A photoswitchable inhibitor of TREK channels controls pain in wild-type intact freely moving animals.

By endowing light control of neuronal activity, optogenetics and photopharmacology are powerful methods notably used to probe the transmission of pain signals. However, costs, animal handling and ethical issues have reduced their dissemination and routine use. Here we report LAKI (Light Activated K+ channel Inhibitor), a specific photoswitchable inhibitor of the pain-related two-pore-domain potassium TREK and TRESK channels. In the dark or ambient light, LAKI is inactive. However, alternating transdermal illumination at 365 nm and 480 nm reversibly blocks and unblocks TREK/TRESK current in nociceptors, enabling rapid control of pain and nociception in intact and freely moving mice and nematode. These results demonstrate, in vivo, the subcellular localization of TREK/TRESK at the nociceptor free nerve endings in which their acute inhibition is sufficient to induce pain, showing LAKI potential as a valuable tool for TREK/TRESK channel studies. More importantly, LAKI gives the ability to reversibly remote-control pain in a non-invasive and physiological manner in naive animals, which has utility in basic and translational pain research but also in in vivo analgesic drug screening and validation, without the need of genetic manipulations or viral infection.

Withaferin A suppresses breast cancer cell proliferation by inhibition of the two-pore domain potassium (K2P9) channel TASK-3.

Withaferin A (WFA), a C5,C6-epoxy steroidal lactone isolated from the medicinal plant Withania somnifera (L.) Dunal, inhibits growth of tumor cells in different cancer types. However, the mechanisms underlying the effect of WFA on tumor cells are not fully understood. In the present study, we evaluated the blockade of TASK-3 channels by WFA in TASK-3-expressing HEK-293 cells. Explore if the WFA-mediated TASK-3 blockade can be used as a pharmacological tool to decrease the cell viability in cancer cells. A combination of functional experiments (patch-clamp, gene downregulation, overexpression and pharmacological inhibition) and molecular docking analysis were used to get insights into the mechanism by which the inhibition of TASK-3 by WFA affects the growth and viability of cancer cells. Withaferin A was found to inhibit the activity of TASK-3 channels. The inhibitory effect of Withaferin A on TASK-3 potassium currents was dose-dependent and independent of voltage. Molecular modeling studies identified putative WFA-binding sites in TASK-3 channel involved the channel blockade. In agreements with the molecular modeling predictions, mutation of residues F125 to A (F125A), L197 to V (L197 V) and the double mutant F125A-L197 V markedly decreased the WFA-induced inhibition of TASK-3. Finally, the cytotoxic effect of WFA was tested in MDA-MB-231 human breast cancer cells transfected with TASK-3 or shRNA that decreases TASK-3 expression. Together, our results show that the cytotoxic effect of WFA on fully transformed MDA-MB-231 cells depends on the expression of TASK-3. Herein, we also provide insights into the mechanism of TASK-3 inhibition by WFA.

Pharmacologic TWIK-Related Acid-Sensitive K+ Channel (TASK-1) Potassium Channel Inhibitor A293 Facilitates Acute Cardioversion of Paroxysmal Atrial Fibrillation in a Porcine Large Animal Model.

