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Mini review article, trek channels in mechanotransduction: a focus on the cardiovascular system.

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  • 1 Laboratory of Neuroscience, CINBIO, University of Vigo, Vigo, Spain
  • 2 Laboratory of Neuroscience, Galicia Sur Health Research Institute (IISGS), Vigo, Spain

Mechano-electric feedback is one of the most important subsystems operating in the cardiovascular system, but the underlying molecular mechanism remains rather unknown. Several proteins have been proposed to explain the molecular mechanism of mechano-transduction. Transient receptor potential (TRP) and Piezo channels appear to be the most important candidates to constitute the molecular mechanism behind of the inward current in response to a mechanical stimulus. However, the inhibitory/regulatory processes involving potassium channels that operate on the cardiac system are less well known. TWIK-Related potassium (TREK) channels have emerged as strong candidates due to their capacity for the regulation of the flow of potassium in response to mechanical stimuli. Current data strongly suggest that TREK channels play a role as mechano-transducers in different components of the cardiovascular system, not only at central (heart) but also at peripheral (vascular) level. In this context, this review summarizes and highlights the main existing evidence connecting this important subfamily of potassium channels with the cardiac mechano-transduction process, discussing molecular and biophysical aspects of such a connection.

Introduction

While great strides have been made in understanding the molecular mechanisms of touch ( 1 – 3 ), cardiovascular mechanotransduction remains a complex and enigmatic process that is not yet fully understood. This review highlights the critical importance of mechano-sensitivity in maintaining proper cardiovascular function, and the challenges associated with elucidating its underlying mechanisms.

Multiple elements can sense the different mechanical forces affecting the cellular body, for example, elements of the extracellular matrix such as integrins, elements of the cytoskeleton, G-protein-coupled receptors or different ion channels ( 4 ). This review is focused on TREK channels, a subfamily of the two-pore-domain potassium (K2P) channels encoded by genes named KCNK, which are capable of detecting mechanical stimuli altering their opening and closing kinetics. These mechano-sensitive ion channels are membrane proteins that allow cells to respond and adapt to physical forces ( 5 ), playing a crucial role in mechano-transduction processes ( 6 , 7 ). Mechanical forces are fundamental in cardiovascular biology, however, the mechanisms that support this physiological process have yet to be elucidated. In this sense, the link between electrical stimulation and mechanical contractions is widely established, and the mechanism by which an electrical stimulus produces muscle contraction is widely accepted ( 8 ). On the contrary, the process by which mechanical forces can influence the electrical properties (mechano-electric feedback) of the cardiovascular cells is still poorly understood ( 9 , 10 ). Mechano-electric feedback is one of the most important subsystems that operate within the cardiovascular system ( 11 ), it can be defined as the process by which mechanical stimuli are converted into electrical signals and plays a key role in the functioning of cardiovascular homeostasis ( 2 , 12 , 13 ). In the heart, different mechano-sensitive structures have been identified, with myocytes being the most relevant ( 14 ), while at peripheral level, smooth muscle fibers (present in veins and arteries) are the main elements.

Roughly speaking (without considering chloride channels) ion channels can be separated into two categories. When activated, certain channels, regardless of their selectivity, can either depolarize or hyperpolarize the cell membrane. Applying this idea to the mechanobiology context, these families are known as depolarizing non-selective cationic channels and hyperpolarizing potassium selective channels. In this context, TRP and Piezo channels are part of the first category. They are a nonselective Na + , Ca 2+ (among others) conductors. TRP channels are usually considered as dominant elements in mechano-sensitivity ( 15 ) and they are part of the mechanosensitive non-selective cardiac current family ( 16 – 19 ). However, they have been shown to be insensitive to membrane stretch ( 20 ) and are not considered primary mechanotransducers ( 21 ). Piezo channels are also considered to be transducers of mechanical stimuli and are widely expressed in the cardiovascular system ( 22 ). and they could work like baroreceptors ( 23 ) even during cardiac development ( 24 ). The second category is made up of TREK channels (TREK-1, TREK-2 and TRAAK) and they are probably the only mechanically-gated potassium channels playing an important role in the process of mechanical transduction ( 25 ). Given their widespread expression throughout the cardiovascular system ( 26 ), these channels are emerging as potential contributors to cardiac mechano-electrical feedback and mechano-associated pathologies. Thus, we reviewed the evidence supporting this possibility.

Mechano-regulation of TREK channels

MS ion channels can be activated by two different mechanisms. The mechanism called tethering needs several cytoskeletal proteins as scaffold proteins to activate the mechano-sensor, this is the case of TRP channels ( 27 ). The other mechanism implies the activation of the channels by the tension in the bilayer itself, without the need for other cellular structures, in this group are TREK channels, see ( 28 ) for controversial.

The molecular mechanism underlying the sensitivity of TREKs to membrane deformation induced by mechanical forces has been extensively investigated, stating that cellular integrity is not essential for mechanical channel activation ( 7 , 29 ), indeed TREK channels are regulated by a mechanism called “selective filter” ( 30 , 31 ) (see Figure 1 ). This consists in a change of conformation in the narrow zone of the pore that regulates the flow of ions, similar to the C-type blockade studied in voltage-dependent potassium channels such as inward rectifier potassium (Kir) channels ( 34 , 35 ). It has been shown that with the pore closed, the helical protein structures would not interfere with the passage of ions ( 36 ), contrary to what occurs in most potassium channels ( 37 – 39 ). Although this selective filter mechanism is widely accepted ( 36 , 40 , 41 ), there are still many open questions ( 42 ) and other gating mechanisms could be present and activated depending on the stimulus ( 43 , 44 ).

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Figure 1 . ( A ) view of the TREK structure in conventional configuration. Red dotted lines indicate the selective filter. ( B ) Topological model proposed for K2P channels, each subunit has two pore forming domain (P loops) and four transmembrane domains (denoted M1-M4). ( C ) Representative response to a mechanical stimuli of TREK showing that it has minimal desensitization in the inside-out configuration. Adapted from ( 32 , 33 ).

Two states, called “Up” and “Down”, have been described for TREK channels and although in both states the pore is open, it has been suggested that only the Up state can be considered conductive and that in the Down one the conductivity is residual ( 36 , 45 ). It has been shown that TREK channels can sense mechanical forces directly through the bilayer and it has been demonstrated that TREK channels have located the mechano-gate in the selectivity filter ( 46 , 47 ). Thus when the membrane is stretched there is a conformational change in the channel's selective filter that favors the entry into the Up state, more conductive when compared with the Down state, notwithstanding this theory has generated some controversy ( 48 ). As mentioned above, two mechanisms enable channels to perceive mechanical forces: direct (Piezo and TREK channels) and indirect (TRP channels). In addition to experimental conditions, while the mechanism underlying the mechanosensitivity of TRP channels is well-established ( 17 , 21 , 27 ), it is apparent that membrane deformation can also bring about mechanical changes in different cytoskeleton proteins, which can contribute to the feedback of tension in the bilayer. Therefore, these mechanisms may not be entirely separate and could potentially complement each other under physiological conditions. For instance, some studies have demonstrated that Piezos are solely responsive to shear stress (frictional force) ( 49 , 50 ), but not to stretch. Furthermore, Piezos can interact with other MS ion channels like TRP channels ( 51 , 52 ) which may conceal their behaviour under certain experimental conditions, leading to further variability.

Role of TREK channels in the cardiovascular system

As we have recently reviewed, TREK-1 is the most expressed TREK channel in heart, both in neuronal and non-neuronal tissue, including the sinoatrial node, cardiomyocytes and purkinje fibers ( 26 ). Several studies have shown how TREK-1 is extensively expressed in heart using molecular techniques, including qRT-PCR and WB ( 53 , 54 ). Confocal imaging also showed TREK-1 arranged in longitudinal stripes at the surface of the cardiomyocytes in rats ( 55 ). Consistently, whole-cell patch-clamp electrophysiological recordings have shown clearly the presence of a potassium current conducted by TREK channels in cardiac cells in both murine and human ( 56 – 58 ). In summary, the presence of TREK-1 in the heart tissue of various mammals including rodents and humans has been widely demonstrated ( Table 1 ). However, the other two members of the TREK subfamily (TREK-2 and TRAAK) have been poorly localized ( 32 , 64 – 68 ).

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Table 1 . Non-systematic but representative summary of the presence of TREK channels in the cardiovascular system.

From a functional point of view, TREK-1 plays a critical role in countering the depolarizing effect of mechano-activated cationic currents, contributing to stimulation-activated central (heart) feedback mechanics in the cardiovascular system ( 69 ). TREK-1 channels also have a potential role in regulating the normal activity of sinoatrial node-hosted pacemakers by preventing the occurrence of ventricular extrasystoles ( 55 , 70 ). Inhibition of TREK-1 channels via PKA during sympathetic stimulation may decrease transmural dispersion of repolarization and prevent the occurrence of arrhythmias ( 58 ), indicating that TREK-1 may have an essential function in the cardiac conduction system ( 71 ). In cardiomyocytes, the refractory period is critical in preventing premature excitation and arrhythmias. The duration or amplitude of the action potential depends on a delicate balance between inward-potassium and outward currents during the action potential plateau. TREK-1, as well as BKCa (large conductance K + channel, both voltage and calcium-gated) or KATP (ATP-sensitive potassium) channels, are the main candidates encoding the cardiac stretch-activated potassium current ( 72 ). However, in contrast to TREK-1, in the human heart, BKCa and KATP channels are poorly expressed, making TREK-1 the primary candidate for encoding cardiac stretch-activated potassium currents in different species, with a single channel conductance of approximately 100 pS ( 9 , 32 , 58 ). These results suggest a clear role for TREK-1 in the repolarization phase of the cardiac action potential ( Figure 2 ).

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Figure 2 . ( A ) representative diagram of a heart showing blood flow (red line) and the different regions of interest: right atrium (RA); superior vena cava (SVC) and inferior vena cava (IVC); right ventricle (RV); pulmonary artery (PA); left atrium (LA); left ventricle (LV) and aorta (Ao). The TREK channels have been schematically represented as 1: sinoatrial node, 2: conduction system (Purkije fibres) and 3: muscle cells. ( B ) Shape of a typical action potential (top) and the conductances that generate it (bottom). Indicating the area where TREK channels are most likely to be involved (green shaded area). ( C ) Effect of TREK channel removal on intrathecal calcium [Ca 2+ ]i activity in mouse cardiomyocytes. Adapted from ( 56 , 73 ).

Finally, the variable distribution of TREK-1 in both endothelial and smooth muscle cells across different regions of the heart could facilitate precise regulation of the depolarization wave that initiates cardiac contraction ( 74 ). For example, TREK-1 is less present in myocytes of the epicardium of adult rats than in endocardial cells ( 12 ).

At the same time, TREK channels play an important role in cardiovascular diseases ( 47 ), so that its experimental withdrawal is expected to be pro-arrhythmic ( 75 ). TREK-1 has been associated with reduced right atrial channel expression in Atrial fibrillation (AF) models ( 46 ). AF is the most common cardiac arrhythmia and results from shortening of atrial effective refractory periods and from a localized deceleration of intra-atrial conduction ( 76 – 78 ). In this context, we recently have shown that verapamil (a class IV antiarrhythmic drug used in pathological conditions such as chronic angina pectoris, cardiac arrhythmias or hypertension) reduces the TREK-1 activity ( 79 ). TREK-1 channels may have a role in other pathophysiological situations, during ischemia, when purinergic agonists such as ATP cause the release of arachidonic acid (AA) ( 80 ), which lowers intracellular pH, the change of pH/AA can be detected by TREK-1 ( 63 ) and could contribute to electrophysiological disturbances in the cardiac mechano-electric feedback ( 81 ). TREK-1, plays a protective role against ischaemia-induced neuronal damage and has been shown to play a critical role in cardiac injury and during remodelling after myocardial infarction. Moreover, in TREK-1 KO animals, TREK-1 increases infarct size induced in experimental models, leading to greater systolic dysfunction than its wild-type counterpart, so that activation of TREK-1 may be an effective strategy to provide cardioprotection against ischaemia-induced damage. In addition, a study on the role of TREK-1 in the control of cardiac excitability found that TREK-1 is essential for normal sinoatrial node cell excitability and serves as a potential target for selectively regulating sinoatrial node cell function ( 56 , 57 ).

Also at peripheral level, the expression pattern of TREK channels in the vascular system has been widely demonstrated. For example, TREK-1, TREK-2 and TRAAK have been detected in various vascular structures such as the pulmonary and femoral artery and the cerebral arteries in both murids and humans, suggesting a putative role for these channels in the vascular system, particularly for TREK-1 ( 81 ). Through WB and RT-PCR, TREK mRNA was detected in rat mesenteric and pulmonary arteries ( 62 ) and TREK-1 has been suggested to influence mechanically induced endothelial signalling by modulating nitric oxide production ( 69 ). In heterologous systems it has been shown that the presence of treprostinil (a tricyclic benzidine analogue of PGI 2 used for treatment of pulmonary arterial hypertension) was able to inhibit TREK-1 and TREK-2, supporting the idea that TREK-1 could contribute to the cardiac mechano-electric feedback with a hyperpolarizing current in response to mechanical forces in the vascular system.

Concluding remarks and perspectives

Two types of currents activated by mechanical stimulation operate in the heart, on the one hand a depolarizing non-selective cationic current and on the other hand a hyperpolarizing outward current mainly transported by potassium. Despite originating some controversy ( 20 ), the family of depolarizing non-selective cationic current is mainly composed of TRP and Piezo channels ( 19 , 20 ), and responds with a wide depolarizing current that occurs mainly in the sarcolemma. Stretch-activated potassium currents are primarily driven by TREK channels, which play an important role in cardiac mechano-electrical feedback, both at the cellular level (e.g., presence in principal cells such as pacemakers and cardiomyocytes) and at the system level through their involvement in the regulation of heartbeat force and rate ( 6 , 19 , 82 ). Overall, there is now some evidence for the ability of TREK channels to control the electrical activity of the heart through central mechano-electrical feedback ( 19 ).. It has been proposed that the main function of TREK-1 is to counteract the depolarising effect induced by currents activated by mechanical stimuli, thus contributing to central mechano-electric feedback in the cardiovascular system ( 55 , 69 ) and controlling, at least in part, the early repolarisation phase and action potential transfer through the ventricular conduction system. Finally, as mentioned above, it appears that TREK channels, especially TREK-1, may play a role in nodal pacemaker activity.

The strong presence of TREK-1 could also indicate a possible role in the mechanical control of the electrical activity of the vascular periphery. However, other players must be considered in the mechano-electric feedback process. Recent work has investigated the role of Piezo 1 channels in the development of cardiac hypertrophy, showing how activation of calcium/calpain signalling through the Piezo 1 pathway contributes to the development of cardiac hypertrophy in murine models. Furthermore, Piezo 1 is a cardiac mechano-sensor that is activated in response to cardiac overload in adult animals, which in turn initiates the myocardial hypertrophic response. On the other hand, it has also been shown that Piezo 1 activation in response to mechanical stimuli triggers chemical signals that contribute to the physiological response of the heart to mechanical stress ( 83 – 85 ). These findings undoubtedly support the relevant role that Piezo channels may play in both mechano-electric feedback and cardiac pathophysiology.

In summary, TREK channels are involved in the regulation of mechanical forces both centrally and peripherally in the cardiovascular system. It should be noted that there may be other currents at play that could contribute to or even counteract TREK activity. More important, recent data have shown that antiarrhythmic drugs can interact with mechanically-gated TREK channels. There is enough evidence supporting the hypothesis that potassium outward currents driven by TREK channels play an important role not only in the normal functioning of the cardiovascular system, where its mechanical sensitivity plays a central aspect, but also in some relevant pathologies such as AF and other cardiac conditions. The expression of TREK-1 channels in the ventricle exhibits regional heterogeneity, similar to that observed in mechano-electrical feedback under physiological conditions. Consequently, this regional variability in TREK-1 channel expression has the potential to modulate mechano-electrical feedback, resulting in altered repolarization of the action potential and consequent arrhythmogenic effects ( 86 ). Although in this review we have focused on the possible role of TREK channels in cardiac mechano-electric feedback as well as their possible role in the phytopathology of the heart, there is no doubt that other MS ion channels such as Piezo channels must be taken into account in the explanation of the molecular mechanism underlying cardiac mechano-electric feedback.

Presently, a significant limitation exists in the investigation of the possible role of TREK channels in the mechano-electric feedback process due to the lack of identified specific TREK channel blockers. Nonetheless, a considerable body of evidence supports the proposition that these channels are unequivocally responsible for potassium current in response to mechanical stimuli, and given their abundant expression in the cardiovascular system, it is highly probable that they are fundamental in the feedback process. Furthermore, as previously remarked, there is clear evidence indicating that TREK channels have a relevant role in cardiac pathophysiology. However, despite the extensive evidence of TREK channel presence in various regions of the cardiovascular system, including sympathetic innervation, it is presently unknown if these channels are also present in the intracardiac ganglion, which is responsible for parasympathetic control of cardiac activity. Moreover, as the pharmacology of TREK channel usage progresses, it is conceivable that more appropriate experimental designs can be employed to elucidate the relationship between TREK channels and mechano-electric feedback more clearly.

Author contributions

Conceptualization, SH-P and JAL; validation, JAL; investigation, SH-P and JAL; resources, JAL; writing of original draft, SH-P and JAL; review and editing of manuscript, SH-P and JAL; supervision, JAL; funding acquisition, JAL All authors have read and agreed to the published version of the manuscript.

This research was funded by the Spanish government M.I.C.I.U. PID2019-109425GB-I00. All the funding was awarded to J. Antonio Lamas.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher's note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

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Keywords: TREK, mechanobiology, cardiovascular system, heart, mechano-feedback

Citation: Herrera-Pérez S and Lamas JA (2023) TREK channels in Mechanotransduction: a Focus on the Cardiovascular System. Front. Cardiovasc. Med. 10:1180242. doi: 10.3389/fcvm.2023.1180242

Received: 6 March 2023; Accepted: 26 April 2023; Published: 23 May 2023.

