TREK-1 Channels Research Articles

Magnetic activation of TREK1 triggers stress signalling and regulates neuronal branching in SH-SY5Y cells.

TWIK-related K+ 1 (TREK1) is a potassium channel expressed in the nervous system with multiple functions including neurotransmission and is a prime pharmacological target for neurological disorders. TREK1 gating is controlled by a wide range of external stimuli including mechanical forces. Previous work has demonstrated that TREK1 can be mechano-activated using magnetic nanoparticles (MNP) functionalised with antibodies targeted to TREK1 channels. Once the MNP are bound, external dynamic magnetic fields are used to generate forces on the TREK channel. This approach has been shown to drive cell differentiation in cells from multiple tissues. In this work we investigated the effect of MNP-mediated TREK1 mechano-activation on early stress response pathways along with the differentiation and connectivity of neuronal cells using the model neuronal cell line SH-SY5Y. Results showed that TREK1 is well expressed in SH-SY5Y and that TREK1-MNP initiate c-Myc/NF-κB stress response pathways as well as Nitrite production after magnetic stimulation, indicative of the cellular response to mechanical cues. Results also showed that TREK1 mechano-activation had no overall effect on neuronal morphology or expression of the neuronal marker βIII-Tubulin in Retinoic Acid (RA)/Brain-derived Neurotrophic factor (BDNF) differentiated SH-SY5Y but did increase neurite number. These results suggest that TREK1 is involved in cellular stress response signalling in neuronal cells, which leads to increased neurite production, but is not involved in regulating RA/BDNF mediated neuronal differentiation.

Unraveling the Role of K2P Channels in Atrial Fibrillation.

Atrial fibrillation (AF) is a condition in which the electrical signals in the upper heart chambers (atria) are rapid and disorganized, producing an irregular and chaotical heartbeat. The sinus rhythm should be between 60 to 100 bpm at rest, while the heart rhythm in AF patients may be over 140 bpm. Either structural and electro-mechanical remodeling of the atrial tissue underlies the perpetuation and evolution of AF from the paroxysmal to persistent form. Unravelling the different pathological pathways involved in AF that lead to arrhythmogenesis and atrial remodeling is needed to discovery new and effective therapeutic approaches. A variety of drugs are available to convert and maintain the AF patient in a normal sinus rhythm; however, these strategies have limited chances of success or fail with the progression of AF to more persistent/permanent forms. Consequently, it is necessary to find new therapeutic targets for the relief of persistent or chronic AF forms, as well as the development of new and more effective pharmacological tools. The atrial specific two-pore domain K+ channels (K2P) constitute the background K+ current on atrial cardiomyocytes and modulate cell excitability emerging as novel targets in this disease and avoiding ventricle side effects. Moreover, several antiarrhythmic drugs used in AF treatment exert their mechanism of action in part by modulation of K2P channels. Thus far, TWIK-1, TREK-1, TASK-1, TASK-2 and TASK-3 channel have been identified as responsible for background currents IK2P current in atrial cells; however, it is not excluded that other K2PX subunits or subfamilies have physiological roles in atria. To date, a great diversity openers, activators and blockers of K2P channel have been identified, particularly those targeting TASK and TREK channels. Several studies have demonstrated that the expression of TWIK-1, TREK-1, TASK-1, TASK-2 and TASK-3 are dysregulated in AF and their pharmacology rescue could suppose a novel therapy in AF. The main objective is to examine the regulation of K2P channels and the current K2P channels pharmacological modulators for AF treatment.

Activation of TREK-1 (K2P2.1) potassium channels protects against influenza A-induced lung injury.

