Activated mTRESK channel (IC50 = 6.8 μM)

Applying A2764 (100 µM) to oocytes expressing mTRESK resulted in a 42.8±11.5% inhibition of background K+ current[1]. In ionomycin-induced mTRESK currents, A2764 (100 µM) exhibits enhanced inhibitory activity versus the IC50 of activated channels. After that, A2764 was used to significantly reduce the current (77.8±3.5%)[1]. At resting conditions, A2764 (100 µM) reduces TRESK current by 42.8±11.5%, while during activity, it inhibits it by 77.8±3.5% [1].

Two-Electrode Voltage-Clamp and Patch-Clamp Measurements.[1]
Two-electrode voltage-clamp experiments were performed 1–3 days after the microinjection of cRNA into Xenopus oocytes, as previously described (Czirják et al., 2004). For each channel type, the oocytes contributing to the n number (the exact n number is indicated in the text or on the figures) were derived from at least two, but usually three, separate frogs. The holding potential was 0 mV. Background potassium currents were measured at the end of 300-millisecond-long voltage steps to −100 mV applied every 4 seconds. The low-potassium recording solution contained the following (in mM): NaCl 95.4, KCl 2, CaCl2 1.8, and HEPES 5, at pH 7.5, adjusted by NaOH. The high-potassium solution contained 80 mM K+ (78 mM NaCl in the low-potassium solution was replaced with KCl). For the measurement of TREK-1, TREK-2, and TRAAK currents, the high-potassium solution contained 40 mM K+. Solutions were applied to the oocytes using a gravity-driven perfusion system. Experiments were performed at room temperature (21°C).
Whole-cell patch-clamp experiments in the voltage-clamp configuration were performed as described previously (Lengyel et al., 2016). The resting membrane potential was recorded in the current-clamp mode with no current injection (I = 0 mode). The rheobase was determined by injecting depolarizing current (in 100 pA increments, up to 1500 pA) for 1 second every 4 seconds. Isolated DRG neurons were used for experiments 1 to 2 days after isolation. For the current-clamp study of DRG neurons, only cells with a membrane potential between −45 and −70 mV were accepted, as described in previous studies (Petruska et al., 2000). Because the focus of this study was to examine the effects of our new cloxyquin analogs on the electrophysiological parameters of isolated DRG neurons, no other selection criteria were used. The cutoff frequency of the eight-pole Bessel filter was adjusted to 200 Hz, and data were acquired at 1 kHz. The pipette solution contained (in mM): 140 KCl, 3 MgCl2, 0.05 EGTA, 1 Na2-ATP, 0.1 Na2-GTP, and 10 HEPES. The low-potassium solution contained (in mM): 140 NaCl, 3.6 KCl, 0.5 MgCl2, 2 CaCl2, 11 glucose, and 10 HEPES. The high-potassium solution contained 30 mM KCl (26.4 mM NaCl of the low-potassium solution was replaced with KCl). The pH of the bath solutions was adjusted to 7.4 with NaOH. Experiments were performed at room temperature (21°C).

Animal Husbandry, Preparation, and Microinjection of Xenopus Oocytes. Generation of a TRESK Knockout Mouse Line: Isolation of DRG Neurons.[1]
Xenopus laevis oocytes were prepared as previously described (Czirják and Enyedi, 2002). For the expression of the different channels, the oocytes were injected with 57 pg to 4 ng of cRNA (depending on the channel type) 1 day after defolliculation. Injection was performed with a Nanoliter Injector. X. laevis frogs were housed in 50-l tanks with continuous filtering and water circulation. Room temperature was 19°C. Frogs were anesthetized with 0.1% tricaine solution and killed by decerebration and pithing.
FVB/Ant (FVB.129P2-Pde6b+Tyrc-ch/Ant) mice were obtained commercially. TRESK knockout (KO) animals were generated by the transcription activator–like effector nuclease (TALEN) technique using plasmids ordered from Addgene. Mouse TRESK (mTRESK)-specific TALEN recognition sites were designed for the genomic sequence of the first exon (corresponding to the N-terminal intracellular domain of the channel) using the 5′-TN19 N14−20 N19A-3′ formula, with the following sequences: left TALEN recognition site, 5′tgaggagccacctgaggcca; right TALEN recognition site, 5′ccctggggaaggccagggga; and an 18 base pair (5′ggagatgctgtcctgagg) FokI nuclease dimerization and cutting sequence in between. mTRESK recognizing TALEN plasmids were assembled according to the protocol of Sanjana et al. (2012). TALEN mRNAs were produced using the Ambion mMESSAGE mMACHINE T7 in vitro transcription kit (Ambion). TALEN mRNAs were microinjected at a concentration of 20–20 ng/μl into the pronuclei of fertilized eggs of FVB/Ant mice. Pups were analyzed with Surveyor assay plus sequencing. In 12 mice, among the 61 born pups the TRESK (KCNK18) gene was changed, and a founder bearing a 33 base pair deletion and also a mutation introducing a stop codon was chosen to establish a colony.
Adult female wild-type and TRESK KO mice (2–3 months of age) were used for the patch-clamp experiments in this study. The animals were maintained on a 12-hour light/dark cycle with free access to food and water in a specific pathogen-free animal facility. Mice were killed humanely by CO2 exposure (CO2 was applied until death of the animals). DRGs were dissected from the thoracic and lumbar levels of the spinal cord and collected in sterile PBS (137 mM NaCl, 2.7 mM KCl, and 10 mM NaH2PO4, pH adjusted to 7.4 with NaOH) at 4°C. Ganglia were incubated in PBS containing 2 mg/ml collagenase enzyme (type I) for 30 minutes with gentle shaking at 37°C. For further details regarding the isolation and culturing of the cells, see Braun et al. (2015). All experimental procedures using animals were conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the National Institutes of Health, local state laws, and institutional regulations. All animal experiments were approved by the Animal Care and Ethics Committee of Semmelweis University (approval ID: XIV-I-001/2154-4/2012).