Reviewed by:

© 2023 Herrera-Pérez and Lamas. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) . The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Salvador Herrera-Pérez [email protected] José Antonio Lamas [email protected]

This article is part of the Research Topic

New Discoveries on Calcium Handling in Cardiovascular Pathology

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  • Published: 15 December 2020

K 2P 2.1 (TREK-1) potassium channel activation protects against hyperoxia-induced lung injury

  • Tatiana Zyrianova 1 ,
  • Benjamin Lopez 1 ,
  • Riccardo Olcese 2 , 3 ,
  • John Belperio 4 ,
  • Christopher M. Waters 5 ,
  • Leanne Wong 1 ,
  • Victoria Nguyen 1 ,
  • Sriharsha Talapaneni 1 &
  • Andreas Schwingshackl 1  

Scientific Reports volume  10 , Article number:  22011 ( 2020 ) Cite this article

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  • Mechanisms of disease

No targeted therapies exist to counteract Hyperoxia (HO)-induced Acute Lung Injury (HALI). We previously found that HO downregulates alveolar K 2P 2.1 (TREK-1) K + channels, which results in worsening lung injury. This decrease in TREK-1 levels leaves a subset of channels amendable to pharmacological intervention. Therefore, we hypothesized that TREK-1 activation protects against HALI. We treated HO-exposed mice and primary alveolar epithelial cells (AECs) with the novel TREK-1 activators ML335 and BL1249, and quantified physiological, histological, and biochemical lung injury markers. We determined the effects of these drugs on epithelial TREK-1 currents, plasma membrane potential (Em), and intracellular Ca 2+ (iCa) concentrations using fluorometric assays, and blocked voltage-gated Ca 2+ channels (Ca V ) as a downstream mechanism of cytokine secretion. Once-daily, intra-tracheal injections of HO-exposed mice with ML335 or BL1249 improved lung compliance, histological lung injury scores, broncho-alveolar lavage protein levels and cell counts, and IL-6 and IP-10 concentrations. TREK-1 activation also decreased IL-6, IP-10, and CCL-2 secretion from primary AECs. Mechanistically, ML335 and BL1249 induced TREK-1 currents in AECs, counteracted HO-induced cell depolarization, and lowered iCa 2+ concentrations. In addition, CCL-2 secretion was decreased after L-type Ca V inhibition. Therefore, Em stabilization with TREK-1 activators may represent a novel approach to counteract HALI.

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Introduction

Oxygen supplementation (hyperoxia; HO) is the most frequently administered therapy in hospitalized patients and the mainstay of treatment for hypoxic respiratory failure, regardless of its etiology 1 . Clinically, supra-physiologic levels of oxygen tension are often tolerated and perceived as a safety net against hypoxemia 2 . As a result, in the US each year approximately 800,000 patients are exposed to HO therapy at a cost of $1.8 billion to the health care budget 3 . Importantly, the degree and duration of HO exposure positively correlate with patient morbidity and mortality rates 4 , 5 , 6 .

Although oxygen therapy can be a life-saving intervention, ample experimental and clinical evidence demonstrates that excessive levels of oxygen supplementation can also initiate and accelerate existing lung injury (HO-induced acute lung injury; HALI). Animal models of HALI have been particularly helpful in investigating the underlying mechanisms 7 , and studies in healthy adults showed that HO exposure causes tracheobronchitis and changes in vital capacity, diffusing capacity, and lung permeability within only six hours, and with a severity that is proportional to the degree of HO exposure 8 , 9 , 10 , 11 , 12 , 13 . Experimentally, a similar dose- and time-dependent inflammatory response to HO can be reproduced in animal models of HALI 14 , 15 , 16 , demonstrating close similarities in lung injury phenotypes between animals and humans 15 , 17 , 18 , 19 , 20 . From these studies we learned that the molecular mechanisms underlying HALI are complex and include extensive alterations in inflammatory cytokine secretion 14 , 21 , 22 . Both alveolar epithelial and endothelial cells are injured by HO, but the epithelial layer is the first line of defense against inhaled HO 23 , 24 , 25 .

Currently, minimizing the duration and amount of HO exposure of patients (“permissive hypoxemia”) represents the only clinical approach to limit HALI, and so far no molecular targets have been identified that translate into improved patient outcomes 26 . However, minimizing HO exposure is complicated by the lack of consensus in defining the lower limits of permissive hypoxemia, which would allow us to clinically differentiate beneficial from injurious levels of HO therapy 27 , 28 .

In our search for new molecular targets against HALI, we recently identified epithelial K 2P 2.1 (TREK-1) K + channels as important regulators in the development and progression of HALI 29 , 30 , 31 , 32 . TREK-1 channels belong to the family of 2-pore domain (K2P) K + channels, which are generally known for their unusual gating properties leading to so-called “leak K + currents” that stabilize the resting plasma membrane potential (Em) 33 , 34 . In general, K2P channels, including TREK-1, are widely expressed in body tissues 35 , 36 , 37 , 38 , 39 , 40 , 41 , but their role in the lung remains largely unknown. Using in vivo and in vitro models of HALI, we previously discovered that HO exposure decreases the expression of TREK-1 channels in mouse lungs and alveolar epithelial cells, and accelerates alveolar inflammation and barrier dysfunction 30 , 42 , 43 . These findings sparked the hypothesis that despite HO-mediated TREK-1 downregulation, pharmacological activation of the remaining subset of TREK-1 channels can protect against HALI. To test this hypothesis, in this study we explored the potential protective effects and underlying mechanisms of two novel TREK-1 activating compounds (ML335, BL1249) using in vivo and in vitro models of HALI.

Intra-tracheal administration of TREK-1 activating compounds protects mice against HO-induced acute lung injury (HALI)

Building on our previous findings that HO downregulates TREK-1 expression 42 , we determined whether pharmacological activation of the remainder subset of TREK-1 channels can counteract the injurious effects of HO on mouse lungs. We treated WT mice with once-daily intra-tracheal ( i.t. ) injections of the TREK-1-activating compounds ML335 or BL1249 44 , 45 , or an equimolar drug vehicle control, for a total of 3 injections over the 72-h HO or room air (RA) exposure period. Histological analysis (Fig.  1 A) and blinded Lung Injury Scoring (LIS; Fig.  1 B) of H&E-stained mouse lung sections revealed that under RA conditions administration of ML335 and BL1249 had no damaging effect on lung histology. As expected, exposure of mice to HO caused significant inflammatory changes (panel d), as also evidenced by an increase in LIS. Importantly, once-daily i.t. injections of ML335 or BL1249 during HO exposure substantially reduced these HO-induced injurious effects (Fig. 1 A panels e and f, and B). Similarly, analysis of physiological parameters of lung injury also revealed protective effects of TREK-1 activation in HO-exposed mice, as evidenced by improvements in quasi-static lung compliance (Fig.  1 C), and a reduction in BAL fluid protein levels and total cell counts (Fig.  1 D, E). These data suggest that pharmacological activation of TREK-1 channels can counteract HALI in an experimental mouse model.

figure 1

TREK-1 activation with the novel compounds ML335 and BL1249 protects form HO-induced lung injury: ( A ) Representative images of H&E-stained lung sections of WT mice exposed to either room air (panels a-c) or HO (panels d-f) for 72 h. All mice received once-daily, intra-tracheal ( i.t. ) injections of ML335, BL1249, or a vehicle control (no drugs) via brief endotracheal intubation. HO exposure caused significant lung injury (panel d), which was ameliorated by concomitant treatment with ML335 or BL1249 (panels e, f). ( B ) Summary of cumulative Lung Injury Scores of n = 5 independent experiments. ( C – E ) The HO-induced decrease in semi-static lung compliance, and increase in BAL fluid total protein and cell count were counteracted by ML335 and BL1249. Data are represented as Box-Whisker plots with medians, 1st and 3rd quartiles, and max and min values; n = 5–9; ^compared to mice injected with a vehicle control and exposed to room air (no drugs), *compared to HO exposed mice; p  ≤ 0.05.

TREK-1 activation decreases inflammatory cytokine concentrations in the BAL fluid of HO exposed mice

To investigate whether the TREK-1-mediated improvements in histological and physiological lung injury parameters are associated with a reduction in inflammatory cytokine concentrations in HO exposed mice, we measured IL-6, IP-10, CCL-2, TNF-α, MIP-1α and IL-10 concentrations in BAL fluid (Fig.  2 ). We found that under room air conditions once-daily i.t. injections of ML335 or BL1249 had no effect on baseline cytokine secretion. Exposure of mice to 72 h HO resulted in an increase in IL-6, IP-10, CCL-2, TNF-α and IL-10 concentrations in the BAL fluid. Importantly, once-daily i.t. injections with ML335 or BL1249 during the 72 h of HO exposure decreased HO-induced IL-6 and IP-10 levels in the BAL fluid, but not CCL-2, TNF-α or IL-10. MIP-1α concentrations were neither affected by HO exposure nor treatment of mice with the TREK-1 activating compounds.

figure 2

Effects of ML335 and BL1249 on BAL fluid cytokine concentrations ( A – F ): HO exposure increased IL-6, IP-10, CCL-2, TNF-α, and IL-10 concentrations, but not MIP-1α. Once-daily i.t . treatment with ML335 or BL1249 decreased HO-induced IL-6 and IP-10 levels, but not CCL-2, TNF-α or IL-10. Data are represented as Box-Whisker plots with medians, 1st and 3rd quartiles, and max and min values; n = 5–9; ^compared to mice exposed to room air and treated with a vehicle control (no drugs), *compared to HO exposed mice; p  ≤ 0.05.

TREK-1 activity regulates inflammatory cytokine secretion from primary mouse AT2 cells

To evaluate whether the protective effects of TREK-1 activation observed in vivo were due to TREK-1-mediated attenuation of inflammatory cytokine secretion from alveolar epithelial cells, we exposed freshly-isolated mouse AT2 cells to HO or RA in the presence or absence of ML335 or BL1249, and quantified inflammatory cytokine secretion in culture supernatants (Fig.  3 ). We chose the shorter (24-h) HO exposure period (compared to 72 h in vivo) for freshly isolated AT2 cells, since in this cell type 72 h of HO exposure resulted in > 60% AT2 cell death (data not shown). Importantly, real-time PCR experiments and immunofluorescence (IF) microscopy imaging confirmed HO-induced TREK-1 downregulation after 24 h in this cell-type (Supplementary Fig.  1 A,B). Similar to our findings in the BAL fluid, HO exposure increased secretion of IL-6 and CCL-2 from freshly isolated mouse AT2 cells, and this effect was counteracted by concomitant treatment of cells with the TREK-1 activators ML335 or BL1249. Furthermore, HO exposure did not induce MIP-1α secretion from AT2 cells, similar to our findings in the BAL fluid. In contrast to our findings in the BAL fluid, HO exposure did not induce IP-10, TNF-α or IL-10 secretion from primary AT2 cells, and treatment with ML335 or BL1249 had no effect on the secretion of these cytokines at baseline or after HO exposure (Fig.  3 ).

figure 3

TREK-1 activation with ML335 and BL1249 regulates cytokine secretion from primary mouse AT2 cells: HO exposure increased IL-6 and CCL-2 secretion, which were inhibited by concomitant treatment with ML335 or BL1249 ( A , C ). In contrast, IP-10, TNF-α, MIP-1α and IL-10 levels were not affected by TREK-1 activation in room air- or HO-exposed AT2 cells ( B , D , E , F ). Data are represented as Box-Whisker plots with medians, 1st and 3rd quartiles, and max and min values; n = 5–9; ^compared to cells treated with a vehicle control and exposed to room air (no drugs), *compared to HO exposed cells; p  ≤ 0.05.

TREK-1 activity regulates inflammatory cytokine secretion from primary human alveolar epithelial cells (HAECs)

To determine whether the TREK-1-mediated protective effects observed in mice and mouse AT2 cells can be reproduced in primary human alveolar epithelial cells (HAEC), we exposed HAEC to 72 h HO in the presence or absence of the TREK-1 activators ML335 or BL1249 (Fig.  4 ). Initial dose–response experiments revealed that BL1249 and ML335 are not cytotoxic at the doses used in this study (Supplementary Fig.  2 ). In contrast to primary mouse AT2 cells, viability of HAECs after 72 h HO exposure remained > 70% (data not shown), and this exposure period closely mimicked our in vivo model. Similar to our findings in primary mouse AT2 cells, HO exposure increased secretion of IL-6 and CCL-2 from HAECs, but did not induce TNF-α or MIP-1α secretion. Of note, overall concentrations of TNF-α and MIP-1α were quite low in these cells. In contrast to primary mouse AT2 cells but similar to BAL fluid, HO also increased secretion of IP-10 and IL-10 from HAECs. Importantly, treatment of HAECs with the TREK-1 activators ML335 or BL1249 inhibited the HO-induced secretion of IL-6, IP-10, CCL-2, and IL-10.

figure 4

TREK-1 activation with ML335 and BL1249 regulates cytokine secretion from primary human alveolar epithelial cells (HAEC): HO exposure increased secretion of IL-6, IP-10, CCL-2 and IL-10, and this effect was counteracted by ML335 or BL1249 ( A , B , C , F ). In contrast, TNF-α and MIP-1α levels were not affected by TREK-1 activation in room air- or HO-exposed HAECs ( D , E ). Data are represented as Box-Whisker plots with medians, 1st and 3rd quartiles, and max and min values; n = 4–8; ^compared to cells treated with a vehicle control and exposed to room air (no drugs), *compared to HO exposed cells; p  ≤ 0.05.

Altogether, these findings suggest that TREK-1 activation regulates HO-induced inflammatory cytokine secretion both in vivo and in vitro, but differences can be observed between overall cytokine concentrations in BAL fluid and cultured primary epithelial cells.

ML335 and BL1249 activate TREK-1 currents in primary AT2 cells

Although the specificity of ML335 and BL1249 for TREK-1 channels has previously been validated in heterologous expression systems 44 , 46 , 47 , the effectiveness of these compounds has never been demonstrated in lung cells. To confirm that both compounds activate TREK-1 currents in a physiologically relevant system and cell type, we used fluorescence-based, K + -sensitive FLIPR assays to demonstrate the effects of ML335 and BL1249 on K + currents in primary mouse AT2 cells (Fig.  5 ). FLIPR assays exploit the permeability of thallium (Tl + ) for open K + channels 48 . After loading AT2 cells with the fluorescent dye, the addition of extracellular Tl + creates a concentration gradient for Tl + to enter the cells. The resultant increase in relative fluorescence is proportional to the open probability of plasma membrane K + channels, and thus represents a measure of the functional activity of K + channels. Therefore, under unstimulated conditions (no drugs), the Tl + -induced fluorescence represents the sum of background K + currents, while after ML335 or BL1249 treatment an increase in fluorescence represents activation of TREK-1-specific K + currents (Fig.  5 ). Our data show that under RA conditions both compounds, ML335 and BL1249, activate TREK-1-specific K + currents (Fig.  5 A). Importantly, these effects were maintained after 24 h of HO exposure (Fig.  5 B).

figure 5

ML335 and BL1249 activate TREK-1 currents in primary mouse AT2 cells: Summary of n = 4–5 independent FLIPR curves (means ± SEM) showing that the TREK-1 activating compounds ML335 or BL1249 induce TREK-1 specific K + currents under both room air and HO conditions ( A , B ). In both room air and HO-treated cells, baseline background K + currents were observed (No drug). *compared to vehicle control (No drug) at room air, ^compared to vehicle control (No drug) after HO exposure; p  ≤ 0.05, n = 4–5, individual experiments were run in triplicates.

Activation of TREK-1 channels hyperpolarizes the plasma membrane potential (Em) of primary AT2 cells

To determine whether the protective effects of ML335 and BL1249 are mediated by TREK-1-induced alterations in the Em, we performed Em-sensitive FLIPR assays on primary mouse AT2 cells under RA and HO conditions (Fig.  6 ). Once cells are loaded with the Em-sensitive fluorescent dye, a decrease in relative fluorescence represents Em hyperpolarization, whereas an increase in fluorescence represents Em depolarization. Since both TREK-1 activating compounds, ML335 and BL1249, had identical effects (i) in our in vivo model, (ii) on inflammatory cytokine secretion, and (iii) on TREK-1 current activation, we used only BL1249 for this part of the study. We found that under room air conditions, activation of TREK-1 channels with BL1249 results in Em hyperpolarization (= a decrease in fluorescence; Fig.  6 A) of primary AT2 cells. Similar to the observed activation of TREK-1 currents with BL1249 (Fig.  5 ), the BL1249-induced Em hyperpolarization also persisted after HO exposure (Fig.  6 B). Importantly, these studies also revealed that HO itself causes Em depolarization when compared to cells kept at RA, as evidenced by a higher baseline fluorescence value in HO-exposed cells (see RED arrows on the Y-axis → in Fig. 6 A, B and summarized in C).

figure 6

TREK-1 activation causes plasma membrane potential (Em) hyperpolarization: Representative curves of Em-sensitive FLIPR assays showing that TREK-1 activation with BL1249 causes Em hyperpolarization in primary mouse AT2 cells under both room air and HO conditions ( A , B ), as indicated by a decrease in fluorescence values. Red arrows on the Y-axis indicate relative fluorescence values reflective of the baseline Em value in room air and HO exposed AT2 cells, demonstrating that HO exposure itself causes Em depolarization (higher baseline fluorescence value in B than A; *BL1249 compared to no drug/vehicle control, p  ≤ 0.05). ( C ) Summary of baseline Em values of RA- vs. HO-exposed AT2 cells averaging n = 6 independent experiments for each condition. Data are represented as Box-Whisker plots with medians, 1st and 3rd quartiles, and max and min values; ^compared to room air exposed AT2 cells; p  ≤ 0.05, individual experiments were run in triplicates.

TREK-1 activation decreases intracellular Ca 2+ (iCa) levels during HO exposure

Since inflammatory cytokine secretion is commonly associated with an increase in iCa concentrations, we used Fluo-4 assays to determine the effects of TREK-1 activation on iCa levels in primary mouse AT2 cells (Fig.  7 ). Importantly, following 24 h of HO exposure, AT2 cells contained higher iCa concentrations than cells kept at RA (PURPLE arrows on the Y-axis → in Fig. 7 A,B and summarized in C). In cells kept at RA, activation of TREK-1 channels and Em hyperpolarization with BL1249 had no effect on iCa levels, likely due to the already low iCa levels in resting cells (Fig.  7 A). In HO-exposed cells, on the other hand, TREK-1 activation with BL1249 decreased the HO-induced elevation in iCa levels (Fig.  7 B).

figure 7

TREK-1 activation decreases intracellular Ca 2+ (iCa) concentrations in HO-exposed primary mouse AT2 cells: ( A , B ) Representative curves of Ca 2+ -sensitive Fluo-4 assays showing that HO-exposed AT2 cells contain higher iCa concentrations than RA-exposed cells, as indicated by an increase in fluorescence values (purple arrows on Y-axes). TREK-1 activation with BL1249 has no effect on iCa concentrations in RA-exposed cells, but decreases iCa levels in HO-exposed cells (*BL1249 compared to no drug/vehicle control, p  ≤ 0.05). A summary of n = 6 independent experiments is shown in C ; data are represented as Box-Whisker plots with medians, 1st and 3rd quartiles, and max and min values; ^compared to room air exposed AT2 cells; p  ≤ 0.05, individual experiments were run in triplicates.

Altogether, these findings highlight the TREK-1 activating effects and resultant Em hyperpolarization caused by ML335 and BL1249 in primary alveolar epithelial cells, and demonstrate that these effects persist under HO conditions, making TREK-1 activation a feasible approach to modulate the Em and iCa concentrations during HO exposure.