Influenza-A virus (IAV) infects yearly an estimated one billion people worldwide, resulting in 300,000-650,000 deaths. Preventive vaccination programs and antiviral medications represent the mainstay of therapy, but with unacceptably high morbidity and mortality rates, new targeted therapeutic approaches are urgently needed. Since inflammatory processes are commonly associated with measurable changes in the cell membrane potential (Em), we investigated whether Em hyperpolarization via TREK-1 (K2P2.1) K+ channel activation can protect against influenza-A virus (IAV)-induced pneumonia. We infected mice with IAV, which after 5 days caused 10-15% weight loss and a decrease in spontaneous activity, representing a clinically relevant infection. We then started a 3-day intratracheal treatment course with the novel TREK-1 activating compounds BL1249 or ML335. We confirmed TREK-1 activation with both compounds in untreated and IAV-infected primary human alveolar epithelial cells (HAECs) using high-throughput fluorescent imaging plate reader (FLIPR) assays. In mice, TREK-1 activation with BL1249 and ML335 counteracted IAV-induced histological lung injury and decrease in lung compliance and improved BAL fluid total protein levels, cell counts, and inflammatory IL-6, IP-10/CXCL-10, MIP-1α, and TNF-α levels. To determine whether these anti-inflammatory effects were mediated by activation of alveolar epithelial TREK-1 channels, we studied the effects of BL1249 and ML335 in IAV-infected HAEC, and found that TREK-1 activation decreased IAV-induced inflammatory IL-6, IP-10/CXCL10, and CCL-2 secretion. Dissection of TREK-1 downstream signaling pathways and construction of protein-protein interaction (PPI) networks revealed NF-κB1 and retinoic acid-inducible gene-1 (RIG-1) cascades as the most likely targets for TREK-1 protection. Therefore, TREK-1 activation may represent a novel therapeutic approach against IAV-induced lung injury.

Activation of TREK1 Channel in the Anterior Cingulate Cortex Improves Neuropathic Pain in a Rat Model.

Objective. To explore the biological function and mechanism of TREK1 in neuropathic pain. Thirty-two healthy rats and rats with sciatic nerve chronic press-fitting model (chronic constriction injury of the sciatic nerve, CCI) were selected. Western blot, immunofluorescence staining, and patch clamp technique were performed to explore the biological functions of TREK1. The expression of TREK1 was decreased in the CCI model. The TREK1 channel current in the CCI model was decreased. After local administration of TREK1 channel activator in the anterior cingulate cortex area, the pain behavior of CCI rats and the expression of TREK1 protein were reversed. The expression of TREK1 was downregulated in the ACC area of CCI rats and the current of TREK1 was decreased, which played an important role in the regulation of neuropathic pain.

Antiarrhythmic calcium channel blocker verapamil inhibits trek currents in sympathetic neurons.

Background and Purpose: Verapamil, a drug widely used in certain cardiac pathologies, exert its therapeutic effect mainly through the blockade of cardiac L-type calcium channels. However, we also know that both voltage-dependent and certain potassium channels are blocked by verapamil. Because sympathetic neurons of the superior cervical ganglion (SCG) are known to express a good variety of potassium currents, and to finely tune cardiac activity, we speculated that the effect of verapamil on these SCG potassium channels could explain part of the therapeutic action of this drug. To address this question, we decided to study, the effects of verapamil on three different potassium currents observed in SCG neurons: delayed rectifier, A-type and TREK (a subfamily of K2P channels) currents. We also investigated the effect of verapamil on the electrical behavior of sympathetic SCG neurons. Experimental Approach: We employed the Patch-Clamp technique to mouse SCG neurons in culture. Key Results: We found that verapamil depolarizes of the resting membrane potential of SCG neurons. Moreover, we demonstrated that this drug also inhibits A-type potassium currents. Finally, and most importantly, we revealed that the current driven through TREK channels is also inhibited in the presence of verapamil. Conclusion and Implications: We have shown that verapamil causes a clear alteration of excitability in sympathetic nerve cells. This fact undoubtedly leads to an alteration of the sympathetic-parasympathetic balance which may affect cardiac function. Therefore, we propose that these possible peripheral alterations in the autonomic system should be taken into consideration in the prescription of this drug.

