Fluorescent Binding Assay for Mu-Opioid Receptor

The fluorescent binding assay employs the native MUR fused at the N-terminus to a SNAP-tag® enzyme and expressed on HEK293 cells. SNAP-tag-mu-opioid is then covalently labeled with terbium cryptate (Lumi4®-Tb), a long lifetime FRET donor. An analog of the potent opioid antagonist naltrexone that contains the d2 dye (red-naltrexone) is used as the fluorescence energy transfer acceptor. Upon ligand binding, a FRET process occurs between the Lumi4-Tb donor (emission at 620 nm) in SNAP-Lumi4-Tb-mu-opioid receptor and the red-naltrexone acceptor (emission at 665 nm). The fluorescence emission from the acceptor is detected in a time resolved manner (TR-FRET). For HTRF assay (Cisbio Bioassays, Bedford, MA), Tag-lite® mu opioid cells suspended in culture medium were dispensed in white 384-well low-volume microplates (Greiner Bio-one Greiner Bio-One North America, Monroe, NC) at 3700 cells/10 µL/well Tag-lite m opioid labeled cells with 60 nM of Tag-lite opioid receptors red-naltrexone, and 5 µL of the wsMUR-TM with 11 further 1∶2 serial dilutions from the µM to the nM range. All samples were mixed with final volume 20 µl and incubated at RT for 2 h. After incubation, HTRF signals were measured using a plate reader (BMG, Cary, NC) after excitation at 337 nm at both 620 and 665 nm emission, HTRF signal was calculated as a two-wavelength signal ratio: [intensity (665 nm)/intensity (620 nm)]. IC₅₀ determination and statistical analysis IC₅₀ values for wsMUR-TM were determined by fitting the dose–responses curves using the Prism program (GraphPad Software, San Diego, CA).