of TASK-3 and TREK-1, namely, tetrahexylammonium chloride. It was found that the effect of muscarinic stimulation was inhibited by M(1)AChR-(pirenzepine, MT-7) and M(3)AChR-specific (4-DAMP) antagonists, phosphoinositide-specific PLCβ (PI-PLC) inhibitors (U73122, ET-18-OCH(3)), but not the phosphatidylcholine-specific PLC (PC-PLC) blocker D609. By comparison, depleting guanosine-5'-triphosphate (GTP) in the intracellular milieu nearly completely abolished the effect of MAChR stimulation. The block of TASK and TREK channels was accompanied by a reduction of the muscarinic effect on I (SO). Current-clamp recordings revealed a membrane depolarization following MAChR stimulation, which was sufficient to switch TC neurons from burst to tonic firing under control conditions but not during block of M(1)AChR/M(3)AChR and in the absence of intracellular GTP. These findings point to a critical role of G proteins and PLC as well as TASK and TREK channels in the muscarinic modulation of thalamic activity modes.

International audience; 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.

International audience; 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.

International audience; Tandem of P domains in a weak inwardly rectifying K(+) channel 1 (TWIK1) is a K(+) channel that produces unusually low levels of current. Replacement of lysine 274 by a glutamic acid (K274E) is associated with stronger currents. This mutation would prevent conjugation of a small ubiquitin modifier peptide to Lys-274, a mechanism proposed to be responsible for channel silencing. However, we found no biochemical evidence of TWIK1 sumoylation, and we showed that the conservative change K274R did not increase current, suggesting that K274E modifies TWIK1 gating through a charge effect. Now we rule out an eventual effect of K274E on TWIK1 trafficking, and we provide convincing evidence that TWIK1 silencing results from its rapid retrieval from the cell surface. TWIK1 is internalized via a dynamin-dependent mechanism and addressed to the recycling endosomal compartment. Mutation of a diisoleucine repeat located in its cytoplasmic C terminus (I293A,I294A) stabilizes TWIK1 at the plasma membrane, resulting in robust currents. The effects of I293A,I294A on channel trafficking and of K274E on channel activity are cumulative, promoting even more currents. Activation of serotoninergic receptor 5-HT(1)R or adrenoreceptor alpha2A-AR stimulates TWIK1 but has no effect on TWIK1I293A,I294A, suggesting that G(i) protein activation is a physiological signal for increasing the number of active channels at the plasma membrane.

International audience; 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.

Glucose sensing by hypothalamic neurons triggers adaptive metabolic and behavioral responses. In orexin neurons, extracellular glucose activates a leak K+current promoting electrical activity inhibition. Sensitivity to external acidification and halothane, and resistance to ruthenium red designated the tandem-pore K+(K2P) channel subunit TASK3 as part of the glucose-induced channel. Here, we show that glucose inhibition and its pH sensitivity persist in mice lacking TASK3 or TASK1, or both subunits. We also tested the implication of another class of K2Pchannels activated by halothane. In the corresponding TREK1/2/TRAAK triple knock-out mice, glucose inhibition persisted in hypothalamic neurons ruling out a major contribution of these subunits to the glucose-activated K+conductance. Finally, block of this glucose-induced hyperpolarizing current by low Ba2+concentrations was consistent with the conclusion that K2Pchannels are not required for glucosensing in hypothalamic neurons.