Preserved oxytocin-induced myometrium contraction and sensitivity to progesterone inhibition following rat uterus thermal insult. Impact on fertility

Women seeking improved fertility often undergo diagnostic hysteroscopy that could cause uterine thermal injury with unclear impact on uterine contraction, embryo implantation and fertility. We tested whether uterine thermal insult adversely affects myometrium function and contraction related receptors, channels, junctional proteins and remodeling enzymes. Female Sprague-Dawley rats were anesthetized, the left uterine horn was infused with 85 ℃ hot saline (thermal Insult) and the right horn was infused with 25℃ warm saline (control) for 3 min. After 7-days recovery, uterine strips were prepared for tissue histology and measurement of contraction, and mRNA and protein levels of oxytocin receptor, progesterone (P4) receptor A (PR-A), membrane K+ channel TREK-1, junctional protein connexin-43 (CX-43) and matrix metalloproteinases MMP-2 and MMP-9. Uterine tissue histology showed cellular swelling and inflammatory cell infiltration immediately following thermal insult, and recovery with no difference from control 7-days later. KCl (96 mM) and oxytocin (10-13-10-7 M) caused significant contraction that was not different in thermal insult vs control uterine strips. Pretreatment with P4 (10-5 M) for 1 h caused marked inhibition of KCl and oxytocin contraction that was insignificantly greater in thermal vs control uterus. RT-PCR showed decreases in oxytocin receptor, PR-A, TREK-1, CX-43, MMP-2 and MMP-9 mRNA in thermal vs control uterus. Western blots showed decreases in oxytocin receptor, no change in TREK-1 and increased PRA, CX-43, MMP-2, and MMP-9 protein levels in thermal vs control uterus. To assess the impact on fertility, female rats were housed with male rats, and on gestational day 19, the litter size, pup weight and crown-rump length, and placenta weight were not different in thermal vs control uterus. Thus, after thermal insult-induced immediate inflammation and reduced heat-sensitive mRNA expression, the uterus undergoes a recovery and adaptation process involving preserved oxytocin-induced contraction, P4 inhibition and TREK-1 channels. The uterus self-healing process appears to require improved PR-A signaling, intercellular communication via CX-43 and tissue remodeling by MMP-2 and MMP-9. The uterine thermal recovery processes could be essential for maintaining fertility and future pregnancy outcome.

TMEM120A/TACAN inhibits mechanically activated PIEZO2 channels.

PIEZO2 channels mediate rapidly adapting mechanically activated currents in peripheral sensory neurons of the dorsal root ganglia (DRG), and they are indispensable for light touch and proprioception. Relatively little is known about what other proteins regulate PIEZO2 activity in a cellular context. TMEM120A (TACAN) was proposed to act as a high threshold mechanically activated ion channel in nociceptive DRG neurons. Here, we find that Tmem120a coexpression decreased the amplitudes of mechanically activated PIEZO2 currents and increased their threshold of activation. TMEM120A did not inhibit mechanically activated PIEZO1 and TREK1 channels and TMEM120A alone did not result in the appearance of mechanically activated currents above background. Tmem120a and Piezo2 expression in mouse DRG neurons overlapped, and siRNA-mediated knockdown of Tmem120a increased the amplitudes of rapidly adapting mechanically activated currents and decreased their thresholds to mechanical activation. Our data identify TMEM120A as a negative modulator of PIEZO2 channel activity, and do not support TMEM120A being a mechanically activated ion channel.