Regulation of inflammatory cytokine secretion by voltage-gated Ca 2+ (Ca V ) channels

Em depolarization, as observed with HO exposure and counteracted by TREK-1 activation, results in opening of Ca V channels in many cell types, and the resultant increase in iCa concentrations is commonly a trigger for downstream inflammatory cytokine secretion 49 , 50 . Therefore, we measured HO-induced cytokine secretion from primary mouse AT2 cells after blocking N- and P/Q-type Ca V channels with ω-conotoxin MVIIC 51 , and L-type Ca V channels with nifedipine 52 (Fig.  8 ). Since in primary AT2 cells HO exposure predominantly induced secretion of IL-6 and CCL-2 (Fig.  3 ), we focused on the role of Ca V channels in the secretion of these two cytokines. Interestingly, while HO-induced IL-6 secretion was not dependent on Ca V channel activity, CCL-2 secretion was inhibited by the L-type Ca V channel blocker nifedipine, but not by the N- and P/Q-type Ca V channel blocker ω-conotoxin MVIIC. Secretion of IP-10, TNF-α, MIP-1α and IL-10 from AT2 cells was not affected by ω-conotoxin MVIIC or nifedipine (data not shown).

figure 8

Effects of voltage-gated Ca 2+ channel (Ca V ) inhibition on cytokine secretion from primary mouse AT2 cells: HO exposure increased IL-6 ( A ) and CCL-2 ( B ) secretion compared to RA-treated cells. Inhibition of N- and P/Q-type Ca V channels with ω-conotoxin MVIIC or L-type Ca V channels with nifedipine revealed that IL-6 secretion occurred independently of Ca V channel activity, whereas CCL-2 secretion was dependent on L-type Ca V channels (inhibited by nifedipine) but not N- and P/Q-type channels (lack of ω-conotoxin MVIIC effect). Data are represented as Box-Whisker plots with medians, 1st and 3rd quartiles, and max and min values; n = 3–6; ^compared to cells treated with a vehicle control and exposed to room air (No drugs), *compared to HO exposed cells; p  ≤ 0.05.

Altogether, these findings suggest that regulation of inflammatory cytokine secretion via TREK-1-induced Em hyperpolarization and inhibition of Ca V channel activation could explain some, but not all, of the TREK-1 protective effects seen in our in vivo model.

In this study we propose activation of TREK-1 K + channels as a potentially new therapeutic approach against HALI, since currently no targeted interventions exist that translate into improved patient outcomes. Recent in vitro studies suggest that overexpression of certain microRNAs (miR16, miR21-5) may protect cultured AT2 cells against HO-induced apoptosis 53 , 54 , 55 , and multiple biomarker studies have aimed at predicting the risk of HALI in patients 56 , 57 . In addition, neutralizing therapies against individual cytokines, including IL-6, TNF-α and CCL-2, have yielded variable results at best in improving inflammatory responses 58 , 59 , 60 , 61 .

Given these challenges, we are particularly interested in identifying strategies that can regulate multiple inflammatory pathways simultaneously, such as the manipulation of the plasma membrane potential (Em). We previously discovered that HO downregulates TREK-1 K + channel expression in lung tissue and alveolar epithelial cells, which correlates with worsening lung injury and alterations in multiple inflammatory cytokines (IL-6, CCL-2, RANTES, and IL-1β) 29 , 30 , 31 , 32 , 42 . Importantly, this HO-induced decrease in TREK-1 expression leaves a remainder subset of TREK-1 channels suitable for pharmacological activation. Although channels of the K2P family are known for their so-called “leak K + currents” (a constant, slow K + efflux that stabilizes the Em), TREK-1 channels are actually thought to be closed at baseline 34 , 62 . This idea is supported by our own data in alveolar epithelial cells (Fig.  5 ) showing that TREK-1 currents can readily be induced by our channel activators ML335 and BL1249 44 , 46 , 47 , thus making TREK-1 channels a feasible target for therapeutic activation.

So far, most of the biophysical characterization of TREK-1 channels has occurred under non-physiological conditions in heterologous expression systems 44 , 63 , 64 , and little is known about their functions in physiologically-relevant models. Our study is the first to (a) report the safety and efficacy of the novel TREK-1 activating compounds ML335 and BL1249 in an in vivo system, and (b) highlight the protective effects of TREK-1 activation in a lung injury model by measuring clinically relevant parameters. The only other reports suggesting a potentially protective role for TREK-1 activation used models of hypoxic-ischemic brain injury and atrial fibrillation/heart failure 65 , 66 , 67 . Interestingly, effects of single nucleotide polymorphisms (SNPs) in the human TREK-1 gene have been reported in the same two organs, and predict resistance to antidepressant medication 68 , and an increased risk for atrial tachycardias 69 . However, until now a similar protective effect for TREK-1 channels has not been reported in any other organ.

The importance of inflammatory mediators in the development and progression of HALI is well-established 14 , 70 , including the cytokines reported in this study: IL-6, IP-10, CCL-2, TNF-α, MIP-1α, and IL-10 17 , 21 . In general, IL-6, IP-10, TNF-α and MIP-1α are known for their proinflammatory properties, while CCL-2 can exert pro- 71 , 72 or anti-inflammatory 73 , 74 effects, and IL-10 is considered a predominantly anti-inflammatory cytokine 75 , 76 . More recently it has become increasingly clear that the inflammatory phenotypes observed in various lung injury models are determined by complex interactions between multiple cytokines. For example, despite the well-documented proinflammatory effects of IL-6 and its association with poor outcomes in ARDS patients 77 , IL-6 also induces anti-inflammatory IL-10 secretion as a counter-regulatory response 78 , and a recent study suggests that IL-6 protects mice from LPS- and mechanical ventilation-induced lung injury 79 . In our HALI model we found increased levels of both IL-6 and IL-10 in the BAL fluid of HO-exposed mice. Interestingly, while TREK-1 activation decreased HO-induced BAL fluid IL-6 levels, IL-10 levels remained elevated even after TREK-1 activation, potentially acting synergistically with the protective effects of TREK-1 activation. It is important to note that both lung resident and immune cells contribute to the cytokine levels measured in BAL fluid, and it is quite likely that in our in vivo model the TREK-1 activators affect cytokine secretion from multiple cell types. In this study we focused on epithelial cells since previously we did not find alterations in TNF-α release from TREK-1-deficient alveolar macrophages, and the single cell RNA-seq database LungGENS only reports low levels of TREK-1 postnatally in endothelial cells 42 , 80 .

Our results report for the first time (1) the expression of functional TREK-1 channels on primary mouse AT2 cells and human alveolar epithelial cells (HAEC), and (2) the effects of Em manipulation via TREK-1 channels on inflammatory cytokine secretion and iCa concentrations in a clinically relevant model of HALI. Since in clinical practice the timing of HO therapy is entirely under the control of the healthcare provider, administration of TREK-1 activators simultaneously with initiation of HO therapy is a clinically feasible approach. Of note, although in animal models the injurious effects of HO on previously healthy lungs have been extensively studied, in humans the exact degree and duration of HO exposure that results in symptomatic and clinically-relevant injury remains a matter of intense discussion 81 .

From studies in macrophages, neutrophils and mast cells, we learned that changes in the Em commonly precede secretory events 82 , 83 , but the molecular mechanisms regulating inflammatory cytokine secretion from lung resident cells remain incompletely understood. Furthermore, studies in lung endothelial cells, revealed that the resting Em can vary among cell phenotypes. Reported Em values in endothelial cells range from − 30 to − 60 mV 84 , 85 , and exposure of pulmonary artery endothelial cells to low oxygen concentrations (hypoxia) has been reported to cause Em depolarization 86 . Similar variations in Em depending on the cellular phenotype have also been documented in lung epithelial cells, including rat AT2 cells (− 30 mV) 87 , 88 , rabbit AT2 cells (− 60 mV) 89 , human bronchial epithelial cells (− 20 to − 45 mV) 90 , 91 , and nasal epithelial cells (− 15 to − 30 mV) 91 , 92 . Limited information form human ex vivo studies point towards Em values between − 15 and − 20 mV in bronchial epithelial cells 91 , 93 . Notably, other studies estimate the resting Em in AT2 cells as low as 0 to − 5 mV 94 , 95 . Despite these ranges in Em for lung resident cells, it is important to realize that the Em of epithelial and endothelial cells is much lower than the Em of excitable cells such as neurons and cardiomyocytes, in which the Em ranges between − 60 and − 90 mV 96 , 97 . Since these latter cell types are more hyperpolarized at baseline (i.e. more negative Em values), they require a much stronger depolarization stimulus for a biological response to occur, such as the opening of voltage-gated Ca 2+ (Ca V ) channels and subsequent Ca 2+ influx. In contrast, in the more depolarized epithelial and endothelial cells, a much smaller Em perturbation can reach the threshold for Ca V channel activation, and trigger downstream responses. Conversely, K + efflux, as caused by TREK-1 activation with BL1249 (Fig.  6 A), moves the Em away from this critical threshold towards more negative (hyperpolarized) Em values, and can counteract depolarization-induced cell activation processes.

HO-mediated depolarization events have been reported in mitochondrial membranes of pulmonary endothelial cells 98 , but our study is the first to show HO-induced Em depolarization in primary epithelial cells. Interestingly, in carotid body cells hypoxia, not hyperoxia, causes Em depolarization and increases iCa 2+ concentrations, while HO inhibits both of these processes 99 , 100 . In contrast to these studies, we demonstrate that primary epithelial cells respond to HO exposure by increasing iCa 2+ levels (Fig.  7 ), and we propose that this response is mediated by HO-induced Em depolarization that can be counteracted by TREK-1 activation (Figs. 6 , 7 ).

Interestingly, although it is well-known that both extracellular Ca 2+ influx and Ca 2+ release from intracellular stores can increase iCa 2+ levels, we found that in primary mouse AT2 cells only secretion of CCL-2, but not IL-6, IP-10, TNF-α, or MIP-1α, was dependent on Ca 2+ influx via Ca V channels (Fig.  8 ). The lack of effect of ω-conotoxin MVIIC on cytokine secretion suggests that Ca 2+ influx via N-, and P/Q-type Ca V channels is unlikely to contribute to these processes. In addition to the novelty and importance of our data, these findings also indicate that Ca 2+ release from intracellular stores is likely to be involved in the observed secretory processes.

Although upregulation of CCL-2 in bronchial and alveolar epithelial cells under inflammatory conditions is well-documented 101 , 102 , 103 , it remains a matter of intense discussion whether CCL-2 secretion in the lung is a Ca 2+ -dependent process, and may ultimately depend on the specific cell type and inflammatory environment. In both immortalized and primary lung epithelial cells, inhibition of Ca 2+ sensing, Ca 2+ influx, and iCa 2+ release all prevent CCL-2 secretion, and in some instances also IL-6 release 104 , 105 . Conversely, it is known that in immune cells CCL-2 itself can increase iCa 2+ concentrations 106 , demonstrating the complex interactions underlying CCL-2 secretion. One study showed that the chemotactic function of CCL-2 can occur in the absence of any changes in iCa 107 , and in an LPS-induced lung injury model inhibition of cellular Ca 2+ sensing receptors (CaSR) decreased IL-6 and TNF-α, but not CCL-2, concentrations in the serum and BAL fluid 104 .

Since in our model inhibition of Ca V channels decreased CCL-2 secretion but no other measured cytokines, we should consider the possibility that TREK-1-induced changes in Em could be directly sensed by a voltage-sensitive protein at the plasma membrane level. For this to occur, such a protein would need to contain one or more transmembrane segments with free charges that can induce a so-called “gating current” following an alteration in Em. Although membrane-bound voltage sensors are well-characterized in the brain and heart 108 , 109 , in the lung this important topic has yet to be explored.

We previously reported an important role for TREK-1 in HALI using a TREK-1-deficient mouse model 42 , which revealed a similar injurious phenotype as can be obtained with HO-induced TREK-1 downregulation 42 . In this study, we now shed some light on how TREK-1 may regulate downstream signaling cascades during HO exposure. Based on the current and our previous studies, we propose that the primary mechanism underlying the HO-mediated effects on TREK-1 signaling consists in a decrease in TREK gene and protein expression levels, rather than potential HO-mediated post-translational modifications of the TREK-1 protein structure. Of note, in HEK293 cells, posttranslational TREK-1 phosphorylation has been reported, and resulted in TREK-1 inhibition 110 . However, even if such changes occurred in the lung, they do not seem to interfere with the activation effects of BL1249 and ML335 on TREK-1 channels. Since BL1249 and ML335 are designed to bind and functionally activate wildtype TREK-1 channels, substantial HO-induced structural/posttranslational changes to the TREK-1 structure are unlikely the cause for our reported outcomes. In fact, one of the key findings of this study is that BL1249 and ML335 can activate TREK-1 channels and ameliorate injury despite any HO-induced changes in the intra- and extracellular cellular environments. Notably, we previously reported TREK-1 expression in both AT1 and AT2 cells from mouse lung slices, as well as mouse alveolar macrophages (AMs), but saw only weak TREK-1 staining in the mouse lung endothelium. Interestingly, in that study we also found that LPS-induced TNF-α release from mouse AMs appears to occur independently of TREK-1 31 , suggesting that epithelial TREK-1 channels are the primary target for BL1249 and ML335 in our HALI model.

In conclusion, we report for the first time the functional expression of TREK- 1K + channels on primary alveolar epithelial cells. We show that pharmacological activation of TREK-1 channels during HO exposure is a novel and clinically feasible approach to protect against HALI by reducing inflammatory cell recruitment and barrier dysfunction in the lungs, which may at least in part be mediated by inhibition of inflammatory cytokine secretion. However, additional studies are required to identify other potential effector mechanisms contributing to TREK-1-mediated protection, which should include ROS production, cell death pathways, and inflammasome activation.

Materials and methods

C57bl/6 wild-type (WT) mice aged 9–12 weeks were obtained from Jackson Laboratories ( www.jax.org ). Mice were housed in same-sex groups of up to 5 mice per cage and provided with food and water ad libitum. For experimental purposes, mice were age- and gender-matched as closely as possible.

Mouse hyperoxia (HO) exposure

Using a rodent HO chamber and a 5-L oxygen concentrator (DeVilbiss Healthcare, #525DS), we exposed mice to HO (F i O 2  = 0.8–0.9 inside the chamber) for 72 h in their native cages with free access to food and water. Temperature, humidity and oxygen concentrations were monitored continuously using commercially available sensors (AcuRite 00325A1 for temperature and humidity; Hudson-RCI5800 for oxygen concentrations). During HO exposure, mice lost less than 10% of weight and appeared overall healthy. No deaths were observed. Control mice were exposed to room air (RA) for the same time period in their native cages.

TREK-1 activating compounds

We used two novel TREK-1 activating compounds, ML335 and BL1249. ML335 has been synthesized and validated by our collaborator Dr. Minor at UCSF 44 , who provided this compound to us as gift. BL1249 has most recently become commercially available (Tocris) 45 . Stock solutions for ML335 (100 mM) and BL1249 (100 mM) were prepared in DMSO. For in vivo experiments, we used a final concentration of 100 μM ML335 and 200 μM BL1249 in sterile PBS. For in vitro experiments in primary cells, we used a final concentration of 100 μM (60 μg/kg) ML335 and 10 μM (100 μg/kg) BL1249 suspended in culture media. Vehicle controls for all experiments contained equimolar amounts of the DMSO.

Intra-tracheal injections

During the 72 h of RA or HO exposure, mice were injected once-daily intratracheally ( i.t. ) via brief endotracheal intubation with either 40μL of the TREK-1 activating compounds ML335, BL1249, or a vehicle control in sterile PBS. Briefly, for i.t. injections, mice underwent brief inhaled isofluorane (2–5%) anesthesia until they lost consciousness, and were then suspended by their incisors on a 3.0 silk suture mounted on a 45 degree-angled stand. The tongue was gently extracted from the mouth and moved to the side using blunt forceps in order to visualize the vocal cords. Using fiberoptic guidance, a 20-gauge angiocatheter was passed through the vocal cords into the subglottic area, and 40 μL of drug or vehicle control were injected with a micropipettor. Mice were then placed back into their native cages and allowed to recover under a warming lamp until fully awake. No perianesthetic deaths were associated with this procedure.

Quasi-static lung compliance measurements

Following RA or HO exposure, a tracheostomy was performed using an 18-gauge steel catheter under general ketamine/xylazine anesthesia (intraperitoneal, 10 mg/kg ketamine; 20 mg/kg xylazine). Quasi-static lung compliance was measured using the Flexivent system (SQIREC). Pressure–volume curves (P–V) were recorded, and each set of P–V curves was preceded by an inflation maneuver to total lung capacity to insure equal standard lung volumes for each experiment. Quasi-static lung compliance was calculated by fitting data derived from the P–V curves to the Salazar-Knowles equation as previously described 111 . Rectal temperatures were maintained in physiologic range using a heat lamp. All experiments were terminal.

Broncho-alveolar lavage (BAL) fluid collection and lung histology

Following Flexivent measurements, BAL fluid was collected from all mice using a 1 ml syringe attached to the tracheostomy catheter. Two wash-outs were performed with 1 ml PBS/0.6 mM EDTA for BAL protein and cell count determination, and 1 ml PBS/0.5% BSA for cytokine assays. All samples were immediately placed on ice. Total BAL protein concentrations were measured using the Bradford assay (BioRad), and total BAL cell counts were performed using a Diff-Quick stain (Fisher Scientific). Thereafter, lung tissue was harvested and processed for histological examination. Briefly, the lungs were gently retrograde perfused via the right ventricle with 10 ml ice-cold PBS to remove red blood cells. Lung tissue was then removed en bloc and immediately perfused and fixed in 4% formalin. Paraffin-embedded sections were cut into 4 µm thick tissues slices using a Microtome, and H&E-stained for histological analysis. Lung Injury Scores (LIS) were determined by an investigator blinded to the experimental conditions on H&E-stained lung sections as previously described, using the following 3 criteria: (1) interstitial and alveolar edema, (2) cellular infiltrate, and 3) parenchymal and perivascular hemorrhage. Each criterion was assigned a score between 0–3, with “0” representing no injury, “1” representing mild injury, “2” representing moderate injury, and “3” representing severe injury. Five randomly assigned high power fields per slide were scored under 40 × magnification on a Motic AE20/21 inverted microscope, and scores were averaged for each criterion. Using the sum of these averages, a composite histological LIS was calculated for each experimental group.

Primary mouse and human alveolar epithelial cells

Primary mouse alveolar type-2 cells (AT2) cells were freshly isolated as previously described 112 . We obtained in average 3–5 × 10 6 AT2 cells per mouse lung with > 90% purity as assessed by immunostaining for pro-SPC. All experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee at the University of California Los Angeles. Freshly isolated AT2 cells were seeded to 70–80% confluence at a density 3.5 × 10 6  cells per well in 6-well tissue culture plates coated with fibronectin. Cells were maintained in DMEM cell culture medium containing 10% FBS, 4 mM glutamine, 1% penicillin/streptomycin, and 0.25 µM amphotericin B. All experimental interventions were started on day 2 after AT2 cell isolation.

