Tritiated 2-methyl-6-(phenylethynyl)pyridine hydrochloride [3H] MPEP was purchased from the American Radiolabeled Chemicals incorporation (ARC), glutamate from Sigma Aldrich, quisqualate and MPEP from Tocris. Sf9 cells adapted in SF-900 II SFM and EX-CELL-420 medium were ordered from Sigma Aldrich, lipofectamine 2000 and DMEM medium from Life Technologies. Detergents were ordered from Anatrace. SNAP-Lumi4-Tb and SNAP Red were obtained from Cisbio Bioassays. Bodipy-FL GTPγS was purchased from ThermoFisher. (R)-5-((3-fluorophenyl) ethynyl)-N-(3-hydroxy-3-methylbutan-2-yl) picolinamide (VU0424465) was synthetized as detailed in the supplementary materials.
Quisqualate
Manufactured by Bio-Techne
Sourced in United Kingdom
Quisqualate is a synthetic amino acid compound used in scientific research. It functions as a selective agonist for the AMPA and kainate subtypes of glutamate receptors. This product is intended for use in in vitro and ex vivo experiments to study the physiological and pharmacological properties of these receptor systems.
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Lab products found in correlation
N 3 chlorophenyl n 4 5 dihydro 1 methyl 4 oxo 1h imidazol 2 yl urea fenobam, by Bio-Techne (2 mentions)
Glutamate, by Bio-Techne (2 mentions)
N methyl d aspartate, by Bio-Techne (2 mentions)
Glutamate, by Merck Group (1 mentions)
Ex cell 420 medium, by Merck Group (1 mentions)
Snap lumi4 tb, by PerkinElmer (1 mentions)
Bodipy fl gtpγs, by Thermo Fisher Scientific (1 mentions)
Snap red, by PerkinElmer (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
6 cyano 7 nitroquinoxaline 2 3 dione cnqx, by Bio-Techne (1 mentions)
S 3 5 dihydroxyphenylglycine, by Bio-Techne (1 mentions)
D 2 amino 5 phosphonopentanoic acid d ap5, by Bio-Techne (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Mk 801, by Bio-Techne (1 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (1 mentions)
Jnj16259685, by Bio-Techne (1 mentions)
3 5 dihydroxyphenylglycine dhpg, by Bio-Techne (1 mentions)
Glutamate pyruvate transaminase, by Roche (1 mentions)
Cpccoet, by Bio-Techne (1 mentions)
9 protocols using quisqualate
1
Radiolabeled MPEP Binding Assay
2
Pharmacological Modulation of Glutamate Receptors
3
Neuronal Culture Reagent Preparation
4
Pharmacological Evaluation of Glutamate Receptors
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Glutamate, quisqualate, (S)-3,5-dihydroxyphenylglycine (DHPG), L -TBA, TBOA, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), D -(−)-2-Amino-5-phosphonopentanoic acid (2S)-2-Amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495), N-[4-(2-Bromo-4,5-difluorophenoxy) phenyl]-L -asparagine (WAY 213613), 2-Amino-5,6,7,8-tetrahydro-4-(4-met¬hoxyphenyl)-7-(naphthalen-1-yl)-5-oxo-4H-chromene-3-carbonitrile (UCPH), MPEP, 7-(Hydroxyimino)-cyclopropan [b]chromen-1a-carboxylate ethyl ester (CPCCOEt), and N-(3-Chlorophenyl)-N′-(4,5-dihydro-1-methyl-4-oxo-1H-imidazol-2-yl)urea (fenobam) were purchased from Tocris Bioscience (Ellisville, MO). THA was obtained from Sigma-Aldrich, (St Louis, MO). 2-Amino-2-(3-cis/trans-carboxycyclobutyl)-3-(9H-thioxanthen-9-yl) propionic acid (LY393053) was obtained from Lilly Research Laboratories, Eli Lilly and Company (Indianapolis, IN).
