Assay buffer consisted of MilliQ water, 135 mM NaCl (AppliChem, Darmstadt, Germany), 1 mM CaCl2 (AppliChem), 5 mM KCl (AppliChem), 1 mM MgCl2 (AppliChem), 11 mM Na-HEPES (pH = 7.4) (Sigma-Aldrich, Taufkirchen, Germany), protease inhibitor cocktail (according to the manufacturer's description, Roche, Basel, Switzerland) and 0.1% Pluronic F-127 (Sigma-Aldrich).
Muscarinic acetylcholine receptor ligands acetylcholine, arecoline, pirenzepine, pilocarpine, atropine and scopolamine were purchased from Sigma-Aldrich and carbachol from Tocris Bioscience (Abingdon, UK). The syntheses of the fluorescent ligands UR-MK342 and UR-CG072 [16 (link)] and the dualsteric M2R ligands UR-SK59 [50 (link)], UR-SK75 [50 (link)] and UNSW-MK259 [51 (link)], showing also high M4R affinity, were described previously. All ligand stock solutions were prepared using cell culture grade DMSO (AppliChem) and stored at −20°C.
Muscarinic acetylcholine receptor ligands acetylcholine, arecoline, pirenzepine, pilocarpine, atropine and scopolamine were purchased from Sigma-Aldrich and carbachol from Tocris Bioscience (Abingdon, UK). The syntheses of the fluorescent ligands UR-MK342 and UR-CG072 [16 (link)] and the dualsteric M2R ligands UR-SK59 [50 (link)], UR-SK75 [50 (link)] and UNSW-MK259 [51 (link)], showing also high M4R affinity, were described previously. All ligand stock solutions were prepared using cell culture grade DMSO (AppliChem) and stored at −20°C.
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