Activation of the TREK-1 Potassium Channel Improved Cognitive Deficits in a Mouse Model of Alzheimer's Disease by Modulating Glutamate Metabolism.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by cognitive dysfunction. The glutamate (Glu) metabolic pathway may be a major contributor to the memory dysfunction associated with AD. The TWIK-related potassium channel-1 (TREK-1) protects against brain ischemia, but any specific role for the channel in AD remains unknown. In this study, we used SAMP8 mice as an AD model and age-matched SAMR1 mice as controls. We explored the trends of changes in TREK-1 channel activity and the levels of AD-related molecules in the brains of SAMP8 mice. We found that the expression level of TREK-1 increased before 3months of age and then began to decline. The levels of Tau and Glu increased with age whereas the acetylcholine level decreased with age. α-Linolenic acid (ALA), an activator of the TREK-1 channel, significantly increased the TREK-1 level, and improved the learning and memory deficits of SAMP8 mice aged 6months. The mechanism in play may involve the Glu metabolic pathway. After activation of the TREK-1 channel, damaged neurons and astrocytes were rescued, the levels of Glu and the N-methyl-D-aspartate receptor were downregulated, and the level of glutamate transporter-1 was upregulated. These findings suggest that TREK-1 plays a crucial role in the pathological progression of AD; activation of the TREK-1 channel improved cognitive deficits in SAMP8 mice via a mechanism that involved Glu metabolism. The TREK-1 potassium channel may thus be a valuable therapeutic target in AD patients.

New insights in the understanding of TREK-1 channel activation by fatty acids

Among the two-pore domain K+ channels (K2P), TREK-1 channel is one of the most studied K2P channel members due to its involvement in cardio- and neuroprotection but it suffers from a poor specific pharmacological profile. TREK-1 channel is polymodulated by voltage, pH changes, membrane stretch and polyunsaturated fatty acids (PUFAs) and generates an outwardly rectifying current. The PUFAs-mediated activation is supposedly correlated to the length of the carbon chain suggesting a link between FAs membrane insertion and a stretch effect through an increase of membrane tension. The aim of this study is to test the effects of different fatty acids, especially polyunsaturated fatty acids, to delineate a possible mechanism of action to explain the activation of ITREK-1. In this study, using the patch-clamp technique in ruptured whole-cell configuration, we compared the effects of FAs, from stearic acid (C18:0, 18 carbons; 0 double bound) to docosahexaenoic acid (C22:6, 22 carbons; 6 double bounds) on ITREK-1 in a HEK293 cell line overexpressing the human TREK-1 channel. C18:0 and C18:3 failed to activate ITREK-1. Surprisingly, it was the shortest PUFA, linoleic acid (LA C18:2) that produced the largest activated ITREK-1. At 0 mV, ITREK-1 was increased 32.4 ± 5.4 folds by LA (C18:2); DHA(C22:6): 29.7 ± 4.1 and AA(C20:4): 14.7 ± 1.9. Since the activation kinetic was fast (in the range of 1.5 min) and the reversibility immediate when FA solution was washed, we supposed that FAs interact directly with TREK-1 rather than FAs membrane insertion changing membrane tension. Some FAs linearized the current-voltage relationship with a calculated linearization index (LI) greater than 0.9, for C18:2, C22:5w6, C22:6, while the rectifier current was maintained for C20:5, C20:4, C20:5, C22:5w3 FAs (LI < 0.75). We propose that the observed linearization results from an activation process involving a K + -flux gating mechanism. Taken together, all these data suggest that the activation of TREK-1 by PUFAs is not a consequence of change in membrane tension but rather more complex than previously assumed. A better understanding of the TREK-1 channels activation mechanisms will help to develop more efficient activators in the perspective of a use to protect the heart following an infarction.

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

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

Novel DCPIB analogs as dual inhibitors of VRAC/TREK1 channels reduced cGAS-STING mediated interferon responses