Background The tandem of P domains in a weak inward rectifying K+ channel (TWIK)-related acid-sensitive K+ channel (TASK-1; hK2P3.1) two-pore-domain potassium channel was recently shown to regulate the atrial action potential duration. In the human heart, TASK-1 channels are specifically expressed in the atria. Furthermore, upregulation of atrial TASK-1 currents was described in patients suffering from atrial fibrillation (AF). We therefore hypothesized that TASK-1 channels represent an ideal target for antiarrhythmic therapy of AF. In the present study, we tested the antiarrhythmic effects of the high-affinity TASK-1 inhibitor A293 on cardioversion in a porcine model of paroxysmal AF. Methods and Results Heterologously expressed human and porcine TASK-1 channels are blocked by A293 to a similar extent. Patch clamp measurements from isolated human and porcine atrial cardiomyocytes showed comparable TASK-1 currents. Computational modeling was used to investigate the conditions under which A293 would be antiarrhythmic. German landrace pigs underwent electrophysiological studies under general anesthesia. Paroxysmal AF was induced by right atrial burst stimulation. After induction of AF episodes, intravenous administration of A293 restored sinus rhythm within cardioversion times of 177±63 seconds. Intravenous administration of A293 resulted in significant prolongation of the atrial effective refractory period, measured at cycle lengths of 300, 400 and 500 ms, whereas the surface ECG parameters and the ventricular effective refractory period lengths remained unchanged. Conclusions Pharmacological inhibition of atrial TASK-1 currents exerts antiarrhythmic effects in vivo as well as in silico, resulting in acute cardioversion of paroxysmal AF. Taken together, these experiments indicate the therapeutic potential of A293 for AF treatment.

Development of a Novel Cell-Based Assay System for High-Throughput Screening of Compounds Acting on Background Two-Pore Domain K+ Channels.

Two-pore domain K+ (K2P) channels are thought to be druggable targets. However, only a few agents specific for K2P channels have been identified, presumably due to the lack of an efficient screening system. To develop a new high-throughput screening (HTS) system targeting these channels, we have established a HEK293-based "test cell" expressing a mutated Na+ channel (Nav1.5) with markedly slowed inactivation, as well as a K+ channel (Kir2.1) that sets the membrane potential quite negative, close to K+ equilibrium potential. We found in this system that Kir2.1 block by 100 µM Ba2+ application consistently elicited a large depolarization like a long-lasting action potential. This maneuver resulted in cell death, presumably due to the sustained Na+ influx. When either the TWIK-related acid-sensitive K+ (TASK)-1 or TASK-3 channel was expressed in the test cells, Ba2+-induced cell death was markedly weakened. Stronger activation of TASK-1 by extracellular acidification further decreased the cell death. In contrast, the presence of K2P channel blockers enhanced cell death. IC50 values for TASK-1 and/or TASK-3 blockers acquired by measurements of relative cell viability were comparable to those obtained using patch-clamp recordings. Both blockers and openers of K2P channels can be accurately assessed with high efficiency and throughput by this novel HTS system.

Antidepressive and anxiolytic effects of ostruthin, a TREK-1 channel activator.

We screened a library of botanical compounds purified from plants of Vietnam for modulators of the activity of a two-pore domain K+ channel, TREK-1, and we identified a hydroxycoumarin-related compound, ostruthin, as an activator of this channel. Ostruthin increased whole-cell TREK-1 channel currents in 293T cells at a low concentration (EC50 = 5.3 µM), and also activity of the TREK-2 channel (EC50 = 3.7 mM). In contrast, ostruthin inhibited other K+ channels, e.g. human ether-à-go-go-related gene (HERG1), inward-rectifier (Kir2.1), voltage-gated (Kv1.4), and two-pore domain (TASK-1) at higher concentrations, without affecting voltage-gated potassium channel (KCNQ1 and 3). We tested the effect of this compound on mouse anxiety- and depression-like behaviors and found anxiolytic activity in the open-field, elevated plus maze, and light/dark box tests. Of note, ostruthin also showed antidepressive effects in the forced swim and tail suspension tests, although previous studies reported that inhibition of TREK-1 channels resulted in an antidepressive effect. The anxiolytic and antidepressive effect was diminished by co-administration of a TREK-1 blocker, amlodipine, indicating the involvement of TREK-1 channels. Administration of ostruthin suppressed the stress-induced increase in anti-c-Fos immunoreactivity in the lateral septum, without affecting immunoreactivity in other mood disorder-related nuclei, e.g. the amygdala, paraventricular nuclei, and dorsal raphe nucleus. Ostruthin may exert its anxiolytic and antidepressive effects through a different mechanism from current drugs.