Primary Human Alveolar Epithelial Cells (HAEC) were purchased from ScienCell (#3200), cultured according to the company’s instructions, and used at a passage numbers < P5. Since these cell suspensions are directly isolated from donated human lung tissue, they contain mixed populations of AT1 and AT2 cells.

HO exposure of cells

HO exposure of cells was performed using a cell culture-compatible HO chamber. HAEC were exposed to 72 h of HO to mimic our in vivo HO protocol. Since in freshly isolated mouse AT2 cells we observed substantial cell death after 72 h of HO exposure (F i O 2 0.8–0.9), we limited HO exposure to 24 h for these cells. Controls for each cell type were cultured at room air for the respective time intervals. During the HO or RA exposure period, cell suspensions were treated with a one-time dose of the TREK-1 activating compounds ML335 (100 μM) or BL1249 (10 μM), or an equimolar DMSO vehicle control. Under all experimental conditions cell viability remained greater than 75% as determined by Trypan Blue staining. To assure that BL1249 and ML335 were not cytotoxic at the doses used, we performed dose–response experiments using two cell viability assays, CCK-8 (APExBIO) and XTT (Biotium).

TREK-1 gene and protein expression

We used real-time PCR and IF microscopy to confirm HO-induced TREK-1 downregulation after 24 h in freshly isolated mouse AT2 cells. Briefly, for PCR experiments total RNA was isolated using a Qiagen RNeasy Mini Kit (Hilden, Germany), 1 μg RNA was reverse transcribed with a High Capacity cDNA Reverse Transcription kit (Applied Biosystems), and amplified by semi-quantitative real-time PCR (TaqMan) with primers specific for TREK-1 (KCNK2; Applied Biosystems). For IF microscopy, mouse AT2 cells were fixed with 4% paraformaldehyde and then incubated with an anti-TREK-1 primary antibody (Alomone, 1:200) at 4 °C overnight, followed by probing with a species-specific secondary antibody (1:1000; Abcam) for one hour at room temperature. Nuclei were counterstained with Fluoro Gel II mounting medium containing DAPI (EMS). All images were recorded using Zen 2009 Light Edition software version 5.5 (Zeiss; https://www.zeiss.com/microscopy/us/products/microscope-software/zen-lite.html ).

Cytokine measurements by ELISA

Cytokine concentrations were quantified in BAL fluid and cell culture supernatants after centrifugation at 8000 rpm for 5 min. Briefly, 100 μL of sample was loaded into 96-well ELISA plate, and analyzed following the manufacturer’s instructions. All samples were run in triplicates and values are displayed in pg/mL. Species-specific ELISA kits were purchased from the following vendors: IL-6 (BD Biosciences), IP-10 (mouse: R&D Systems; human: BD Biosciences), CCL-2 (BD Biosciences), TNF-α (BD Biosciences), MIP-1α (R&D Systems), IL-10 (R&D Systems).

FLIPR and Fluo-4 assays for K + flux, plasma membrane potential (Em), and intracellular Ca 2+ (iCa) measurements

K + channel activity and Em measurements were performed using commercially available FLIPR assays (Molecular Devices, #R8222 and #R8126, respectively), and Fluo-4 assays (Invitrogen, #F36206) for iCa measurements. All three assays were performed following the manufacturer’s instructions. Briefly, for all assays 30,000 cells/well were seeded into dark-walled, clear-bottom 96-well plates (Grenier Bio-One, #655090), and cultured in growth medium overnight. The next day, cells were washed once and incubated at 37 °C with the respective loading dye for 60 min for K + channel activity assays, and 30 min for Em and Fluo-4 assays. In all assays, fluorescence traces were recorded for 1 min to reach a stable baseline before the addition of any drugs. All plates were analyzed using a BioTek Synergy-2 fluorescence plate reader. Data points were collected and integrated every 7 s. To determine whether an increase in iCa concentrations was due to Ca 2+ influx via voltage-gated Ca 2+ (Ca V ) channels, we blocked N- and P/Q-type Ca V channels with ω-conotoxin MVIIC (1 μM), and L-type Ca V channels with nifedipine (10 μM).

Statistical analysis

Quasi-static lung compliance, BAL protein and cell counts, LIS values, cytokine concentrations, and FLIPR and Fluo-4 data are represented as Box-Whisker plots with median values, 1st and 3rd quartiles, and maximum and minimum values. FLIPR curves in Figs. 5 A,B, 6 A,B, and 7 A,B show mean + SEM values. Data were analyzed using the unpaired student t-test, multivariate analysis of variance (ANOVA), and pairwise comparison of means using the Tukey–Kramer method to adjust for multiple comparisons. All statistical analyses were performed using GraphPad Prism 7 software (version 6.04, La Jolla, CA; https://www.graphpad.com/ ), and p values p  ≤ 0.05 were considered significant.

Study approval

Approval for all experiments was obtained from the “University of California Los Angeles Animal Research Committee (ARC). All experiments were performed in accordance with our institutional protocols, guidelines and recommendations.

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Acknowledgements

We thank Dr. Michela Ottolia (UCLA) and her lab for ongoing discussions and their intellectual input. We also thank Dr. Daniel Minor (UCSF) for providing us with the ML335 compound and for sharing with us his knowledge about the pharmacokinetics and pharmacodynamics of the compound. This study was supported by the following Grants: NIH HL118118-3 (AS); NIH HL131526 (CMW); NIH HL134346 (RO).

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Department of Pediatrics, University of California Los Angeles, 10833 Le Conte Ave, MDCC 12-475, Los Angeles, CA, 90095, USA

Tatiana Zyrianova, Benjamin Lopez, Leanne Wong, Victoria Nguyen, Sriharsha Talapaneni & Andreas Schwingshackl

Department of Anesthesiology and Perioperative Medicine, University of California Los Angeles, Los Angeles, CA, USA

Riccardo Olcese

Department of Physiology, University of California Los Angeles, Los Angeles, CA, USA

Department of Pulmonary and Critical Care Medicine, University of California Los Angeles, Los Angeles, CA, USA

John Belperio

Department of Physiology, University of Kentucky, Lexington, KY, USA

Christopher M. Waters

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T.Z.: experimental design and execution, manuscript writing and editing. B.L.: experimental design and execution. R.O.: experimental design and manuscript editing. J.B.: experimental design and manuscript editing. C.M.W.: experimental design and manuscript editing. L.W.: experimental execution. V.N.: experimental execution. S.T.: experimental execution. A.S.: experimental design, manuscript writing and editing.

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Zyrianova, T., Lopez, B., Olcese, R. et al. K 2P 2.1 (TREK-1) potassium channel activation protects against hyperoxia-induced lung injury. Sci Rep 10 , 22011 (2020). https://doi.org/10.1038/s41598-020-78886-y

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Published : 15 December 2020

DOI : https://doi.org/10.1038/s41598-020-78886-y

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TREK-1, a K+ channel involved in polymodal pain perception

Affiliation.

  • 1 Laboratoire de Pharmacologie Médicale EA 3848 INSERM/Faculté de Médecine/CHU, Clermont-Ferrand, France.
  • PMID: 16675954
  • PMCID: PMC1478167
  • DOI: 10.1038/sj.emboj.7601116

The TREK-1 channel is a temperature-sensitive, osmosensitive and mechano-gated K+ channel with a regulation by Gs and Gq coupled receptors. This paper demonstrates that TREK-1 qualifies as one of the molecular sensors involved in pain perception. TREK-1 is highly expressed in small sensory neurons, is present in both peptidergic and nonpeptidergic neurons and is extensively colocalized with TRPV1, the capsaicin-activated nonselective ion channel. Mice with a disrupted TREK-1 gene are more sensitive to painful heat sensations near the threshold between anoxious warmth and painful heat. This phenotype is associated with the primary sensory neuron, as polymodal C-fibers were found to be more sensitive to heat in single fiber experiments. Knockout animals are more sensitive to low threshold mechanical stimuli and display an increased thermal and mechanical hyperalgesia in conditions of inflammation. They display a largely decreased pain response induced by osmotic changes particularly in prostaglandin E2-sensitized animals. TREK-1 appears as an important ion channel for polymodal pain perception and as an attractive target for the development of new analgesics.

Publication types

  • Research Support, Non-U.S. Gov't
  • Ganglia, Spinal / cytology
  • In Situ Hybridization
  • Mice, Knockout
  • Nerve Fibers, Unmyelinated / metabolism
  • Neurons, Afferent / cytology
  • Neurons, Afferent / metabolism
  • Nociceptors / metabolism*
  • Pain / metabolism*
  • Pain Measurement
  • Patch-Clamp Techniques
  • Perception / physiology*
  • Potassium Channels, Tandem Pore Domain / genetics
  • Potassium Channels, Tandem Pore Domain / metabolism*
  • RNA, Messenger / metabolism
  • Potassium Channels, Tandem Pore Domain
  • RNA, Messenger
  • potassium channel protein TREK-1

Screen Rant

10 star trek sequels to past episodes.

TNG, DS9, and Voyager have a surprising number of episodes that are sequels to earlier episodes of Star Trek.

  • Star Trek episodes occasionally feature sequels to earlier episodes, allowing for a cohesive continuity in the franchise.
  • The Berman era shows frowned upon serialization but had a surprising number of sequel episodes to maintain continuity.
  • Star Trek: The Next Generation, Deep Space Nine, and Voyager all have standalone episodes that can be enjoyed by new viewers.

There are a surprising number of Star Trek episodes that are essentially sequels to earlier episodes, either within the same show, or on other shows in the same era. In Star Trek: The Next Generation, Star Trek: Voyager , and earlier seasons of Star Trek: Deep Space Nine, the common practice was that each episode of Star Trek was a single, stand-alone story that didn't require too much foreknowledge of earlier events in the Star Trek timeline . In a time before streaming, when it was harder to watch a show from the very beginning, any Star Trek episode could be someone's first, so this ensured that new viewers weren't lost.

While serialization was generally frowned upon in the 1980s and 1990s trio of Star Trek shows, episodes occasionally cropped up that directly referenced events of earlier episodes. Guest characters returned, sometimes years later, to tie up the loose ends that were left dangling in their earlier appearances. Characters' memories were triggered by being in similar situations. Unlike multi-part episodes or the miniature arcs that later happened on DS9 and Voyager , sequel episodes happen long after the consequences of earlier episodes have had time to develop , sometimes after years have passed.

Star trek enterprise archer the next generation picard captain burnham

How To Watch All Star Trek TV Shows In Timeline Order

10 star trek: voyager season 6, episode 17 - "spirit folk", sequel to star trek: voyager season 6, episode 11 - "fair haven".

In the 19th-century Irish town of Fair Haven, rumors abound that mysterious "Outsiders" may be magical spirit folk. Lt. Tom Paris (Robert Duncan McNeill) controls the weather, Ensign Harry Kim (Garrett Wang) once turned Maggie O'Halloran (Henriette Ivanans) into a cow, and Captain Kathryn Janeway (Kate Mulgrew) may have bewitched Michael Sullivan (Fintan McKeown). Of course, Fair Haven is Tom's holodeck program that ran constantly before a massive power drain erased all but 10% of its original code in "Fair Haven". "Spirit Folk" sees Fair Haven's residents witness a few too many "miracles" as their home is rebuilt, cluing them in to the truth of their existence.

With only six episodes between each installment, "Fair Haven" has the shortest amount of time until its sequel, "Spirit Folk".

9 Star Trek: The Next Generation Season 6, Episode 19 - "Lessons"

Sequel to star trek: the next generation season 5, episode 25 - "the inner light".

Star Trek TNG Inner Light Picard

In Star Trek: The Next Generation 's outstanding "The Inner Light", an alien probe causes Captain Picard to live the life of Kamin, a family man on the dying planet Kataan, within the span of about 20 minutes. Picard's experience as Kamin is profound, but rarely addressed on-screen until "Lessons". Jean-Luc's romance with the musical head of stellar sciences, Lt. Commander Nella Daren (Wendy Hughes), prompts Picard to confess that he does know how to play the Ressikan flute. After bonding with Nella through musical duets, Picard reveals he acquired the flute from the probe, and knowledge of how to play it from living as Kamin , garnering Nella's sympathy.

8 Star Trek: Voyager Season 7, Episodes 9 & 10 - "Flesh & Blood"

Sequel to star trek: voyager season 4, episodes 18 & 19 - "the killing game".

janeway confronts hirogen voyager flesh and blood

The predatory Hirogen commandeer the USS Voyager in "The Killing Game", turning the holodecks into their personal playgrounds to hunt Voyager's crew as unwilling prey. Despite the fact that holodecks were used to harm Captain Janeway and Voyager's crew , Janeway believes that Starfleet hologram technology can actually help the Hirogen continue their sacred hunt without killing anyone else. The Hirogen believe the hunt is meaningless if their prey don't feel pain, so "Flesh & Blood" reveals that the Hirogen programmed their holograms to be sentient . Star Trek: Voyager season 7 explores the personhood of holograms, so it's up for debate whether that's any different than hunting organic people.

7 Star Trek: The Next Generation Season 6, Episode 26 - "Descent, Part 1"

Sequel to star trek: the next generation season 5, episode 23 - "i, borg".

Jonathan Del Arco portraying Hugh in Star Trek TNG

Star Trek: The Next Generation closes its sixth season with "Descent, Part 1", the first half of a two-part episode that sees the USS Enterprise crew face off against a new type of Borg, detached from the greater Borg Collective. These new, emotional Borg call themselves as "I" instead of "we", and call each other by name -- in other words, they're individuals. A year earlier, in "I, Borg", Lt. Commander Geordi La Forge (LeVar Burton) repaired and befriended a lone drone, Hugh (Jonathan del Arco). The concept of individuality was introduced to the Borg after Hugh returned to the Collective , but instead of becoming hopeful individuals, other Borg respond to liberation with vengeance.

star-trek-enemy-aliens-became-heroes

8 Star Trek Enemy Aliens Who Became Heroes

6 star trek: voyager season 5, episode 3 - "false profits", sequel to star trek: the next generation season 3, episode 8 - "the price".

Janeway and Tuvok talk to Ferengi in Voyager False Profits

In Star Trek: The Next Generation 's "The Price", negotiations over ownership of a stable wormhole near Barzan II attract the attention of Ferengi delegates Arridor (Dan Shor) and Kol (J.R. Quinonez). While the wormhole is said to connect to the Gamma Quadrant, a test flight finds Kol and Arridor stranded in the Delta Quadrant after the surprisingly un stable wormhole vanishes before they can turn around. Seven years later, in Star Trek: Voyager 's "False Profits", the similarly stranded USS Voyager crew is surprised to discover Arridor and Kol (Leslie Jordan) so far from home, but less surprised that the Ferengi are swindling the pre-warp Takarians by using replicator technology to pose as important religious figures.

5 Star Trek: Voyager Season 5, Episode 18 - "Course: Oblivion"

Sequel to star trek: voyager season 4, episode 24 - "demon".

Janeway's duplicate in the Star Trek: Voyager episode

Star Trek: Voyager' s "Course: Oblivion" opens with the long-awaited wedding of Tom Paris and B'Elanna Torres (Roxann Dawson) , and the celebration of upgrades that will shorten the USS Voyager's journey home to only 2 years. All is not well for long, however, as a mysterious radiation sickness befalls Voyager's crew, and one by one they, along with the ship, suffer total cellular degradation. Over a year ago, in "Demon", Voyager encountered biomemetic "silverblood" organisms that were allowed to replicate the USS Voyager and its crew , but Voyager left the Demon-class natives on their home planet. Surely the silverbloods didn't forget that they're not the real Voyager crew and set a course for Earth ... right?

4 Star Trek: Deep Space Nine Season 3, Episode 9 - "Defiant"

Sequel to star trek: the next generation season 6, episode 24 - "second chances".

Star Trek TNG Thomas Riker Second Chances

In Star Trek: Deep Space Nine 's "Defiant", Commander Will Riker (Jonathan Frakes) pays a visit to the Deep Space Nine station while ostensibly on vacation, and befriends Major Kira Nerys (Nana Visitor). Kira agrees to give Riker a tour of the USS Defiant, but as soon as they're aboard, Riker turns against Kira, revealing he isn't Will, but Thomas . Several years earlier, the USS Enterprise discovered a second Will Riker had materialized from a reflected transporter beam, who elected to rename himself Thomas Riker to pursue his own Starfleet career. DS9 reveals that hadn't worked out, so Thomas Riker joined the Maquis and intended to steal the Defiant by posing as Will.

3 Star Trek: Voyager Season 7, Episode 19 - "Q2"

Sequel to star trek: voyager season 3, episode 11 - "the q & the grey".

In "The Q and the Grey", Q (John de Lancie) comes to the USS Voyager with intentions to procreate with Captain Janeway, believing that a Q-human hybrid will reunite the fractured Q Continuum. Repulsed by the idea, Janeway convinces Q that the Continuum would be better served by Q's union with Miss Q (Suzie Plakson) . The product of that union arrives four years later in "Q2", as the intolerable adolescent Q Junior (Keegan de Lancie), who has only sown chaos instead of bringing peace to the Continuum. Bringing Junior to Voyager is a test, as most things are with Q, to see if Junior can learn anything from "Aunt Kathy" and Starfleet ideals.

That family resemblance is genuine: Q Junior is played by John de Lancie's actual son, Keegan de Lancie.

Star Trek John de Lancie Q TNG Voyager DS9 Picard

Every Q Star Trek Appearance Ranked Worst To Best

2 star trek: the next generation season 6, episode 12 - "ship in a bottle", sequel to star trek: the next generation season 2, episode 3 - "elementary, my dear data".

Lt. Reginald Barclay (Dwight Schultz) accidentally releases the holographic Professor James Moriarty (Daniel Davis) from an unusual program that's been continually running within the ship's computer. Moriarty has been trapped inside the computer, fully conscious while awaiting the development of technology that would let him leave the holodeck. Moriarty insists on leaving the holodeck and the Enterprise, despite being acutely aware he's a hologram. This holographic Moriarty gained sentience four years earlier , when Lt. Commander Data (Brent Spiner) solved Sherlock Holmes' mysteries too easily, and the holodeck altered Holmes' adversary Moriarty to become Data's intellectual equal.

1 Star Trek: The Next Generation Season 7, Episode 15 - "Lower Decks"

Sequel to star trek: the next generation season 5, episode 19 - "the first duty".