The enzyme cyclic GMP-AMP synthase (cGAS) senses cytosolic DNA and catalyzes the formation of 2′3′-cyclic-GMP-AMP (cGAMP), which in turn triggers interferon (IFN) production. Inappropriate activation of cGAS and production of cGAMP have been linked to a diversity of autoimmune diseases. The volume-regulated anion channels (VRACs) have been recently demonstrated to permeate cGAMP, thus making the channel essential for the activation of the cGAS-cGAMP-STING axis. DCPIB, a prominent inhibitor of VRAC channel, has been recently reported to also significantly activate TREK1 channel. Herein, in this study, we have designed and synthesized a series of novel DCPIB derivatives and investigated their potential regulatory effects on VRAC/TREK1 channels. Our results manifested that compound 6u was a dual inhibitor of VRAC/TREK1 channels with IC50s of 7.11 ± 0.94 μM and 4.43 ± 0.90 μM, respectively. On top of that, our data demonstrated that 6u impaired interferon production in a concentration-dependently manner by dampening cGAS-cGAMP-STING pathway without any cytotoxicity when it comes to herpes simplex virus type 1 (HSV1) infection. To sum up, our study not only discovered a novel DCPIB analog with dual inhibitory effects on VRAC/TREK1 channels but also provided a new strategy for the design and development of newly potent VRAC inhibitors, which benefits the treatment of cGAS-STING related autoimmune and inflammatory diseases.

The Protective Effects of Hydrogen Sulfide New Donor Methyl S-(4-Fluorobenzyl)-N-(3,4,5-Trimethoxybenzoyl)-l-Cysteinate on the Ischemic Stroke.

In this paper, we report the design, synthesis and biological evaluation of a novel S-allyl-l-cysteine (SAC) and gallic acid conjugate S-(4-fluorobenzyl)-N-(3,4,5-trimethoxybenzoyl)-l-cysteinate (MTC). We evaluate the effects on ischemia-reperfusion-induced PC12 cells, primary neurons in neonatal rats, and cerebral ischemic neuronal damage in rats, and the results showed that MTC increased SOD, CAT, GPx activity and decreased LDH release. PI3K and p-AKT protein levels were significantly increased by activating PI3K/AKT pathway. Mitochondrial pro-apoptotic proteins Bax and Bim levels were reduced while anti-apoptotic protein Bcl-2 levels were increased. The levels of cleaved caspase-9 and cleaved caspase-3 were also reduced in the plasma. The endoplasmic reticulum stress (ERS) was decreased, which in turns the survival rate of nerve cells was increased, so that the ischemic injury of neurons was protected accordingly. MTC activated the MEK-ERK signaling pathway and promoted axonal regeneration in primary neurons of the neonatal rat. The pretreatment of MEK-ERK pathway inhibitor PD98059 and PI3K/AKT pathway inhibitor LY294002 partially attenuated the protective effect of MTC. Using a MCAO rat model indicated that MTC could reduce cerebral ischemia-reperfusion injury and decrease the expression of proinflammatory factors. The neuroprotective effect of MTC may be due to inhibition of the over-activation of the TREK-1 channel and reduction of the current density of the TREK1 channel. These results suggested that MTC has a protective effect on neuronal injury induced by ischemia reperfusion, so it may have the potential to become a new type of neuro-ischemic drug candidate.

Membrane phospholipids control activity of the mechanosensitive K2P channel TREK1

Tandem pore (K2P) potassium ion channels play vital roles in human physiology, chiefly responsible for maintaining cellular resting membrane potential. Modulation of K2P channel activity controls cellular excitability, altering the degree of input signal required to reach action potential thresholds. For the mechanosensitive subfamily of K2Ps, including the TREK1, TREK2, and TRAAK channels, a wide range of diverse and dissimilar inputs alter channel activity, including pH, heat, mechanical stretch, lipids, post-translational modification, and an array of pharmacologically active agents including volatile anesthetics, antidepressants, and neuroprotective agents.

Involvement of TREK1 channels in the proliferation of human hepatic stellate LX-2 cells

