TR-FRET measurement
TR-FRET measurements were performed as previously described . Briefly, ST-hmGlu5 HEK293T seeded at 5 x 104 cells per well in 96-wells black bottom plates were incubated for 1h-1h30 with 100nM of SNAP–Lumi4-Tb and/or 60 nM of SNAP–green. Measurements were performed at room temperature using the Pherastar FS (BMG LabTech). Following SNAP–Lumi4-Tb excitation (337 nm), the decays of SNAP–green or SNAP–Lumi4-Tb emission were measured every 10µs at 520 nm and 620 nm respectively. TR-FRET ratios and SNAP–Lumi4-Tb decays were calculated following Scholler et al. recommendation for mGlu receptors with the corresponding time intervals:\(\frac{\Sigma\ [50us-100us]}{\Sigma[1200us-1600us]}\). For cells incubated only with SNAP–green, the fluorescence was measured at 520 nm with a 485 nm excitation. Each independent experiment was performed in triplicate, which average corresponds to n = 1 and data were normalized to the maximum response at Vrest except for LY341495 dose-response curves which were normalized to the maximum response at Vdepol. Basic calculations from the raw data files were performed on Microsoft Excel (Office 2016), statistical analyses and graphics on GraphPad Prism 8.1.