Star Trek: The Next Generation 's "Lower Decks" shifts the perspective from the USS Enterprise's senior staff to four junior officers: the go-getting Ensign Sam Lavelle (Dan Gauthier), Vulcan engineer Ensign Taurik (Alexander Enberg), Nurse Alyssa Ogawa (Patti Yasutake), and Ensign Sito Jaxa (Shannon Fill). Sito accepts a dangerous espionage mission as part of a personal redemption arc , after previously appearing as a Starfleet Academy cadet. Sito, like Cadet Wesley Crusher (Wil Wheaton), was a member of the elite Nova Squadron, which collectively agreed to cover up the irresponsible actions of Cadet Nick Locarno (Robert Duncan McNeill) that led to the death of teammate Joshua Albert.

Star Trek: Lower Decks season 4, episode 10 "Old Friends, New Planets" could be considered the third installment of this arc, with the return of Nick Locarno and the memory of Sito Jaxa having a profound impact on Lt. Beckett Mariner (Tawny Newsome).

Although serialization was generally frowned upon in the Berman era of Star Trek shows, the surprising number of sequel episodes proves that there was still a relatively cohesive continuity in Star Trek: The Next Generation , Star Trek: Deep Space Nine , and Star Trek: Voyager . DS9 embraced serialization, as the events of individual DS9 episodes built on each other, shaping the story of the Dominion War. Star Trek: Voyager also started to take on a continuity of its own, with character relationships progressing in miniature arcs. Thanks to the use of sequel episodes to maintain continuity, Star Trek showed that actions have consequences, intended or otherwise.

Star Trek: The Next Generation , Star Trek: Deep Space Nine , and Star Trek: Voyager are streaming on Paramount+.

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TV broadcasting in the trains of the Moscow Metro

  • News & Events
  • Press release

Tomsk, Russia, January 23, 2020. The Elecard company took part in the large-scale project on content preparation and broadcasting of 12 channels in the Moscow underground trains.

The Moscow Metro is the backbone of the Moscow transport system. It consists of 15 lines and 269 stations, on which more than 12 thousand trains are passed daily. There are 6200 video screens installed in the subway cars, which broadcast TV channels, information videos, and commercials. The monitors are designed for a large audience: more than 7 million people use the metro every day.

Elecard, SoftLab-NSK, and Stream Labs developed and implemented the hardware and software complex for preparation, broadcasting, and monitoring of TV channels in metro trains of 12 lines. This solution is based on the transcoder Elecard CodecWorks and high-density servers. CodecWorks supports H.265/HEVC, which is especially important in this project, as the communication bandwidth in the rolling stock is limited. HEVC allows broadcasting video of higher quality due to a higher degree of compression, as opposed to the AVC format. The solution guarantees stable broadcasting, as it includes redundancy of all system components. If an error occurs, the schema will automatically switch to the reserve source, and viewers will not notice a failure.

The Stream Labs provided the solution for the head-end station monitoring. The technologies of the SoftLab-NSK were used to implement the playout system. The solution supports targeted advertising adjusted for the content availability and duration. A playlist with a specific set of informational videos and commercials is prepared for each subway line. The system is integrated with external systems of advertising content delivery.

“ Metropolitan is a unique facility that has its own specifics in terms of standards and operation. It is important to understand and consider this in our work. We provided exclusive technical support during testing and implementation of the solution ," said Nikolay Milovanov, Elecard CEO.

" We came up with the task to implement broadcasting of media content with targeted advertising in Moscow Metro. We turned to Elecard with request to provide a high-quality software solution. The implemented solution allows us to broadcast not only commercials but also sports ans cultural events. On behalf of the company I would like to express my gratitude to Elecard employees for the project implementation and professional support at all stages of the system integration ," comments Maxim Shemegon, Development Director of the Moscow Metro State Unitary Enterprise.

Elecard CodecWorks is a professional software solution for real-time decoding, encoding, and transcoding into MPEG-2/AVC/HEVC with up to 16K resolution supporting multi-screen encoding and HLS/MPEG-DASH adaptive streaming technologies. CodecWorks has passed through comprehensive testing and guarantees high performance and continuous content delivery suitable for projects of any scale and complexity.

About Elecard Elecard provides software products for encoding, decoding, processing, receiving, and transmission of video and audio data in different formats (H.265/HEVC, H.264/AVC, MPEG-4, MPEG-2). Elecard is based in the United States, Canada, Russia, and Vietnam. For more information, please visit www.elecard.com. The company offers a wide range of reference designs for professional digital TV broadcasting market, which includes streaming, transcoding, video-on-demand servers, professional software products, and software development kits. Elecard is based in the United States, Russia, and Vietnam. For more information, please visit www.elecard.com.

Contacts: Tel.: +7 (3822) 488-585 [email protected]

About the Moscow Metro The Moscow Metro State Unitary Enterprise is an organization that provides rail transportation services for passengers in Moscow. It includes the Moscow Metro and the monorail. The Moscow Metro is the basis of the Moscow transport system, which consists of 15 lines and 269 stations.

Contacts: Tel.: +7 (495) 539-54-54

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NEWS... BUT NOT AS YOU KNOW IT

The Star Trek episode ‘banned’ after predicting a ‘united’ Ireland

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The Star Trek episode The High Ground.

A Star Trek episode released in 1990 has only ever been screened in Ireland once over concerns about a single line .

The original season of the sci-fi series first hit screens in 1966 and ran for three years.

It was then followed by Star Trek: The Next Generation , which aired from 1987 until 1994.

However nearly 35 years after it aired, one episode from the franchise’s second series is still ‘banned’ .

Titled The High Ground, the 12 th episode of the third season sees a crew member of the Federation Starfleet starship USS Enterprise-D taken hostage by terrorists who hope Federation involvement will help them win concessions for their cause.

In one scene android character Data, played by actor Brent Spiner, spoke about the ‘Irish unification of 2024’ as an example of violence successfully achieving a political aim.

The Star Trek episode The High Ground.

Originally shown in the US in 1990, there was so much concern over the exchange that the episode was not broadcast on the BBC or Irish public broadcaster RTÉ.

At the time, US TV shows often debuted internationally several years after their original broadcast.

Two years later, satellite broadcaster Sky reportedly aired an edited version, cutting the crucial scene.

The episode was then shown by the BBC on September 29 2007, however BBC Archives has said it believes that is the only time it’s ever been aired.

The decision not to air the episode came at a time when deadly conflict continued to rage in Northern Ireland, with the Provisional IRA – a paramilitary group with the aim of ending British rule in Northern Ireland – one of its main protagonists.

Now, in 2024, Sinn Féin, which emerged as the political wing of the IRA, is the largest party in the devolved Stormont assembly.

Reflecting on the episode, writer Melinda M Snodgrass told the BBC she had no clue at the time how the episode would still be so divisive decades later.

The Star Trek episode The High Ground.

‘We became aware of it later… and there isn’t much you can do about it,’ she said.

‘Writing for television is like laying track for a train that’s about 300 feet behind you. You really don’t have time to stop.’

But she added: ‘Science fiction is incredibly important because it allows people to discuss difficult topics – but at arm’s length.’

The episode, which was based on the theme of terrorism, saw the Starship Enterprise’s chief medical officer Dr Beverly Crusher is abducted by the separatist Ansata group, who use murder and violence to pursue their aim of independence.

In it, Data commented: ‘I’ve been reviewing the history of armed rebellion, and it appears that terrorism is an effective way to promote political change.’

Captain Jean-Luc Picard, played by Patrick Stewart responded: ‘Yes it can be, but I have never subscribed to the theory that political power flows from the barrel of a gun.’

The Star Trek episode The High Ground.

However, the android then added: ‘Yet there are numerous examples of when it was successful. The independence of the Mexican state from Spain, the Irish unification of 2024, and the Kenzie rebellion.’

The exchange then saw Data ask whether it would be ‘accurate to say that terrorism is acceptable when all options for peaceful settlement have been foreclosed?’.

‘Data, these are questions that mankind has been struggling with throughout history. Your confusion is only human,’ the Captain shared.

Snodgrass said her script’s parallels to what was unfolding in Northern Ireland at the time was deliberate.

‘I was a history major before I went to law school and I wanted to get into that; discuss the fact that one man’s freedom fighter is another man’s terrorist,’ she said.

‘I mean, these are complicated issues. And when do people feel like their back is so much against the wall that they have no choice but to turn to violence? And is that actually ever justified?’

A Republicans holds an Irish Flag as he stands next to a line of police during clashes in the Oldpark area of north Belfast, northern Ireland, on August 9, 2015.

She added that what they wanted to say at the time was: ‘If we’re talking and not shooting, we’re in a better place.’

The episode was initially due to air in the UK in 1992, two years before the IRA ceasefire and six before the Good Friday Agreement.

From 1988 until 1994, a ban was enforced on broadcasting the voices of members of certain groups from Northern Ireland on TV and radio.

The BBC’s press office said it had spoken to ‘a number of people’ about why a ban may have been implemented but was unable to get this information ‘as it dates quite far back’.

Star Trek is streaming on Netflix.

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Mainstay entertainment and paradigm sign tisha campbell, breaking news.

Kenneth Mitchell Dies: ‘Star Trek: Discovery’, ‘Captain Marvel’ & ‘Jericho’ Actor Was 49

By Denise Petski

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Kenneth Mitchell

Kenneth Mitchell , who played several characters in Star Trek: Discovery , and also was known for his roles in Jericho and Captain Marvel, has died from complications of ALS, his family revealed Saturday. He was 49.

“With heavy hearts we announce the passing of Kenneth Alexander Mitchell, beloved father, husband, brother, uncle, son and dear friend,” his family shared on X/Twitter.

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Gary Graham Dies: ‘Alien Nation’ & ‘Star Trek’ Actor Was 73

Mitchell announced publicly that he’d been diagnosed with ALS (amyotrophic lateral sclerosis), also known as Lou Gehrig’s disease, in 2020 in an interview with People.

“The moment that they told us it was [ALS], it was like I was in my own movie,” Mitchell told the publication. “That’s what it felt like, like I was watching that scene where someone is being told that they have a terminal illness. It was just a complete disbelief, a shock.”

Mitchell played three Klingon characters in Star Trek: Discovery’ s first two seasons. He portrayed Kol in Season 1, Kol-Sha and Tenavik in Season 2. In Season 3, as the disease progressed, he played Aurellio, a character who used a hoverchair, created to incorporate his need for a wheelchair, into the series.

He also voiced three characters in the first season of Star Trek: Lower Decks Season 1, a black ops operative and a Romulan guard.

StarTrek.com also posted a tribute to Mitchell.

“Being a part of Star Trek keeps me inspired and gives me purpose,” Mitchell told Syfy Wire in 2020. “Hopefully, that will keep going.”

Mitchell is survived by his wife, Susan, their children, Lilah and Kallum, his parents and in-laws and several nieces and nephews.

The family asks that any gifts be directed toward ALS research or toward his children. A GoFundMe campaign has been set up for the children.

View this post on Instagram A post shared by Kenneth Mitchell (@mr_kenneth_mitchell)

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Kenneth Mitchell, ‘Star Trek: Discovery’ and ‘Captain Marvel’ Actor, Dies at 49

By Caroline Brew

Caroline Brew

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LOS ANGELES, CA - SEPTEMBER 19:  Actor Kenneth Mitchell arrives for the Premiere Of CBS's "Star Trek: Discovery"  held at The Cinerama Dome on September 19, 2017 in Los Angeles, California.  (Photo by Albert L. Ortega/Getty Images)

Kenneth Mitchell , known for his multiple roles on “Star Trek: Discovery,” died from ALS complications on Saturday. He was 49.

“For five and a half years, Ken faced a series of awful challenges from ALS. And in truest Ken fashion, he managed to rise above each one with grace and commitment, to living a full and joyous life in each moment,” a statement reads on Mitchell’s official Instagram page. “He lived by the principals that each day is a gift and we never walk alone.”

In 2020, Mitchell announced to People that he was diagnosed with ALS in 2018. The actor had been using a wheelchair since 2019.

“I think it, over time, became the theme of us accepting this with grace,” he said. “Trying to see the beauty in it, in a way. I’ll never forget, one of my ‘Star Trek’ co-stars told me, because they had dealt with some trying times with illnesses and stuff, and I remember them communicating to me, saying, ‘You have a choice. You can look at this in many different ways, but maybe try to look at this like a gift where you get to experience life in a way that most people don’t.'”

He also revealed to People that he had to give up his part as the lead in a television show, which would require him to move to Newfoundland. “Being lead of the show, I really wanted that responsibility. But in the end, it just wasn’t the right thing to continue on,” he said.

Donations to Mitchell’s family can be made to this GoFundMe campaign .

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Kenneth Mitchell, Star Trek and Captain Marvel actor, dies aged 49

Canadian actor who played several Star Trek characters died after complications from ALS, according to a statement on Instagram

The Canadian actor Kenneth Mitchell, known for roles in Star Trek: Discovery and the Marvel film Captain Marvel , has died following complications from amyotrophic lateral sclerosis, or ALS.

Mitchell, who was 49 years old, died on Saturday, according to a statement released by his verified Instagram account.

“With heavy hearts we announce the passing of Kenneth Alexander Mitchell, beloved father, husband, brother, uncle, son and dear friend to many,” the statement said .

“For five and a half years Ken faced a series of awful challenges from ALS. And in truest Ken fashion, he managed to rise above each one with grace and commitment to living a full and joyous life in each moment,” it added.

“He lived by the principles that each day is a gift and that we never walk alone. His life is a shining example of how full one can be when you live with love, compassion, humour, inclusion, and community,” it continued.

In a statement on the official Star Trek website, the franchise also mourned the death of the actor who played multiple roles in Star Trek: Discovery including Klingons Kol, Kol-Sha, and Tenavik, as well as Aurellio.

“The entire Star Trek family sends their condolences to Mitchell’s family, friends, loved ones, and fans around the world,” it added.

In addition to his Star Trek roles, Mitchell also starred in the Marvel film Captain Marvel, as well as the post-apocalyptic television series Jericho, among other projects.

Mitchell is survived by his wife, Susan May Pratt, and their two children, and has requested any gifts be directed towards ALS research or in support of his children, the Instagram statement said.

With an average of 5,000 people diagnosed every year in the US, ALS is a progressive neurodegenerative disease that affects nerve cells in the brain and spinal cord. Symptoms include difficulty walking, slurred speech as well as muscle weakness which eventually impacts chewing, swallowing, speaking and breathing.

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The 'banned' Star Trek episode that promised a united Ireland

W hen sci-fi writer Melinda M Snodgrass sat down to write Star Trek episode The High Ground, she had little idea of the unexpected ripples of controversy it would still be making more than three decades later.

"We became aware of it later... and there isn't much you can do about it," she says, speaking to the BBC from her home in New Mexico. "Writing for television is like laying track for a train that's about 300 feet behind you. You really don't have time to stop."

While the series has legions of followers steeped in its lore, that one particular episode of Star Trek: The Next Generation has lived long and prospered in infamy.

It comes down to a scene in which the android character Data, played by actor Brent Spiner, talks about the "Irish unification of 2024" as an example of violence successfully achieving a political aim.

Originally shown in the US in 1990, there was so much concern over the exchange that the episode was not broadcast on the BBC or Irish public broadcaster RTÉ.

At the time, US TV shows often debuted internationally several years after their original broadcast.

Satellite broadcaster Sky reportedly aired an edited version in 1992, cutting the crucial scene. But The High Ground was not shown by the BBC until 02:39 GMT, 29 September 2007 - and BBC Archives says it is confident this is its only transmission.

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The decision not to air the episode reflects a time when a bloody conflict continued to rage in Northern Ireland, with the Provisional IRA - a paramilitary group with the stated aim of ending British rule in Northern Ireland - one of its main protagonists.

Now it is 2024 - and Sinn Féin, which emerged as the political wing of the IRA, is the largest party in the devolved Stormont assembly .

The party's leader in Northern Ireland, Michelle O'Neill, became first minister last month and has predicted a referendum on Irish unity within a decade.

She strikes a very different tone to Sir Keir Starmer, favourite to be the UK's next prime minister, who has said such a poll is "not even on the horizon" .

On social media, people have been sharing screenshots of Data's prediction and drawing links to Sinn Féin's electoral success.

Back when Ms Snodgrass was writing the script, she did not think it would cause any problems. "Science fiction is incredibly important because it allows people to discuss difficult topics - but at arm's length," she says.

In the episode, Data's line does not come out of the blue.

The High Ground is based on the theme of terrorism, after the Starship Enterprise's chief medical officer Dr Beverly Crusher is abducted by the separatist Ansata group, who use murder and violence to pursue their aim of independence.

"I've been reviewing the history of armed rebellion, and it appears that terrorism is an effective way to promote political change," says Data.

"Yes it can be," responds Captain Jean-Luc Picard, played by Patrick Stewart, "but I have never subscribed to the theory that political power flows from the barrel of a gun."

"Yet there are numerous examples of when it was successful," Data says. "The independence of the Mexican state from Spain, the Irish unification of 2024, and the Kenzie rebellion."

"I'm aware of them," says Picard, to which Data asks: "Would it then be accurate to say that terrorism is acceptable when all options for peaceful settlement have been foreclosed?"

"Data, these are questions that mankind has been struggling with throughout history. Your confusion is only human."

The story has parallels with the situation in Northern Ireland at the time - something Ms Snodgrass says was deliberate.

"I was a history major before I went to law school and I wanted to get into that; discuss the fact that one man's freedom fighter is another man's terrorist," she says.

"I mean, these are complicated issues. And when do people feel like their back is so much against the wall that they have no choice but to turn to violence? And is that actually ever justified?

"I think what I wanted to say was: if we're talking and not shooting, we're in a better place."

In 1992, when the episode was due to air in the UK, the IRA ceasefire of 1994 and 1998 Good Friday Agreement were still years away.

In April of that year, the Baltic Exchange bombing carried out by the IRA in the heart of London's financial centre killed three people, and injured more than 90.

Such was the atmosphere from 1988 to 1994, a ban was enforced on broadcasting the voices of members of certain groups from Northern Ireland on television and radio. Restrictions were seen as specifically targeting Sinn Féin.

It resulted in the bizarre situation where prominent politicians including Martin McGuinness and Gerry Adams had their voices dubbed by actors (Mr Adams, famously, was voiced at times by Oscar-nominated actor Stephen Rea).

Reflecting on the Star Trek episode, Prof Robert Savage of Boston College says: "It was amazing it was censored."

His latest book - Northern Ireland, the BBC, and Censorship in Thatcher's Britain - covers the period when the episode was pulled.

"The argument I think the robot [Data] asks you is basically just: does terrorism work? If there are no alternatives, if you've tried every other avenue to try to affect change, is it acceptable? To use terrorism?

"And it's a very human question. But [Jean-Luc Picard] doesn't answer the question! That would have unsettled somebody like Thatcher," Prof Savage adds.

There is some murkiness about how a decision was reached not to broadcast the programme at the time.

BBC Archives confirmed the 2007 broadcast of the episode and was "satisfied" any other screening would have been listed.

The BBC's press office said it had spoken to "a number of people" about why a ban may have been implemented, but was unable to get this information "as it dates quite far back".