Activation of hepatic stellate cells (HSCs) causes hepatic fibrosis and results in chronic liver diseases. Although activated HSC functions are facilitated by an increase in the cytosolic Ca2+ concentration ([Ca2+]cyt), the pathophysiological roles of ion channels are largely unknown. In the present study, functional analyses of the two-pore domain K+ (K2P) channels, which regulate the resting membrane potential and [Ca2+]cyt, were performed using the human HSC line, LX-2. Expression analyses revealed that TREK1 (also known as KCNK2 and K2P2.1) channels are expressed in LX-2 cells. Whole-cell K+ currents were activated by 10 μM arachidonic acid and the activation was abolished by 100 μM tetrapentylammonium, which are pharmacological characteristics of TREK1 channels. The siRNA knockdown of TREK1 channels caused membrane depolarization and reduced [Ca2+]cyt. In addition, TREK1 knockdown downregulated the gene expression of collage type I and platelet-derived growth factor. Furthermore, TREK1 knockdown inhibited the proliferation of LX-2 cells. In conclusion, the activity of TREK1 channels determines the resting membrane potential and [Ca2+]cyt, which play a role in extracellular matrix production and cell proliferation in HSCs. This study may help elucidate the molecular mechanism underlying hepatic fibrosis in HSCs and provide a potential therapeutic target for hepatic fibrosis.

'C-type' closed state and gating mechanisms of K2P channels revealed by conformational changes of the TREK-1 channel.

ABSTRACTTwo-pore domain potassium (K2P) channels gate primarily within the selectivity filter, termed ‘C-type’ gating. Due to the lack of structural insights into the nonconductive (closed) state, ‘C-type’ gating mechanisms remain elusive. Here, molecular dynamics (MD) simulations on TREK-1, a K2P channel, revealed that M4 helix movements induce filter closing in a novel ‘deeper-down’ structure that represents a ‘C-type’ closed state. The ‘down’ structure does not represent the closed state as previously proposed and instead acts as an intermediate state in gating. The study identified the allosteric ‘seesaw’ mechanism of M4 helix movements in modulating filter closing. Finally, guided by this recognition of K2P gating mechanisms, MD simulations revealed that gain-of-function mutations and small-molecule activators activate TREK-1 by perturbing state transitions from open to closed states. Together, we reveal a ‘C-type’ closed state and provide mechanical insights into gating procedures and allosteric regulations for K2P channels.

Distinct classes of potassium channels fused to GPCRs as electrical signaling biosensors

SummaryLigand-gated ion channels (LGICs) are natural biosensors generating electrical signals in response to the binding of specific ligands. Creating de novo LGICs for biosensing applications is technically challenging. We have previously designed modified LGICs by linking G protein-coupled receptors (GPCRs) to the Kir6.2 channel. In this article, we extrapolate these design concepts to other channels with different structures and oligomeric states, namely a tetrameric viral Kcv channel and the dimeric mouse TREK-1 channel. After precise engineering of the linker regions, the two ion channels were successfully regulated by a GPCR fused to their N-terminal domain. Two-electrode voltage-clamp recordings showed that Kcv and mTREK-1 fusions were inhibited and activated by GPCR agonists, respectively, and antagonists abolished both effects. Thus, dissimilar ion channels can be allosterically regulated through their N-terminal domains, suggesting that this is a generalizable approach for ion channel engineering.

Activity of TREK-2-like Channels in the Pyramidal Neurons of Rat Medial Prefrontal Cortex Depends on Cytoplasmic Calcium.

Simple SummaryThe pyramidal neurons of rat prefrontal cortex express potassium channels identified as a non-canonical splice variant of the TREK-2 channel. The main function of TREK channels is to regulate the resting membrane potential. We showed that cytoplasmic Ca2+ upregulates the activity of TREK-2-like channels. Previous studies have indicated that the activation of TREK-2 channels is mediated by PI(4,5)P2, a polyanionic lipid in the inner leaflet of the plasma membrane. While TREK channels are believed to not be regulated by calcium, our work shows otherwise. We propose a model in which calcium ions enable the formation of PI(4,5)P2 nanoclusters, which stabilize active conformation of the channel.TREK-2-like channels in the pyramidal neurons of rat prefrontal cortex are characterized by a wide range of spontaneous activity—from very low to very high—independent of the membrane potential and the stimuli that are known to activate TREK-2 channels, such as temperature or membrane stretching. The aim of this study was to discover what factors are involved in high levels of TREK-2-like channel activity in these cells. Our research focused on the PI(4,5)P2-dependent mechanism of channel activity. Single-channel patch clamp recordings were performed on freshly dissociated pyramidal neurons of rat prefrontal cortexes in both the cell-attached and inside-out configurations. To evaluate the role of endogenous stimulants, the activity of the channels was recorded in the presence of a PI(4,5)P2 analogue (PI(4,5)P2DiC8) and Ca2+. Our research revealed that calcium ions are an important factor affecting TREK-2-like channel activity and kinetics. The observation that calcium participates in the activation of TREK-2-like channels is a new finding. We showed that PI(4,5)P2-dependent TREK-2 activity occurs when the conditions for PI(4,5)P2/Ca2+ nanocluster formation are met. We present a possible model explaining the mechanism of calcium action.

