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.