A spokesman for Sky said he had looked into it, but could not confirm it had broadcast an edited version of the episode in 1992 - or what its reasoning might have been for doing so.

RTÉ noted that TV guides from the time show it had broadcast Star Trek: The Next Generation, but did not have further information in its acquisitions system, and could not find anyone from the time to speak to.

"I think this would probably have stirred a memory if I had been made aware of this at the time, but I am afraid it rings no bells at all," said Lord John Birt, who was director general of the BBC from 1992 to 2000, and before this served as deputy director general.

If the episode had been removed, it would probably have been a decision made at operational level in Network Television, he said.

More than three decades on, the picture in Northern Ireland has changed.

Ms Snodgrass says she was "thrilled" when the Good Friday Agreement was signed, adding it had allowed Northern Ireland to prosper.

She notes Games of Thrones, a television series based on books by George RR Martin (who she knows well and has co-authored work with ) was filmed in the region in recent years - something which has given a big boost to the economy .

"[At the time] 2024 seemed a long way away. I probably should have made it, you know, 2224! I just pulled that number and it didn't occur to me that suddenly we would be here."

Star Trek: The Next Generation featured Sir Patrick Stewart as Captain Jean-Luc Picard, in what has become one of his most iconic roles

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  • v.23(13); 2004 Jul 7

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

C heurteaux.

1 Institut de Pharmacologie Moléculaire et Cellulaire, CNRS, Institut Paul Hamel, Sophia-Antipolis, Valbonne, France

L Lang-Lazdunski

M lazdunski, associated data.

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.

Introduction

Two-pore-domain potassium channels (K 2P channels) form a novel class of K + channels identified in various types of neurons ( Kim et al , 1995 ; Wei et al , 1996 ; Lesage and Lazdunski, 2000 ; Talley et al , 2003 ). They are open at membrane potentials across the physiological range and are therefore likely to contribute to the background or leak currents that help set the resting membrane potential and oppose depolarizing influences. They are key components in shaping the characteristics of neuronal excitability. TREK-1 ( Fink et al , 1996 ) is expressed throughout the central nervous system ( Fink et al , 1996 ; Lauritzen et al , 2000 ; Maingret et al , 2000b ; Hervieu et al , 2001 ; Talley et al , 2001 ) and is an important member of this family. It is the probable mammalian homolog of the Aplysia S-type K + channel ( Siegelbaum et al , 1982 ; Patel et al , 1998 ), a channel involved in simple forms of learning and memory. TREK-1 is activated by membrane stretch and intracellular acidification ( Patel et al , 1998 ; Maingret et al , 1999b ). TREK-1 is opened by arachidonic acid and other polyunsaturated fatty acids (PUFAs) as well as lysophospholipids (LPLs) ( Patel et al , 1998 ; Maingret et al , 2000b ). On the other hand, PUFAs and LPLs are potent protective agents against forebrain ischemia and seizures, and it has been proposed that this effect results, at least in part, from their action on TREK channels ( Lauritzen et al , 2000 ; Blondeau et al , 2001 , 2002 ). TREK-1 probably has a central role in the control of excitability by a variety of neurotransmitters. TREK-1 is potently inhibited by neurotransmitters that produce an increase in intracellular cAMP ( Patel et al , 1998 ) and also by those that activate the Gq protein pathway ( Lesage et al , 2000 ; Chemin et al , 2003 ). The inhibition of TREK channels by glutamate via the activation of group I Gq-coupled metabotropic glutamate receptors requires PTX-insensitive G proteins coupled to phospholipase C ( Chemin et al , 2003 ). TREK-1 is also activated by volatile anesthetics and suggested to be a target in the action of these drugs ( Patel et al , 1999 ). This paper definitively shows that TREK-1 plays a major role in the PUFAs/LPLs-induced neuroprotection against epilepsy and ischemia and that TREK-1-deficient mice display resistance to anesthesia.

Generation and characterization of TREK-1 null mice

The TREK-1 gene of mice was disrupted through homologous recombination using a Cre/loxp-based strategy ( Figure 1A ). The CRE-mediated excision of exon 3 led to the deletion of the first transmembrane domain of the TREK-1 channel. Heterozygous matings produced offspring with normal Mendelian ratios ( Figure 1B and C ). Homozygous (Trek1 −/− ) mutant mice were healthy, fertile and did not display any visible morphological differences. PCR amplification of testicular cDNA (a tissue where TREK-1 is abundant; Hervieu et al , 2001 ; Talley et al , 2001 ) showed that the null mutant only expressed a truncated transcript ( Figure 1D ). Sequencing of this transcript confirmed that it results from the deletion of the 311 nucleotides of the targeted exon ( Figure 1D ). The brain morphology of Trek1 −/− mice appeared normal. In brain regions known to express the KCNK2 gene, no TREK-1 messenger RNA was detected by in situ hybridization using a probe recognizing the 3′-end of the mRNA ( Figure 1E ). The absence of the TREK-1 protein in null mutants was confirmed by the lack of immunoreactivity to specific anti-TREK-1 antibody ( Maingret et al , 2000a ) in brain areas such as the cortex or the hippocampus where it is highly expressed ( Figure 1F ).

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Disruption of the KCNK2 gene. ( A ) Targeting vector (▸=loxp), native (WT) and recombined floxed (Flox) alleles. External probes used to characterize homologous recombination are designated as P1 and P2. Arrowheads (1–3) display locations of the primers used for PCR analysis of the different products. Double-headed arrows indicate the expected size of restriction fragments for Southern analysis (bg= Bgl II; b= Bam HI; e= Eco RI). ( B ) Southern blot analysis of Eco RI- and Bam HI-digested tail DNA from wild-type (+/+), heterozygous (+/−) or homozygous (−/−) KCNK2 mice probed with P1 and P2, respectively. ( C ) PCR amplification from tail genomic DNA. ( D ) PCR amplification from +/+, +/− and −/− mouse testis cDNA with primers surrounding the deletion. ( E ) In situ hybridization analysis shows the lack of mRNA expression in Trek −/− mouse brain on X-ray films. ( F ) Immunocytochemical TREK-1 staining in neocortex (Cx) and hippocampal CA3 subfield sections using a specific α-TREK-1 antibody ( Lauritzen et al , 2000 ).

The TREK-1 mutation did not interfere with the mRNA expression in brain and cerebellum of other K 2P channels and of the GABAα6 subunit whose deletion causes an increased expression of TASK-1, another K 2P channel ( Brickley et al , 2001 ) ( Figure 2A ). There was no compensatory upregulation of genes for other neuronal K 2P channels such as TWIK-1, TREK-2, TRAAK, TASK-1, TASK-3 or the GABAα6 subunit in Trek1 −/− mice ( P <0.01).

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Characterization of TREK-1 null mice. ( A ) Relative expression of TREK-1, TREK-2, TRAAK, TASK-1, TASK-3 and GABAα6 mRNA levels in brain and cerebellum from Trek1 −/− and Trek1 +/+ mice. Mean levels of gene expression, normalized to cyclophilin D, are displayed in arbitrary units on the vertical axis ( n =3 mice, P <0.01, Student's t -test). ( B ) Primary behavioral test battery showing the lack of abnormal phenotype in TREK-1-deficient mice. Results are expressed as mean±s.e.m. Statistical significance was set at P <0.05 (Student's t -test or a Mann–Whitney test).

Primary behavioral testings (see Supplementary Materials and methods ) showed that the TREK-1-deficient mice did not display any abnormal phenotype in appearance ( Figure 2B ). There was no difference in skin color, body tone or body weight. Trek1 −/− mice did not display any abnormalities in body position, respiration or spontaneous activity. Stereotypies or tremor were not observed. There was no difference in frequency and volume of defecation or urination. Locomotor activity of the Trek1 −/− mutant was not different from Trek1 +/+ control in the open field test as well as in the rotarod. No difference was seen in the touch escape response or in the positional passivity test. Recordings of reflexes and autonomic functions did not show any significant differences. Scorings were comparable in the visual placing test, grip strength, corneal and pinna reflex and in the righting reflex. No significant difference was seen between Trek1 +/+ and Trek1 −/− mice in the object recognition test.

For comparative purpose, we have also deleted the TRAAK gene (see Supplementary Materials and methods and Supplementary Figure 1 ) to be able to evaluate the respective properties of Trek1 −/− and Traak −/− mice. The TRAAK channel is closely related to the TREK-1 channel. Like TREK-1, it is a background outward rectifier K + channel, opened by membrane stretch, cell swelling and activated by PUFAs and LPLs. However, unlike TREK-1, the TRAAK channel is not activated by intracellular acidification ( Maingret et al , 1999b ) nor volatile anesthetics ( Patel et al , 1999 ) and not inhibited by neurotransmitters that increase cAMP via a protein kinase A-dependent phosphorylation process ( Fink et al , 1998 ; Maingret et al , 1999a ) or by those that activate the Gq protein pathway ( Chemin et al , 2003 ).

Electrophysiological recordings

To test whether TREK-1 currents could be recorded in neurons from wild-type mice and were absent in neurons from TREK-1 null mice, we performed patch clamp recordings in striatal neurons in culture. These neurons were chosen because they strongly express TREK-1 but not TREK-2 or TRAAK channels ( Hervieu et al , 2001 ; Talley et al , 2001 ), two K 2P channels that are also activated by membrane stretch, PUFAs and LPLs ( Lesage and Lazdunski, 2000 ; Lesage et al , 2000 ; Patel and Honoré, 2001 ). In the striatum, the primary type accounting for 85% of the neurons is the GABAergic medium-size spiny neuron ( Kita and Kitai, 1988 ). Using an antibody against GABA, we have checked that most neurons in our culture were indeed GABAergic (data not shown). The resting membrane potential of the striatal neurons from Trek1 +/+ and Trek1 −/− mice was not significantly different (Student's t -test, P =0.0586) with −47.2±1.6 mV ( n =30) and −51.5±7.5 mV ( n =26), respectively. Neurons with resting membrane potential less negative than −30 mV were discarded. Using the inside-out configuration and in the presence of K + channels blockers (TEA, 4-AP and glibenclamide), a native TREK-1-like current was regularly recorded in cultures from wild-type mice. This current was reversibly activated by 10 μM arachidonate (AA) ( Figure 3A ) and by internal acidification ( Figure 3B ), as previously described ( Maingret et al , 1999b , 2000b ). The conductance was 55.8±0.9 pS at +50 mV ( n =6), which is close to the conductance of the cloned TREK-1 ( Patel et al , 1998 ). The outwardly rectifying current reversed around the potassium equilibrium potential ( Figure 3C ). Like TREK-1 ( Patel et al , 1998 ), the native current was also activated by membrane stretch ( Figure 3D ). The effect of volatile anesthetics was also studied on the TREK-like current recorded in striatal cultures from wild-type mice ( Figure 3E , inset) and in TREK-1-transfected COS cells ( Supplementary Figure 2A and B ). Halothane in striatal neurons ( Figure 3E , inset) as well as halothane and sevoflurane in COS cells ( Supplementary Figure 2A and B ) highly stimulated a TREK-1 channel activity. The loss of functional TREK-1 channels in TREK-1 null mutants was demonstrated by outside-out patch clamp recordings in striatal neurons. Figure 3E and F shows that in the presence of TEA and 4-AP to block voltage-dependent K + channels, there was no expression of basal current in wild-type neurons and in null mutants. Upon perfusion with the TREK-1 activator AA (20 μM), a robust TREK-1-like current was recorded in Trek1 +/+ neurons, whereas no significant variation was observed in Trek1 −/− neurons. This electrophysiological analysis confirmed (i) that the TREK-1 deletion had taken place and (ii) that there was no compensatory upregulation of genes for other neuronal K 2P channels.

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Patch clamp recordings in striatal neurons from Trek1 +/+ and Trek1 −/− mice. ( A ) Activation of the TREK-like current by 10 μM AA. ( B ) Activation of the TREK-like current by internal acidification to pH 5.5. Currents in (A, B) were recorded in inside-out configuration at 0 mV. ( C ) Single channel currents recorded as in (A) at various potentials as indicated. ( D ) Activation by membrane stretch recorded as in (A) at various negative pressures as indicated. ( E ) Typical TREK-like current recorded in outside-out configuration before (control) and after activation by 10 μM AA in striatal neurons from wild-type mice (WT). Values are average of two consecutive current traces elicited with voltage ramps starting from 0 mV down to −120 mV, from a holding potential of 0 mV. Inset: Effect of 2 mM halothane (hal) on TREK-like activity recorded at 0 mV in outside-out configuration. ( F ) Same recordings in neurons from TREK-1 knockout mice (KO).

Role for the TREK-1 channel in the control of epileptogenesis

The high level of TREK-1 channel expression in the cortex and thalamic nuclei and its colocalization on GABAergic cortical and hippocampal interneurons, which are inhibitory to pyramidal cell activity ( Hervieu et al , 2001 ; Talley et al , 2001 ), suggest a possible involvement of the TREK-1 channel in the control of epileptic seizures. To analyze the seizure susceptibility of Trek-1-deficient mice, we used the response to kainic acid (KA, an agonist of glutamate receptor) and to pentylenetetrazol (PTZ, a GABA A receptor antagonist), as an overall index of neuronal network excitability. Trek1 +/+ and Trek1 −/− mice were injected intraperitoneally with epileptogenic doses of KA (22 mg/kg) or PTZ (40–55 mg/kg) and the degree of seizures was scored ( Tsirka et al , 1995 ). Trek1 −/− mice were much more vulnerable to KA-induced seizures than Trek1 +/+ mice as assessed by either seizure score or mortality rate ( Figure 4A ). More than 75% of the mutant mice died within 3 days of KA administration, compared with 3% of Trek1 +/+ mice, and the average maximum intensity of seizures observed in Trek1 −/− mice increased by 33%. A comparison of electroencephalogram (EEG) patterns in the hippocampus of Trek1 +/+ and Trek1 −/− mice is shown in Figure 4F . A spectral analysis of EEG activity shows that 45 min following KA treatment (22 mg/kg), Trek1 −/− mice developed generalized convulsive seizures with the appearance of bilateral spike-wave discharges with spike frequencies and amplitudes higher than in Trek1 +/+ mice ( Figure 5A and B ).

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Increased susceptibility to epileptic agents in TREK-1-deficient mice. ( A, B ) Seizure behavior and mortality rate in wild-type and mutant TREK-1 mice after KA (A) or PTZ injection (B). ( C, D ) Seizure behavior and mortality rate in wild-type and mutant TRAAK mice after KA (C) or PTZ (D) injection. Seizures were scored for 2 h after intraperitoneal injection with KA (22–28 mg/kg) or PTZ (40–55 mg/kg). Seizures were ranked as follows: 1, immobility; 2, myoclonic jerks of the neck and head with brief twitching movements; 3, unilateral clonic activity; 4, bilateral forelimb tonic and clonic activity; 5, generalized tonic–clonic activity with loss of postural tone including death from continuous convulsions. Values represent mean±s.e.m. of the maximum seizure intensity recorded for each mouse ( n =20 per genotype). * Significantly different from vehicle-treated wild type (KA treatment 22 mg/kg), ** P <0.001, *** P <0.0001, ANOVA followed by Tukey's multiple comparison test. ( E ) Increased expression of c -fos protein in CA3 pyramidal neurons in Trek1 −/− mice 120 min after KA treatment (22 mg/kg). ( F ) EEG following KA (22 mg/kg) showing the increased KA susceptibility of Trek1 −/− mice as compared to Traak −/− mice ( G ) ( n =10 per genotype).

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Spectral profiles of EEG recordings following KA (22 mg/kg) injection in Trek and Traak mice. ( A ) Increased KA susceptibility of Trek1 −/− mice. ( B ) No anticonvulsive effect of LIN injection in Trek1 −/− mice. ( C ) No difference in KA susceptibility between vehicle-treated Traak −/− (KO) and Traak +/+ (WT) mice. Spectral profiles of EEG recordings ( n =10 per genotype and treatment) are shown 15 and 45 min following KA injection in vehicle-treated Trek1 +/+ and Trek1 −/− mice and 45 min following KA injection in LIN (500 nmol/kg)-treated Trek1 +/+ and Trek1 −/− mice. Spectral profiles of EEG recordings in vehicle-treated Traak mice are shown 45 min following KA injection.

Trek1 −/− mice also showed an increased sensitivity to PTZ-induced seizures ( Figure 4B ). Unlike the slow progression of motor symptoms observed in the KA-induced seizures, PTZ induced abrupt general tonic–clonic seizures within 5 min of injection. At a dose of 55 mg/kg, more than 90% of Trek1 −/− mice died from continuous tonic–clonic convulsions, whereas 60% of Trek1 +/+ mice survived ( Figure 4B ).

Activation of c -fos , in regions susceptible to kainate injection, is routinely used as a biochemical marker of neuronal excitability ( Smeyne et al , 1992 ). The expression of the c -fos protein was drastically enhanced in Trek1 −/− mice compared to Trek1 +/+ mice, particularly in CA3 subfield at 120 min after KA injection ( Figure 4E ).

A comparative study was carried out with the TRAAK channel. TRAAK-deficient mice did not display an increased sensitivity to epilepsy ( Figures 4C, D and G and ​ and5C). 5C ). Taken together, all these results show that, unlike TRAAK null mice, TREK-1-deficient mice are hypersensitive to kainate and PTZ-induced seizures and point to TREK-1 as a key target for epileptogenesis.

TREK-1 channel in brain and spinal chord ischemia and its major role in the neuroprotection provided by PUFAs and LPLs

Linolenic acid (LIN) or lysophosphatidylcholine (LPC) at a dose of 500 nmol/kg injected 30 min before the KA administration induced a potent decrease of the seizure activity in Trek1 +/+ mice but had no effect in Trek1 −/− mice ( Figure 6A and B ). The seizure score or the mortality rate shows that LIN- or LPC-injected Trek1 +/+ mice were much less vulnerable to KA-induced seizures than vehicle-injected Trek1 +/+ mice, while LIN- or LPC-injected Trek1 −/− mice were not protected ( Figure 6A ). More than 78% of the mutant mice treated with LIN or LPC died within 3 days of KA22 administration, compared with 3% of LIN- or LPC-injected Trek1 +/+ mice, and the average maximum intensity of seizures observed in treated Trek1 −/− mice increased by 38%. EEG patterns in the hippocampus of Trek1 +/+ and Trek1 −/− mice treated with LIN ( Figure 6B ) and their spectral analysis of EEG activity ( Figure 5B ) confirm the lack of efficiency of LIN treatment in null mutant mice. The same protocol applied to TRAAK mice showed no difference in the neuroprotective effect of LIN or LPC between Traak +/+ and Traak −/− mice (data not shown). This strongly suggests that the antiepileptic effect of PUFAs or LPLs is directly related to the activation of the TREK-1 channel.