Aristolochic acid, a plant extract used in the treatment of pain and linked to Balkan endemic nephropathy, is a regulator of K2P channels.

BACKGROUND AND PURPOSE: Aristolochic acid (AristA) is found in plants used in traditional medicines to treat pain. We investigated the action of AristA on TREK and TRESK, potassium (K2P) channels, which are potential therapeutic targets in pain. Balkan endemic nephropathy (BEN) is a renal disease associated with AristA consumption. A mutation of TASK-2 (K2P 5.1) channels (T108P) is seen in some patients susceptible to BEN, so we investigated how both this mutation and AristA affected TASK-2 channels. EXPERIMENTAL APPROACH: Currents through wild-type and mutated human K2P channels expressed in tsA201 cells were measured using whole-cell patch-clamp recordings in the presence and absence of AristA. KEY RESULTS: TREK-1- and TREK-2-mediated currents were enhanced by AristA (100 µM), whereas TRESK was inhibited. Inhibition of TRESK did not depend on the phosphorylation of key intracellular serines but was completely blocked by mutation of bulky residues in the inner pore (F145A_F352A). The TASK-2_T108P mutation markedly reduced both current density and ion selectivity. A related mutation (T108C) had similar but less marked effects. External alkalization and application of flufenamic acid enhanced TASK-2 and TASK-2_T108C current but did not affect TASK-2_T108P current. AristA (300 µM) produced a modest enhancement of TASK-2 current. CONCLUSIONS AND IMPLICATIONS: Enhancement of TREK-1 and TREK-2 and inhibition of TRESK by AristA may contribute to therapeutically useful effects of this compound in pain. Whilst AristA is unlikely to interact directly with TASK-2 channels in BEN, loss of functional TASK-2 channels may indirectly increase susceptibility to AristA toxicity.

Hydroxy-α sanshool induces colonic motor activity in rat proximal colon: a possible involvement of KCNK9.

Various colonic motor activities are thought to mediate propulsion and mixing/absorption of colonic content. The Japanese traditional medicine daikenchuto (TU-100), which is widely used for postoperative ileus in Japan, accelerates colonic emptying in healthy humans. Hydroxy-α sanshool (HAS), a readily absorbable active ingredient of TU-100 and a KCNK3/KCNK9/KCNK18 blocker as well as TRPV1/TRPA1 agonist, has been investigated for its effects on colonic motility. Motility was evaluated by intraluminal pressure and video imaging of rat proximal colons in an organ bath. Distribution of KCNKs was investigated by RT-PCR, in situ hybridization, and immunohistochemistry. Current and membrane potential were evaluated with use of recombinant KCNK3- or KCNK9-expressing Xenopus oocytes and Chinese hamster ovary cells. Defecation frequency in rats was measured. HAS dose dependently induced strong propulsive "squeezing" motility, presumably as long-distance contraction (LDC). TRPV1/TRPA1 agonists induced different motility patterns. The effect of HAS was unaltered by TRPV1/TRPA1 antagonists and desensitization. Lidocaine (a nonselective KCNK blocker) and hydroxy-ß sanshool (a geometrical isomer of HAS and KCNK3 blocker) also induced colonic motility as a rhythmic propagating ripple (RPR) and a LDC-like motion, respectively. HAS-induced "LDC," but not lidocaine-induced "RPR," was abrogated by a neuroleptic agent tetrodotoxin. KCNK3 and KCNK9 were located mainly in longitudinal smooth muscle cells and in neural cells in the myenteric plexus, respectively. Administration of HAS or TU-100 increased defecation frequency in normal and laparotomy rats. HAS may evoke strong LDC possibly via blockage of the neural KCNK9 channel in the colonic myenteric plexus.

Identification of the muscarinic pathway underlying cessation of sleep-related burst activity in rat thalamocortical relay neurons.