5
Monitoring Glutamate Receptor Activity
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HEK293 cells were transiently transfected with mGlu5 receptors by electroporation. Receptors were cotransfected with EAAC1, a glutamate transporter, to avoid the influence of extracellular glutamate. The receptor activity was monitored through measurements of inositol monophosphate (IP1) production. IP1 production was determined using the IP-One HTRF kit (CisBio Bioassays) according to the manufacturer’s recommendations [48 (link)]. All points were performed in triplicate. Mutant receptors were obtained using the Quick-Change strategy (Stratagene, La Jolla, CA, USA) and all mutations were verified by sequencing. Experimental data were fitted using the Hill equation with Prism software (GraphPad, La Jolla, CA, USA). Quisqualate and MPEP were purchased from Tocris Bioscience (Bristol, UK). Alloswitch-1 was synthesized in the laboratory of A. Llebaria following described literature procedures [29 (link)]. All solutions were prepared just before experiments.
6
Cryo-EM Structures of AMPA Receptor States
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Cryo-EM grids were prepared using an FEI Vitrobot Mark IV. For the resting state, protein was incubated with 300 μM ZK200775 (Tocris, catalogue no. 2345) for at least 30 min on ice before freezing. For the active state, GluA1/γ3 homomeric complex, protein was first incubated with 300 μM cyclothiazide (Tocris, catalogue no. 0713) for at least 30 min on ice and then quickly mixed with 1 M l -glutamate stock solution to a final concentration of 100 mM before loading onto the grids. For desensitized structures, 10 mM quisqualate (Tocris, catalogue no. 0188) was quickly added to protein to a final concentration of 1 mM before loading. Quantifoil Au 1.2/1.3 (300 mesh) or Quantifoil Cu 1.2/1.3 (300 mesh) grids were glow-discharged for 30 s before use. A 3 μl sample was applied to the grids, blotted for 4.5–6 s at 4 °C with 100% humidity and plunge-frozen in liquid ethane.
All cryo-EM data were collected on an FEI Titan Krios operated at 300 kV, equipped with a K3 detector (Gatan) and a GIF Quantum energy filter (slit width 20 eV). Videos at 1.5–2.5 μm underfocus were taken in counting mode with a pixel size of 1.06 Å per pixel or 0.826 Å per pixel. A combined total dose of 50 e/Å2 was applied with each exposure and 50 frames were recorded for each video. All datasets were collected using EPU2.
All cryo-EM data were collected on an FEI Titan Krios operated at 300 kV, equipped with a K3 detector (Gatan) and a GIF Quantum energy filter (slit width 20 eV). Videos at 1.5–2.5 μm underfocus were taken in counting mode with a pixel size of 1.06 Å per pixel or 0.826 Å per pixel. A combined total dose of 50 e/Å2 was applied with each exposure and 50 frames were recorded for each video. All datasets were collected using EPU2.
7
Pharmacological Modulation of mGlu, AT1, and NMDA Receptors
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(S)-3,5-Dihydroxyphenylglycine (DHPG, HelloBio ® ); Quisqualate (Tocris); LY341495 (Tocris ® ); 2-methyl-6-(phenylethynyl)-pyridine hydrochloride (MPEP, HelloBio ® ); human angiotensin II (HelloBio ® ); N-Methyl-D-Aspartate (NMDA, Tocris ® ). 7-(Hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt, Tocris ® ); Tetrodotoxin (TTX) Citrate (Latoxan ® ), Bicuculline (Hellobio ® ). Except for dose response curves, all the mGlu5, AT1 and mGlu1 ligands are applied at saturating concentrations.
8
Neurotransmitter Receptor Assay Protocol
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We purchased L-glutamate, L-aspartate, glycine, GABA, AMPA, NMDA, IVM, PTX, and FIP from Sigma-Aldrich. Ibotenate, kainate, and quisqualate were purchased from Tocris Bioscience (Bio-Techne Corp.). NA was a kind gift from Bayer Animal Health (Leverkusen, Germany). All chemicals except IVM, PTX, and FIP (DMSO; final concentration ≤ 0.1%) were dissolved in recording solution.
9
Synthesis and Characterization of Novel Optogluram Compounds
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All chemicals were reagent grade (from Roth, Merck, or Sigma, Germany). Quisqualate, DCG-IV, VU0364770 and L-AP4 were purchased from Tocris Bioscience (Bristol, UK).
Optogluram [N-(4-((2-chlorophenyl)diazenyl)-3-methoxyphenyl)picolinamide] and LSP4-2022 were synthesized following the experimental procedures previously reported 23, 24 .
Optogluram [N-(4-((2-chlorophenyl)diazenyl)-3-methoxyphenyl)picolinamide] and LSP4-2022 were synthesized following the experimental procedures previously reported 23, 24 .
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