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Increased vulnerability of TREK-1-deficient mice to ischemia and loss of the neuroprotective effect of LIN and LPC in Trek1 −/− mice. ( A ) Effect of LIN or LPC injection (500 nmol/kg) 10 min before KA treatment. ( B ) EEG recordings (15 and 45 min after KA treatment) in Trek1 +/+ and Trek1 −/− mice with or without LIN (500 nmol/kg). ( C ) Increased mortality rate in vehicle (Veh)-, LIN- or LPC-treated Trek1 −/− mice following 30 min global ischemia ( n =20 per genotype). LIN and LPC were injected at a concentration of 500 nmol/kg 30 min before ischemia. * Significantly different from vehicle-treated wild type (KA treatment 22 mg/kg), # significantly different from vehicle-treated wild type (KA treatment 28 mg/kg), ** P <0.001, *** P <0.0001, ### P <0.0001, ANOVA followed by Tukey's multiple comparison test.

Another important cause of neuronal damage is ischemia. Trek1 +/+ and Trek1 −/− mice were submitted to a transient bilateral occlusion of common carotid arteries (CCAs) during systemic hypotension (mean arterial blood pressure (MABP) 30±3 mmHg) maintained for 30 min. Trek1 +/+ mice presented no sign of hyperexcitability in the days following a 30 min period of ischemia. In contrast, most of the knockout mice developed seizures of progressive severity during the same time of reperfusion. More than 70% of Trek1 −/− mice died in the 3 days after ischemia compared with 34% of Trek1 +/+ mice ( Figure 6C ; P <0.001). LIN or LPC (500 nmol/kg) injected 30 min before the induction of global ischemia had no effect in Trek1 −/− mice, while it protected the Trek1 +/+ mice against neuronal death and significantly increased their survival ( Figure 6C ). This observation strongly suggests that the neuroprotective effect of PUFAs or LPLs against global ischemia is directly related to the activation of the TREK-1 channel. The specificity of the TREK-1 channel in neuroprotection against ischemic injury is strengthened by results obtained with TRAAK-deficient mice, which did not display an increased sensitivity to ischemia ( Figure 6C ).

We also analyzed the role of TREK-1 in spinal cord ischemia. It is a devastating complication with resulting paraplegia, observed after repair of thoracic or abdominal aortic aneurysms or dissection ( Kouchoukos and Dougenis, 1997 ). Combined occlusion of the aortic arch and left subclavian artery was performed to induce spinal cord ischemia in mice ( Lang-Lazdunski et al , 2000 ). Values of mean femoral arterial blood pressure (MABP) recorded for 5 h throughout the procedure did not differ significantly between Trek1 +/+ and Trek1 −/− mice ( Table Ia ). The susceptibility to spinal cord ischemia was much higher in null allele mice. A total of 75% of Trek1 −/− mice died within the first 3 h following 10 min ischemia compared with 14% of Trek1 +/+ mice up to 24 h after the procedure ( Table Ib ; P <0.001). All surviving Trek1 +/+ mice recovered without any neurological deficit and failed to develop any form of neurological deficit during the subsequent 48 h. In contrast, surviving Trek1 −/− mice developed severe hind limb paralysis at the onset of reperfusion. They remained paralyzed during the first hours of reperfusion and retained deficits in motor function during the subsequent 48 h ( Table Ib ). Within 5 min following aortic crossclamping, Trek1 −/− mice had vesical relaxation with urination, which did not occur in Trek1 +/+ mice, further indicating a lower tolerance to spinal cord ischemia. Autopsies of Trek1 −/− mice did not reveal any severe abnormality in heart, lungs or major vessels.

Comparison of susceptibility to spinal cord ischemia in wild-type and TREK-1-deficient mice

TREK-1 channel in the mechanism of action of volatile anesthetics in vivo

Another interesting property of the TREK-1 channel concerns its sensitivity to activation by general volatile anesthetics ( Patel et al , 1999 ), and we hypothesized ( Patel et al , 1999 ) that TREK-1 might be involved in the mechanism of action of these agents. The comparative sensitivity to different volatile anesthetics of Trek1 +/+ and Trek1 −/− mice was assessed by comparing the onset of anesthetic action, the loss of righting reflex (LORR) and the inspired minimum alveolar anesthetic concentration (MAC) values for each anesthetic in both. MAC is the minimum steady-state alveolar concentration of an inhalational anesthetic required to suppress a strong motor reaction to the noxious stimulus of tail-clamping in 50% of mice ( Quasha et al , 1980 ). Figure 7A shows that knockout mice had a decreased sensitivity to chloroform and halothane, which are the most potent activators of the TREK-1 channel in vitro ( Patel et al , 1999 ). Interestingly, the same type of results was obtained with sevoflurane and desflurane ( Figure 7B ), the most widely used agents in clinical anesthesia as well as isoflurane ( Supplementary Figure 2C ). The period of time necessary for the induction of anesthesia was longer, the concentrations required for LORR lower and the partial pressures of all anesthetics tested (i.e. MAC) were higher in Trek1 −/− mice. There was no significant difference in the respiratory rate between either genotype before induction of anesthesia and at the MAC value ( Table II ). In contrast with volatile anesthetics, no difference was seen between Trek1 +/+ and Trek1 −/− mice upon injection of the barbiturate pentobarbital ( Figure 7C ), which produces anesthesia by acting on different GABA A receptor subunits ( Yamakura et al , 2001 ) and in vitro it has no effect on TREK-1 channel activity ( Figure 7C ), unlike halothane and sevoflurane ( Supplementary Figure 2A and B ). Pentobarbital did not affect the latency or the duration of LORR ( Figure 7C ) in null mutants. This latter result supports the idea that the differences observed are specific to volatile anesthetics and related to the TREK-1 channel.

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Effects of different anesthetics on LORR and MAC in Trek1 +/+ and Trek1 −/− mice. LORR measurements after inhalation of volatile anesthetics. Latency to LORR is defined as the period of time (s) from inhalation to the LORR. Concentration for LORR corresponds to average concentrations of volatile anesthetics ( A ) chloroform and halothane and ( B ) sevoflurane and desflurane for the recovery from LORR. ( C ) LORR measurements (latency and duration of LORR expressed in minutes) after pentobarbital injection (30 mg/kg). Lack of effect of pentobarbital (2.4 mM) on TREK-1 channel expressed in transfected COS cells. I – V curves in steady-state control condition and after a 5 min application of pentobarbital (2.4 mM). I – V curve was elicited by a voltage ramp (1 s duration from −130 to +100 mV). Data represent mean±s.e.m. ( n =20 per genotype and anesthetic agent). Statistical significance (Student's t -test): ** P <0.001, *** P <0.0001. Logistic regression probability of no movement fitted for volatile anesthetic concentrations. MAC and its 95% confidence interval (horizontal line) are shown on each graph.

Respiratory rate (beats/min) of wild-type and TREK-1-deficient mice before the induction of anesthesia and at the MAC value

Potassium channels play a major role in the control of K + homeostasis and in physiological and pathological functions that are associated with modifications of the electrical membrane potential. Many subtypes of K + channels have been cloned in the past decades ( Salkoff et al , 1992 ; Jan and Jan, 1997 ; Pongs, 1999 ; Kurachi et al , 1999 ). The mammalian two-pore-domain K + channel family ( Lesage and Lazdunski, 2000 ; Patel and Honoré, 2001 ; Lesage, 2003 ), and particularly the TREK-1 channel, has been proposed to play a key role in brain and spinal chord injuries ( Lauritzen et al , 2000 ; Blondeau et al , 2002 ; Lang-Lazdunski et al , 2003 ). The lipid and mechano-gated TREK-1 channel is closely related to pathophysiological conditions, such as ischemia and epilepsy. It is activated by arachidonic acid and other PUFAs, LPLs, cell volume expansion and internal acidosis. During the process of ischemia, arachidonic acid is released from the plasma and intracellular pH is decreased. These condition changes could potently activate the lipid-sensitive mechano-gated K 2P channels, an activation that would occur to protect the neuronal cell against excessive and deleterious neuronal excitability and Ca 2+ entry. On the other hand, the TREK-1 channel is inhibited by the activation of group I metabotropic glutamate receptors, known to be involved in brain disorders, including ischemia, epilepsy and neurodegenerative disorders ( Bockaert et al , 1993 ; Bordi and Ugolini, 1999 ; Fagni et al , 2000 ). Group I metabotropic glutamate receptor antagonists are neuroprotectors, while agonists amplify the excitotoxic neuronal degeneration induced by glutamate ( Nicoletti et al , 1996 ; Gasparini et al , 2002 ). In fact, injections of PUFAs and LPLs protect against brain and spinal chord ischemia as well as epileptic seizures ( Lauritzen et al , 2000 ; Blondeau et al , 2001 , 2002 ; Lang-Lazdunski et al , 2003 ). Riluzole, another activator of TREK-1 channel ( Duprat et al , 2000 ), is also neuroprotective against ischemia ( Pratt et al , 1992 ; Ettaiche et al , 1999 ; Lang-Lazdunski et al , 1999 ). Although the opening of lipid-sensitive mechano-gated K 2P channels has been presumed to be the significant factor in neuroprotection, the lack of specific blockers did not allow until now a direct demonstration of this property. Using mice with disrupted TREK-1 and TRAAK genes, the present study provides evidence for a major role of the TREK-1 channel in surviving excessive neuronal excitability and in resistance to forebrain and spinal cord ischemia. The absence of an increased sensitivity to ischemia and epilepsy in Traak −/− mice demonstrates that the extreme vulnerability of Trek1 −/− mice is not a nonspecific effect due to the lack of an important K + channel on neuronal excitability. Consequently, the TREK-1 channel can be considered to play a key role in the regulation of neuronal excitability. The high expression of the TREK-1 protein both pre- and postsynaptically in the cortex and thalamic nuclei is consistent with a potential role for this channel in prevention of epileptic seizures. The high levels of TREK-1 expression in the hippocampus, a structure susceptible to damage during ischemia, and its modulation by neurotransmitter receptor activation are supplementary arguments for a major role of this channel in the control of excitotoxicity. Its activation in the neurons would be expected to hyperpolarize synaptic terminals, decreasing glutamate release and/or producing a postsynaptic hyperpolarization, which would favor the blockade of the NMDA receptor-associated channel by Mg 2+ and also counterbalance glutamate-induced depolarization on other types of ionotropic glutamate receptors ( Lauritzen et al , 2000 ). Without excluding a localization of the TREK-1 protein in glutamatergic neurons ( Lauritzen et al , 2000 ), the TREK-1 channel has been described to be colocalized in GABAergic interneurons, specifically from striatum (this work), cerebellum, cortex and hippocampus ( Hervieu et al , 2001 ). The phenotype of extreme vulnerability of TREK-1 null mutants against epilepsy and ischemia is consistent with the absence of TREK-1 channel in GABAergic interneurons, known to serve inhibitory functions in CNS and be involved in ischemic and epileptic disorders ( Treiman, 2001 ; Wang, 2003 ). In the light of the role of TREK-1 channels in setting resting membrane potential, this is suggestive that TREK-1 may set the membrane potential of interneurons and thereby contribute to their often distinctive neurophysiological properties.

The beneficial effects of PUFAs on human health have long been advocated ( Leaf and Kang, 1996 ; Nair et al , 1997 ; Leaf et al , 1999 ; Nordoy, 1999 ; Stoll et al , 1999 ) and indeed the effects of PUFAs on neuroprotection against epilepsy and ischemic paradigms in animals are spectacular ( Lauritzen et al , 2000 ; Lang-Lazdunski et al , 2003 ). The results presented here strengthen the idea that neuroprotection induced by PUFAs (and LPLs) against seizures and ischemia is related to their action on the TREK-1 channel since this neuroprotection disappears in Trek1 −/− mice and open the way for a novel neuroprotective strategy.

The possibility that a significant part of the effects of general anesthetics might result from potassium channel activation and especially K 2P channels has been previously suggested ( Patel et al , 1999 ). This work definitively shows that the deletion of the TREK-1 gene induces a resistance to volatile anesthetics. This resistance is actually the greatest found for any ion channel knockout tested, including knockouts of GABA A receptors ( Campagna et al , 2003 ), also believed to be potential targets of volatile anesthetics. One might of course wonder why the deletion of TREK-1 does not completely abolish sensitivity to volatile anesthetics. An important reason is that volatile anesthetics such as halothane, desflurane and sevoflurane also activate other K 2P channels such as TREK-2 and TASK channels ( Patel et al , 1999 ; Lesage et al , 2000 ), which are still expressed in the Trek1 −/− mice. It will be important in the future to analyze multiple K 2P channel knockouts, which would then be expected to display extreme resistance to volatile anesthetics. Further experiments using selective Cre mice to abolish specifically the gene in a tissue or a cell type will also permit a more detailed analysis of the cellular mechanisms that underlie the behavioral responses.

In conclusion, this work provides evidence for a major involvement of the TREK-1 channel in the control of the neuronal excitability and neuroprotective effects induced by PUFAs and LPLs against ischemia and epileptic seizures. TREK-1 appears to be an innovative target for the development of novel therapeutic neuroprotective strategies for brain pathologies.

Materials and methods

All experiments were conducted according to the policies on the care and use of laboratory animals of the Society of Neurosciences.

Generation of TREK-1-deficient mice

Trek-1 genomic clones were isolated from a 129 mouse genomic library by using a TREK-1 cDNA probe and subcloned into pBluescript SK (Stratagene). The floxed targeting vector was generated from a 7.5 kb Bgl II/ Eco RI restriction fragment containing exons 1–3 of the KCNK2 gene. The vector was designed to allow CRE-mediated deletion of exon 3, which encodes the TM1 domain of the channel. The first loxp sequence was inserted in the 5′ flanking intron of exon 3. Similarly, the PGK-neomycin resistance cassette (neo) was inserted together with a second loxp sequence in the 3′ flanking intron of exon 3. Both loxp sequences were in the same orientation to allow CRE-mediated simultaneous excision of Exon 3 and neo cassette. A copy of the diphteric toxin gene was subcloned adjacent to the homologous region for negative selection of the ES clone. The targeting vector (50 μg) was linearized prior to electroporation into 129-derived embryonic stem cells. After drug selection (G-418, 350 μg/ml), one positive clone (1/288) was identified by Southern blot and PCR analysis. Five highly chimeric males were generated by injection of the targeted ES cells into C57Bl/6J blastocysts. They were mated with C57Bl/6J females and germline transmission was assessed by Southern blot and PCR analysis of tail DNA from the agouti pups. TREK-1 floxed mice were then crossed with mice carrying the CRE recombinase gene under the control of the ubiquitous CMV promoter (D Metzger). Heterozygous TREK-1-deficient mice were then backcrossed with C57Bl/6J congenic mice over 11 generations. All animals (+/+ and −/−) were 8- to 10-week-old males of N6F2 to N11F2 backcross generation.

Kainate and pentylenetetrazol administration

After intraperitoneal injection of KA at 22 or 28 mg/kg, mice ( n= 20 per group) were monitored for 2 h for onset and extent of seizures. Seizure severity was blindly scored ( Tsirka et al , 1995 ). PTZ was injected similarly at 40 or 55 mg/kg and seizures were scored based on the highest degree of seizure within 15 min of the PTZ injection. The seizure index was calculated by averaging the points for seizure activity in each group ( n= 20 per genotype and treatment). EEGs were recorded for 2 h on conscious mice ( n= 10 per genotype and treatment) using four small platinum electrodes (diameter 0.28 mm) placed in the hippocampus (1.2 mm lateral, 1.6 mm posterior to the bregma, 1.6 mm inside) and in the anterior neocortex (2 mm lateral, 0.5 mm anterior to the bregma, 1.5 mm inside). The signals were amplified, digitized and quantified using the Galileo system (Sirius BB, Medical Equipment International).

Forebrain ischemia model (2 VO+hypotension)

Global ischemia ( n= 20 per genotype and treatment) was induced by occluding both CCAs with aneurysm clips (Aesculap, Germany) during a 30 min episode of systemic hypotension induced by withdrawal of blood to maintain an MABP of 30±3 mmHg ( Sheng et al , 1999 ).

Spinal cord ischemia model

Mice were subjected to crossclamping of the aortic arch, left subclavian artery and internal mammary artery for 10 min ( Lang-Lazdunski et al , 2000 ). Motor function was blindly evaluated in the hind limbs using a rating scale of 0 (normal function) to 6 (total absence of movement) ( Lang-Lazdunski et al , 2000 ).

Behavioral studies of sensitivity to anesthetic agents

Loss of righting reflex . Unrestrained mice ( n =10 per genotype and volatile anesthetic) were placed in a chamber maintained at 33–35°C. Carbon dioxide pressure (<0.05 atm) and rectal temperature (36.5±1.2°C) were controlled. Each volatile anesthetic (chloroform, halothane, isoflurane, sevoflurane and desflurane) was administered with a calibrated vaporizer in 100% oxygen as the carrier gas with a fresh gas flow of 2 l/min at initial concentrations of 3.0, 1.2, 1.0, 1.8 and 5%, respectively. Concentrations of the volatile anesthetic were continuously measured by using a calibrated infrared analyzer (RGM 5250, Ohmeda, Louisville). After equilibration for 20 min at each initial anesthetic concentration, mice were blindly scored for LORR. The concentration of the anesthetics was then decreased in 10–20% increments and allowed to re-equilibrate at each concentration. Mice were observed continuously for recovery of the righting reflex. The concentration reported for LORR was calculated by averaging the two concentrations at which the mouse either retained or lost the righting reflex. Data were reported as mean±s.e.m. Differences were evaluated using an unpaired t -test.

Tail-clamp/withdrawal assay . MAC was determined using the tail-clamp technique ( Quasha et al , 1980 ). Mice ( n =20 per genotype and volatile agent) were first exposed for 20 min to a constant anesthetic concentration of almost 50% anesthetic induction values used in clinical practice. A hemostatic clamp was applied for 45 s to the midportion of the tail. Mice were scored blind for a motor withdrawal in response to clamping the tail. A mouse was considered to have moved if it made a purposeful muscular movement of the hind limb and/or the body. The anesthetic concentration was decreased in steps of 0.1% for each anesthetic, and the testing sequence was repeated after 20 min of exposure to each concentration. Concentration–response data were fitted to a logistic equation, yielding half-effect concentrations (median MAC values), slopes and estimates of their respective standard errors. Median MAC values were given with their respective 95% confidence interval limits. All P -values were two-tailed, and a P -value <0.05 was considered significant.

Sleep time assay (i.e. duration of the LORR) . Mice ( n =20 per genotype and anesthetic agent) were blindly tested for the duration of LORR (i.e. sleep time) in response to an intraperitoneal injection of pentobarbital (30 mg/kg). Mean sleep times for each agent were compared in null allele and wild-type mice using an unpaired t -test.