Apoptotic volume decrease (AVD) is prerequisite to apoptotic events that lead to cell death. In a previous study, we demonstrated in kidney proximal cells that the TASK2 channel was involved in the K+ efflux that occurred during regulatory volume decrease. The aim of the present study was to determine the role of the TASK2 channel in the regulation of AVD and apoptosis phenomenon. For this purpose renal cells were immortalized from primary cultures of proximal convoluted tubules (PCT) from wild type and TASK2 knock-out mice (task2-/-). Apoptosis was induced by staurosporine, cyclosporin A, or tumor necrosis factor alpha. Cell volume, K+ conductance, caspase-3, and intracellular reactive oxygen species (ROS) levels were monitored during AVD. In wild type PCT cells the K+ conductance activated during AVD exhibited characteristics of TASK2 currents. In task2-/- PCT cells, AVD and caspase activation were reduced by 59%. Whole cell recordings indicated that large conductance calcium-activated K+ currents inhibited by iberiotoxin (BK channels) partially compensated for the deletion of TASK2 K+ currents in the task2-/- PCT cells. This result explained the residual AVD measured in these cells. In both cell lines, apoptosis was mediated via intracellular ROS increase. Moreover AVD, K+ conductances, and caspase-3 were strongly impaired by ROS scavenger N-acetylcysteine. In conclusion, the main K+ channels involved in staurosporine, cyclosporin A, and tumor necrosis factor-alpha-induced AVD are TASK2 K+ channels in proximal wild type cells and iberiotoxin-sensitive BK channels in proximal task2-/- cells. Both K+ channels could be activated by ROS production.

TREK1 is the most extensively studied of the mammalian two-pore-forming K+channels and is widely expressed in the brain. Honore reviews the functional properties of TREK1 and describes recent results indicating its important roles in CNS function and disease. Two-pore-domain K+ (K2P) 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, K2P 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 K2P channel, has a key role in the cellular mechanisms of neuroprotection, anaesthesia, pain and depression.

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 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.

The acid- and volume-sensitive TASK2 K + channel is strongly expressed in renal proximal tubules and papillary collecting ducts. This study was aimed at investigating the role of TASK2 in renal bicarbonate reabsorption by using the task2 –/– mouse as a model. After backcross to C57BL6, task2 –/– mice showed an increased perinatal mortality and, in adulthood, a reduced body weight and arterial blood pressure. Patch-clamp experiments on proximal tubular cells indicated that TASK2 was activated during \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document} transport. In control inulin clearance measurements, task2 –/– mice showed normal NaCl and water excretion. During i.v. NaHCO 3 perfusion, however, renal Na + and water reabsorption capacity was reduced in –/– animals. In conscious task2 –/– mice, blood pH, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document} concentration, and systemic base excess were reduced but urinary pH and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document} were increased. These data suggest that task2 –/– mice exhibit metabolic acidosis caused by renal loss of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document} . Both in vitro and in vivo results demonstrate the specific coupling of TASK2 activity to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document} transport through external alkalinization. The consequences of the task2 gene inactivation in mice are reminiscent of the clinical manifestations seen in human proximal renal tubular acidosis syndrome.

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Several papers reported the role of TASK2 channels in cell volume regulation and regulatory volume decrease (RVD). To check the possibility that the TASK2 channel modulates the RVD process in kidney, we performed primary cultures of proximal convoluted tubules (PCT) and distal convoluted tubules (DCT) from wild-type and TASK2 knockout (KO) mice. In KO mice, the TASK2 coding sequence was in part replaced by the lac-Z gene. This allows for the precise localization of TASK2 in kidney sections using β-galactosidase staining. TASK2 was only localized in PCT cells. K+ currents were analyzed by the whole-cell clamp technique with 125 mM K-gluconate in the pipette and 140 mM Na-gluconate in the bath. In PCT cells from wild-type mice, hypotonicity induced swelling-activated K+ currents insensitive to 1 mM tetraethylammonium, 10 nM charybdotoxin, and 10 μM 293B, but blocked by 500 μM quinidine and 10 μM clofilium. These currents were increased in alkaline pH and decreased in acidic pH. In PCT cells from TASK2 KO, swelling-activated K+ currents were completely impaired. In conclusion, the TASK2 channel is expressed in kidney proximal cells and could be the swelling-activated K+ channel responsible for the cell volume regulation process during osmolyte absorptions in the proximal tubules.