[1]. Chemically Modified Derivatives of the Activator Compound Cloxyquin Exert Inhibitory Effect on TRESK (K2P18.1) Background Potassium Channel.Mol Pharmacol. 2019 Jun;95(6):652-660.

Cloxyquin has been reported as a specific activator of TRESK [TWIK-related spinal cord K+ channel (also known as K2P18.1)] background potassium channel. In this study, we have synthetized chemically modified analogs of cloxyquin and tested their effects on TRESK and other K2P channels. The currents of murine K2P channels, expressed heterologously in Xenopus oocytes, were measured by two-electrode voltage clamp, whereas the native background K+ conductance of mouse dorsal root ganglion (DRG) neurons was examined by the whole-cell patch-clamp method. Some of the analogs retained the activator character of the parent compound, but, more interestingly, other derivatives inhibited mouse TRESK current. The inhibitor analogs (A2764 and A2793) exerted state-dependent effects. The degree of inhibition by 100 µM A2764 (77.8% ± 3.5%, n = 6) was larger in the activated state of TRESK (i.e., after calcineurin-dependent stimulation) than in the resting state of the channel (42.8% ± 11.5% inhibition, n = 7). The selectivity of the inhibitor compounds was tested on several K2P channels. A2793 inhibited TWIK-related acid-sensitive K+ channel (TASK)-1 (100 µM, 53.4% ± 13, 5%, n = 5), while A2764 was more selective for TRESK, it only moderately influenced TREK-1 and TWIK-related alkaline pH-activated K+ channel. The effect of A2764 was also examined on the background K+ currents of DRG neurons. A subpopulation of DRG neurons, prepared from wild-type animals, expressed background K+ currents sensitive to A2764, whereas the inhibitor did not affect the currents in the DRG neurons of TRESK-deficient mice. Accordingly, A2764 may prove to be useful for the identification of TRESK current in native cells, and for the investigation of the role of the channel in nociception and migraine. SIGNIFICANCE STATEMENT: TRESK background potassium channel is a potential pharmacological target in migraine and neuropathic pain. In this study, we have identified a selective inhibitor of TRESK, A2764. This compound can inhibit TRESK in native cells, leading to cell depolarization and increased excitability. This new inhibitor may be of use to probe the role of TRESK channel in migraine and nociception.[1]

C15H20CL2N2O

315.238101959229

350.071

861038-72-4

146013302

Typically exists as Light yellow to yellow solids at room temperature

2

3

6

21

261

0

ZVRDPULCSHGKBD-UHFFFAOYSA-N

InChI=1S/C15H19ClN2O.2ClH/c1-3-18(4-2)10-11-19-14-8-7-13(16)12-6-5-9-17-15(12)14;;/h5-9H,3-4,10-11H2,1-2H3;2*1H

2-(5-chloroquinolin-8-yl)oxy-N,N-diethylethanamine;dihydrochloride

A 2764 diHCl; A-2764 diHCl; A2764 dihydrochloride; 2-((5-Chloroquinolin-8-yl)oxy)-N,N-diethylethanamine dihydrochloride; A2764 (dihydrochloride); 2-((5-Chloroquinolin-8-yl)oxy)-N,N-diethylethanaminedihydrochloride; 2-(5-chloroquinolin-8-yl)oxy-N,N-diethylethanamine;dihydrochloride; A2764 dihydrochloride?; TRESK inhibitor A2764;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ≥ 100 mg/mL (~284.33 mM)
DMSO : ~41.67 mg/mL (~118.48 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.91 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.91 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)