Onset of volatile and intravenous anesthetic action (i.e. latency to the LORR) Mice ( n =10 per genotype and anesthetic agent) were exposed to 8% chloroform, 4% halothane, 8% sevoflurane, 3% isoflurane or 10% desflurane in the same chamber used for LORR and tail-clamp assays. Onset of anesthetic action was defined as the time interval between the beginning of the anesthetic inhalation or the injection of the intravenous agent and the LORR.

Electrophysiology on COS cells

COS cells were seeded at a density of 20 000 cells per 35-mm dish 24 h before transfection. Cells were transiently transfected by the classical DEAE–dextran method with 0.1 μg pCI-mTREK-1+0.05 μg pCI-CD8. Transfected cells were visualized 48 h after transfection using anti-CD8 beads. The external solution contained (in mM) 140 NaCl, 5 KCl, 2 MgCl 2 , 2 CaCl 2 and 10 HEPES, adjusted to pH 7.4 with NaOH. The pipette solution contained (in mM) 140 KCl, 4 MgCl 2 , 5 EGTA and 10 HEPES (pH 7.2). The cell under study was continuously superfused with a microperfusion system (0.1 ml/min) at room temperature.

Electrophysiology on mouse striatal neurons

Primary culture of mouse striata was carried out according to Weiss et al (1986) . Cells were plated in culture dishes previously coated with polyornithin and 50% fetal calf serum. Culture medium was DMEM plus glucose (1.5 g/l) for the first 24 h, then B27 plus uridine (2 μM) and 5-fluoro-2′-deoxyuridine (2 μM). Patch clamp measurements were performed 2 or 3 days after plating. In outside-out configuration, the internal solution contained (in mM) 155 KCl, 3 MgCl 2 , 5 EGTA, 10 HEPES and 5 ATP-K + (pH 7.2) and the external solution contained (in mM) 120 NaCl, 5 KCl, 3 MgCl 2 , 1 CaCl 2 , and 10 HEPES. We daily prepared and added to the external solutions 10 mM tetra-ethyl-ammonium chloride, 3 mM 4-aminopyridine, 10 μM glibenclamide and 5 mM glucose (pH at 7.4). TREK current anesthetic sensitivity was assessed in striatal neurons and in TREK-1-expressing COS cells ( Patel et al , 1999 ).

DNA extraction

Tail biopsy was lysed with proteinase K (200 μg/ml) for 5–12 h at 56°C in buffer containing 100 mM Tris (pH 8.5), 200 mM NaCl, 5 mM EDTA and 0.2% SDS. Proteinase K was heat inactivated at 95°C for 5–10 min and the lysate was then either diluted in water for PCR amplification or centrifuged to get rid of undigested material prior to ethanol precipitation for subsequent digestion by restriction enzymes.

Southern blot

For Southern blotting, genomic DNA was digested overnight with the appropriate restriction enzyme, precipitated, size fractionated on a 0.6% agarose gel and transferred onto a nylon membrane in 0.4 M NaOH. 32 P-labelled probe hybridization was carried out overnight at 65°C in 0.5 M Na 2 Pi/5% SDS, pH 6.8.

PCR analysis

PCR reactions were performed on 1 μl of a 20–30 times water dilution of the crude tail lysate in 15 μl final volume containing 67 mM Tris–HCl (pH 8.8), 16 mM (NH 4 ) 2 SO 4 , 0.01% Tween 20, 1.5 mM MgCl 2 , 200 mM dNTP and 0.2 μl Taq polymerase (Eurobio). Conditions were as follows: for TREK-1, 94°C/3 min≫(94°C/20 s≫58°C/20 s≫72°C/35 s) × 33, oligos (see Figure 1 ) #1 (5′GGT GCC AGG TAT GAA TAG AG3′), #2 (5′TTC TGA GCA GCA GAC TTG G3′), #3 (5′GTG TGA CTG GGA ATA AGA GG3′); for TRAAK, 94°C/3 min≫(94°C/30 s>63.5°C/25 s>72°C/35 s) × 35, primers #1 (5′CCCTGCTCCTTCTTCCC3′), #2 (3′ATTCTTCCTTCCTCCCTTCC5′), #3 (5′TGGACGAAGAGCATCAGGG3′), #4 (5′GAGGAGCAGCCAACTTTAGC3′) (see Supplementary Figure 1 ).

In situ hybridization

Perfused brain sections were hybridized with specific oligonucleotide 3′-end-labelled probes (nucleotides 726–694 and 1536–1504 of the cloned mouse TREK-1; GenBank accesssion number {"type":"entrez-nucleotide","attrs":{"text":"U73488.2","term_id":"4584798","term_text":"U73488.2"}} U73488.2 ).

Immunohistochemistry

Immunostainings were performed on floating brain sections (50 μm) using the anti-rabbit α-TREK-1 ( Lauritzen et al , 2000 ) and c-fos (Oncogene) rabbit polyclonal antibodies. Sections were floated in a solution of the primary antibody overnight at 4°C (1:200 dilution). Biotinylated secondary antibodies were amplified using a rabbit IgG Vector Elite ABC kit (Vector laboratories) with 3-diaminobenzidine as substrate.

TaqMan assays (real-time quantitative RT–PCR analysis)

Total RNA from the brain and cerebellum of Trek1 −/− and Trek1 +/+ mice was isolated by using the Trizol method (InVitrogen). Reverse transcription was performed with 2 μg of total RNAs, treated for 30 min with RQ1 DNase I (Promega) and reverse-transcribed with Superscript II reverse transcriptase (InVitrogen). Real-time PCR analysis (SYBR Green Mastermix Plus, Eurogentec) was performed to estimate the level of expression of TREK-1, TREK-2, TRAAK, TASK-1, TASK3, TWIK-1 and GABAα6 subunit in the brain and cerebellum of Trek1 −/− and Trek1 +/+ mice. Primers for the seven different amplicons were as follows:

TREK-1 forward TTTTCCTGGTGGTCGTCCTC;

TREK-1 reverse GCTGCTCCAATGCCTTGAAC;

TREK-2 forward CCGGAATTACTCTCTGGATGAAGA;

TREK-2 reverse CATGGCTGTGCTGGAGTTGT;

TRAAK forward CCCCAGTGAGAATCTGGCC;

TRAAK reverse GGGCACAGCCACGCTC;

TASK-1 forward CGGCTTCCGCAACGTCTAT;

TASK-1 reverse TTGTACCAGAGGCACGAGCA;

TASK-3 forward GACGCCCTCGAGTCGGACCA;

TASK-3 reverse CTCTGAGACGGACTTCTTC;

TWIK-1 forward TGTCCTTCTCCTCCGTCACTG;

TWIK-1 reverse AGGCCACAAAAGGCTCACTTT;

GABAα6 forward CGCCCCCTGTGGCAA;

GABAα6 reverse TACTTGGAGTCAGAATGCACAACA;

CYCLOPHILIN forward GGCTCTTGAAATGGACCCTTC;

CYCLOPHILIN reverse CAGCCAATGCTTGATCATATTCTT.

Real-time PCR assays for each gene target were performed on cDNA samples in 96-well plates on an ABI Prism 7700 Sequence Detection System (PE Biosystems). PCR data were captured using Sequence Detector Software. Data were analyzed using the comparative CT method where the amount of target was normalized to an endogeneous reference (cyclophilin D) and calibrated to the amount of target in wild-type mice (User Bulletin No. 2 Applied Biosystems). Experiments were performed in triplicate. Standard curves were generated for each set of primers using serial dilutions of mouse brain cDNA to ensure a high efficiency of amplification.

Supplementary Material

Supplementary Materials

Acknowledgments

This work was supported by the Centre National de la Recherche Scientifique (CNRS) and the Paul Hamel Institute. We are grateful to the Fondation de la Recherche Médicale and the Association Française contre les Myopathies for fellowships to M Mazzuca and C Laigle and to the Ministere de la Recherche et de la Technologie (ACI ‘Biologie du développement & physiologie intégrative'). We thank Dr J Barhanin and Dr E Honoré for fruitful discussions and Dr L Rash for a critical reading of the manuscript. We thank G Jarretou for his remarkable help in histological analysis, M Jodar for expert work in neuronal cultures and F Aguila and V Briet for their skillful technical assistance.

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Kenneth Mitchell, ‘Star Trek: Discovery’ actor, dies at 49

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Kenneth Alexander Mitchell, a Canadian actor known for roles on the series “Star Trek: Discovery ,” died on Saturday. He was 49.

His death was confirmed in statements Sunday on the Star Trek website and his personal Instagram page.

For more than five years, Mr. Mitchell had been battling ALS, or amyotrophic lateral sclerosis, the Instagram post read.

In “Star Trek: Discovery,” Mr. Mitchell played Klingon characters Kol, Kol-Sha and Tenavik, as well as Aurellio, a character who used a hovercraft wheelchair . In an Instagram post, he described Aurellio’s creation as a “special collaboration with my Discovery family that injected me with heaps of Love & Inspiration during my tough battle with ALS.”

Mr. Mitchell also appeared in the film “Captain Marvel” (2019), in which he played Joseph Danvers, the father to superhero Carol Danvers/Captain Marvel, and he had roles in the TV series “Nancy Drew” and “The Detectives.”

View this post on Instagram A post shared by Kenneth Mitchell (@mr_kenneth_mitchell)

In 2020, Mr. Mitchell opened up about his ALS diagnosis in an emotional interview with People , in which he shared that he was using a wheelchair.

“The moment that they told us it was [ALS], it was like I was in my own movie,” he said, holding back tears. “That’s what it felt like, like I was watching that scene where someone is being told that they have a terminal illness. It was just a complete disbelief, a shock.”

ALS affects the nerve cells that make the muscles function in the upper and lower parts of the body, and causes the cells to stop working and die, according to the Centers for Disease Control and Prevention . This rare neurological disease is also known as motor neuron disease or Lou Gehrig’s disease. The CDC estimates that there are about 30,000 cases of ALS in the United States.

Sandra Bullock’s partner died of ALS. What to know about the rare disease.

Last August, Mr. Mitchell marked five years living with ALS, thanking his friends and family in an Instagram post. “There is so much beauty in that. This disease is absolutely horrific … yet despite all the suffering, there is so much to be grateful for,” he wrote alongside a photo of himself on a beach.

Mr. Mitchell was born Nov. 25, 1974, in Toronto. He is survived by his wife, the actress Susan May Pratt; their two children, Lilah and Kallum; his parents, Diane and David Mitchell; and a brother.

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COMMENTS

  1. KCNK2

    KCNK2. Potassium channel subfamily K member 2, also known as TREK-1, is a protein that in humans is encoded by the KCNK2 gene. [5] [6] [7] This gene encodes K 2P 2.1, a lipid-gated ion channel belonging to the two-pore-domain background potassium channel protein family. This type of potassium channel is formed by two homodimers that create a ...

  2. Molecular regulations governing TREK and TRAAK channel functions

    Gene heterogeneity. TREK-1 (K 2p 2.1), 6 TREK-2 (K 2p 10.1), 17, 18 and TRAAK (TWIK Related Arachidonic acid Activated K + channel) (K 2p 4.1), 19 compose the TREK subfamily of K 2p channels. TREK-1 shares 63% identity and 78% homology with TREK-2. The identity falls to 45% and homology to 69% with TRAAK and to 50-55% homology with the other K 2P subunits. 17, 18 Recent findings have ...

  3. Star Trek Channel

    Star Trek Natures Hunger: "Scorned at the Captain's Table" — Season 7, Episode 1. Posted on December 31, 2015. Posted in Fan Film.

  4. Structural models of TREK channels and their gating mechanism

    Our goal is to build structural models of TREK-1 channel that can be used as a starting point for hypothesis-driven structural and functional experiments. If experimental tests confirm crucial aspects of the models, they can further be used as templates for modeling other channels in K2P family. Predicting the structure of TREK channels is a ...

  5. TREK channel activation suppresses migraine pain phenotype

    Therefore, targeting TREK channel intrinsic activity to reduce TG neuron excitability should be considered as an alternative strategy to treat migraine. Limitations of the study. This study was carried out in rodent models exhibiting an allodynic phenotype which is commonly used as a readout for the migraine-like phenotype studies in rodents ...

  6. Where to Watch

    Star Trek: Discovery Seasons 1 through 4 are currently streaming exclusively on Paramount+ in the U.S., the UK, Switzerland, South Korea, Latin America, Germany, France, Italy, Australia and Austria.Seasons two and three also are available on the Pluto TV "Star Trek" channel in Switzerland, Germany and Austria. In Canada, the series airs on Bell Media's CTV Sci-Fi Channel.

  7. TREK Channel Family Activator with a Well-Defined Structure-Activation

    TWIK-related K + (TREK) channels are potential analgesic targets. However, selective activators for TREK with both defined action mechanism and analgesic ability for chronic pain have been lacking. Here, we report (1 S ,3 R )-3-((4-(6-methylbenzo[ d ]thiazol-2-yl)phenyl)carb …

  8. Frontiers

    In this situation both channel types, if coexpressed, would tend to drive the cold receptor far from the threshold and hence we would expect the receptor to be hyperpolarized and silent. When the temperature is reduced (from 30 to 10°C), TREK channel activity would fall to zero and the activity of cold sensitive TRPs would strongly increase.

  9. TREK Channel Family Activator with a Well-Defined Structure-Activation

    TWIK-related K+ (TREK) channels are potential analgesic targets. However, selective activators for TREK with both defined action mechanism and analgesic ability for chronic pain have been lacking. Here, we report (1S,3R)-3-((4-(6-methylbenzo[d]thiazol-2-yl)phenyl)carbamoyl)cyclopentane-1-carboxylic acid (C3001a), a selective activator for TREK, against other two-pore domain K+ (K2P) channels ...

  10. Frontiers

    From a functional point of view, TREK-1 plays a critical role in countering the depolarizing effect of mechano-activated cationic currents, contributing to stimulation-activated central (heart) feedback mechanics in the cardiovascular system ().TREK-1 channels also have a potential role in regulating the normal activity of sinoatrial node-hosted pacemakers by preventing the occurrence of ...

  11. K2P2.1 (TREK-1) potassium channel activation protects against hyperoxia

    This idea is supported by our own data in alveolar epithelial cells (Fig. 5) showing that TREK-1 currents can readily be induced by our channel activators ML335 and BL1249 44,46,47, thus making ...

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

    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 p …

  13. TrekMovie.com

    TrekMovie.com is the source for Star Trek news and information, covering the latest updates on movies, TV shows, books, comics, merchandise, and more. Whether you are a fan of Picard, Riker, Seven ...

  14. TREK-1, a K+ channel involved in polymodal pain perception

    The TREK-1 channel is a temperature-sensitive, osmosensitive and mechano-gated K+ channel with a regulation by Gs and Gq coupled receptors. This paper demonstrates that TREK-1 qualifies as one of the molecular sensors involved in pain perception. TREK-1 is highly expressed in small sensory neurons, is present in both peptidergic and ...

  15. The Purified Mechanosensitive Channel TREK-1 Is Directly Sensitive to

    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.

  16. Trek (TV channel)

    Trek is a French themed television channel owned by Mediawan Thematics. History. Trek began broadcasting on February 2, 2015, replacing Escales. Trek was removed of Orange on 9 July 2020, and of Canal+ on 1 September 2020. Programming. The channel broadcasts programs dedicated to adventure, feats and thrills. ...

  17. 10 Star Trek Sequels To Past Episodes

    In Star Trek: The NextGeneration's outstanding "The Inner Light", an alien probe causes Captain Picard to live the life of Kamin, a family man on the dying planet Kataan, within the span of about 20 minutes.Picard's experience as Kamin is profound, but rarely addressed on-screen until "Lessons". Jean-Luc's romance with the musical head of stellar sciences, Lt. Commander Nella Daren (Wendy ...

  18. TV broadcasting in the trains of the Moscow Metro

    The Elecard company took part in the large-scale project on content preparation and broadcasting of 12 channels in the Moscow underground trains.

  19. The Star Trek episode 'banned' after predicting a 'united' Ireland

    A Star Trek episode released in 1990 has only ever been screened in Ireland once over concerns about a single line. The original season of the sci-fi series first hit screens in 1966 and ran for ...

  20. TREK-1 is a heat-activated background K+ channel

    Fig. 1. TREK-1 is a temperature-sensitive K + channel in Xenopus oocyte. (A) The two-microelectrode voltage-clamp technique was used to voltage-clamp oocytes expressing TREK-1.I-V curves of an oocyte expressing TREK-1 maintained at 12 and 37°C. The holding potential is -80 mV and increment voltage steps of 20 mV are applied every 5 s from -130 to 90 mV.

  21. Kenneth Mitchell Dead: 'Star Trek: Discovery' Actor Was 49

    Kenneth Mitchell, who played several characters in Star Trek: Discovery, and also was known for his roles in Jericho and Captain Marvel, has died from complications of ALS, his family revealed ...

  22. Kenneth Mitchell Dead: 'Star Trek: Discovery' Star Was 49

    Kenneth Mitchell, known for his multiple roles on "Star Trek: Discovery," died from ALS complications on Saturday. He was 49. "For five and a half years, Ken faced a series of awful ...

  23. Residents Outside Moscow Protest Power Outage, Demand Heating Amid

    The Telegram news channel Ostorozhno Moskva published a video of several local residents who gathered in the town's central square to demand the authorities restore their heating, as well as ...

  24. Kenneth Mitchell, Star Trek and Captain Marvel actor, dies aged 49

    The Canadian actor Kenneth Mitchell, known for roles in Star Trek: Discovery and the Marvel film Captain Marvel, has died following complications from amyotrophic lateral sclerosis, or ALS.

  25. The 'banned' Star Trek episode that promised a united Ireland

    At the time, US TV shows often debuted internationally several years after their original broadcast. Satellite broadcaster Sky reportedly aired an edited version in 1992, cutting the crucial scene.

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

    TREK-1 channel in brain and spinal chord ischemia and its major role in the neuroprotection provided by PUFAs and LPLs. Linolenic acid (LIN) or lysophosphatidylcholine (LPC) at a dose of 500 nmol/kg injected 30 min before the KA administration induced a potent decrease of the seizure activity in Trek1 +/+ mice but had no effect in Trek1 −/ ...

  27. Elektrostal

    History. It was known as Zatishye (Зати́шье) until 1928. [citation needed] In 1938, it was granted town status.[citation needed]Administrative and municipal status. Within the framework of administrative divisions, it is incorporated as Elektrostal City Under Oblast Jurisdiction—an administrative unit with the status equal to that of the districts.

  28. Kenneth Mitchell, 'Star Trek: Discovery' actor, dies at 49

    Kenneth Alexander Mitchell, a Canadian actor known for roles on the series "Star Trek: Discovery," died on Saturday. He was 49. His death was confirmed in statements Sunday on the Star Trek ...

  29. The 'banned' Star Trek episode that promised a united Ireland

    At the time, US TV shows often debuted internationally several years after their original broadcast. Satellite broadcaster Sky reportedly aired an edited version in 1992, cutting the crucial scene.