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

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

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

TASK Channel Deletion Reduces Sensitivity to Local Anesthetic-induced Seizures.

BACKGROUND: Local anesthetics (LAs) are typically used for regional anesthesia but can be given systemically to mitigate postoperative pain, supplement general anesthesia, or prevent cardiac arrhythmias. However, systemic application or inadvertent intravenous injection can be associated with substantial toxicity, including seizure induction. The molecular basis for this toxic action remains unclear. METHODS: We characterized inhibition by different LAs of homomeric and heteromeric K channels containing TASK-1 (K2P3.1, KCNK3) and TASK-3 (K2P9.1, KCNK9) subunits in a mammalian expression system. In addition, we used TASK-1/TASK-3 knockout mice to test the possibility that TASK channels contribute to LA-evoked seizures. RESULTS: LAs inhibited homomeric and heteromeric TASK channels in a range relevant for seizure induction; channels containing TASK-1 subunits were most sensitive and IC50 values indicated a rank order potency of bupivacaine > ropivacaine >> lidocaine. LAs induced tonic-clonic seizures in mice with the same rank order potency, but higher LA doses were required to evoke seizures in TASK knockout mice. For bupivacaine, which produced the longest seizure times, seizure duration was significantly shorter in TASK knockout mice; bupivacaine-induced seizures were associated with an increase in electroencephalogram power at frequencies less than 5 Hz in both wild-type and TASK knockout mice. CONCLUSIONS: These data suggest that increased neuronal excitability associated with TASK channel inhibition by LAs contributes to seizure induction. Because all LAs were capable of evoking seizures in TASK channel deleted mice, albeit at higher doses, the results imply that other molecular targets must also be involved in this toxic action.

The neuroprotective impact of the leak potassium channel TASK1 on stroke development in mice.

Oxygen depletion (O(2)) and a decrease in pH are initial pathophysiological events in stroke development, but secondary mechanisms of ischemic cell death are incompletely understood. By patch-clamp recordings of brain slice preparations we show that TASK1 and TASK3 channels are inhibited by pH-reduction (42+/-2%) and O(2) deprivation (36+/-5%) leading to membrane depolarization, increased input resistance and a switch in action potential generation under ischemic conditions. In vivo TASK blockade by anandamide significantly increased infarct volumes at 24 h in mice undergoing 30 min of transient middle cerebral artery occlusion (tMCAO). Moreover, blockade of TASK channels accelerated stroke development. Supporting these findings TASK1(-/-) mice developed significantly larger infarct volumes after tMCAO accompanied by worse outcome in functional neurological tests compared to wild type mice. In conclusion, our data provide evidence for an important role of functional TASK channels in limiting tissue damage during cerebral ischemia.

Zinc activates TREK-2 potassium channel activity.

TWIK-related K(+) channel (TREK)-2 is thought to contribute to setting the resting membrane potential and to tuning action potential properties. In the present study, the effects of divalent metal ions (Ba(2+), Co(2+), Ni(2+), Pb(2+), and Zn(2+)) were examined on TREK-2 expressed in Xenopus oocytes using the two-electrode voltage clamping technique. Pb(2+) inhibited TREK channel activity (IC(50) = 15.6 microM), whereas Zn(2+) enhanced it in a dose-dependent manner (EC(50) = 87.1 microM). Ba(2+) slightly inhibited TREK currents but only at high concentrations. Co(2+) and Ni(2+) had no significant effect. The structural element(s) contributing to the zinc enhancement effect were studied using a series of chimeras consisting of Zn(2+)-activated TREK-2 and Zn(2+)-inhibited TWIK-related acid-sensing K(+) channel-3. The structural elements were localized to the first pore and the preceding extracellular loop of TREK-2, in which multiple residues, including His121, His156, Asp158, and Asn177, are likely to be involved in the zinc activation effect. Stimulation by Zn(2+) may be used as a criterion of TREK-2, distinguishing it from other two-pore K(+) channels.