Effects of ionic strength on gating and permeation of TREK-2 K2P channels.

In addition to the classical voltage-dependent behavior mediated by the voltage-sensing-domains (VSD) of ion channels, a growing number of voltage-dependent gating behaviors are being described in channels that lack canonical VSDs. A common thread in their mechanism of action is the contribution of the permeating ion to this voltage sensing process. The polymodal K2P K+ channel, TREK2 responds to membrane voltage through a gating process mediated by the interaction of K+ with its selectivity filter. Recently, we found that this action can be modulated by small molecule agonists (e.g. BL1249) which appear to have an electrostatic influence on K+ binding within the inner cavity and produce an increase in the single-channel conductance of TREK-2 channels. Here, we directly probed this K+-dependent gating process by recording both macroscopic and single-channel currents of TREK-2 in the presence of high concentrations of internal K+. Surprisingly we found TREK-2 is inhibited by high internal K+ concentrations and that this is mediated by the concomitant increase in ionic-strength. However, we were still able to determine that the increase in single channel conductance in the presence of BL1249 was blunted in high ionic-strength, whilst its activatory effect (on channel open probability) persisted. These effects are consistent with an electrostatic mechanism of action of negatively charged activators such as BL1249 on permeation, but also suggest that their influence on channel gating is complex.

Are TREK Channels Temperature Sensors?

Internal human body normal temperature fluctuates between 36.5 and 37.5°C and it is generally measured in the oral cavity. Interestingly, most electrophysiological studies on the functioning of ion channels and their role in neuronal behavior are carried out at room temperature, which usually oscillates between 22 and 24°C, even when thermosensitive channels are studied. We very often forget that if the core of the body reached that temperature, the probability of death from cardiorespiratory arrest would be extremely high. Does this mean that we are studying ion channels in dying neurons? Thousands of electrophysiological experiments carried out at these low temperatures suggest that most neurons tolerate this aggression quite well, at least for the duration of the experiments. This also seems to happen with ion channels, although studies at different temperatures indicate large changes in both, neuron and channel behavior. It is known that many chemical, physical and therefore physiological processes, depend to a great extent on body temperature. Temperature clearly affects the kinetics of numerous events such as chemical reactions or conformational changes in proteins but, what if these proteins constitute ion channels and these channels are specifically designed to detect changes in temperature? In this review, we discuss the importance of the potassium channels of the TREK subfamily, belonging to the recently discovered family of two-pore domain channels, in the transduction of thermal sensitivity in different cell types.

Convergence of Multiple Stimuli to a Single Gate in TREK1 and TRAAK Potassium Channels

Inhibitory potassium channels of the TREK1/TRAAK family are integrators of multiple stimuli, including temperature, membrane stretch, polyunsaturated fatty acids and pH. How these signals affect the gating of these channels is the subject of intense research. We have previously identified a cytoplasmic domain, pCt, which plays a major role in controlling channel activity. Here, we use pharmacology to show that the effects of pCt, arachidonic acid, and extracellular pH converge to the same gate within the channel. Using a state-dependent inhibitor, fluoxetine, as well as natural and synthetic openers, we provide further evidence that the “up” and “down” conformations identified by crystallography do not correspond to open and closed states of these channels.