Cell-based GCaMP fluorescence calcium flux assay

All DNA constructs used in this assay were cloned into a modified pME18 s vector with no fluorescent marker, flanked by AscI/NotI restriction enzyme sites for efficient cloning. Each transfection condition contained 0.5 μg of a plasmid encoding GCaMP6s (Addgene #40753) and 1.5 μg of the plasmid encoding the appropriate olfactory receptor, diluted in 250 μl OptiMEM (Gibco). In experiments with heteromeric olfactory receptors, the total amount of DNA was 1.5 μg, in a ratio of 1:1 of Orco:OR. These were diluted in a solution containing 7 μl Lipofectamine 2000 (Invitrogen) and 250 μl OptiMem, followed by a 20-min incubation at room temperature. HEK293 cells were maintained in high-glucose DMEM supplemented with 10% (v/v) FBS and 1% (v/v) GlutaMAX at 37 °C with 5% (v/v) carbon dioxide. Cells were detached using trypsin and resuspended to a final concentration of 1 × 10⁶ cells/ml. Cells were added to each transfection condition, mixed and added to 2 × 16 wells in a 384-well plate (Grenier CELLSTAR). Four to six hours later, a 16-port vacuum manifold on low vacuum was used to remove the transfection medium, replaced by fresh FluoroBrite DMEM (Gibco) supplemented with 10% (v/v) FBS and 1% (v/v) GlutaMAX. Twenty-four hours later, this medium was replaced with 20 μl reading buffer (20 mM HEPES/NaOH (pH 7.4), 1× HBSS (Gibco), 3 mM Na₂CO₃, 1 mM MgSO₄, and 2 or 5 mM CaCl₂) in each well. The calcium concentration was optimized for each receptor to account for their differences in baseline activity: for experiments with MhOR5 and MhOR5 mutants, reading buffer contained 2 mM CaCl₂, while 5 mM CaCl₂ was used for MhOR1, Orco and Orco–AgOR28 heteromers. The fluorescence emission at 527 nm, with excitation at 480 nm, was continuously read by a Hamamatsu FDSS plate reader. After 30 s of baseline recording, an optimized amount of odorant solution—10 μl for all MhOR-containing experiments or 20 μl for all Orco-containing experiments—was added to the cells and read for 2 min. All solutions were warmed to 37 °C before beginning.

Seven ligand concentrations were used for each transfection condition in sequential dilutions of 3, alongside a control well of only reading buffer. Ligands were dissolved in DMSO to 150 mM, then diluted with reading buffer to a highest final-well concentration of 0.5 mM (DMSO never exceeded 0.5%). Water-soluble ligands (arabinose, caffeine, denatonium, glucose, MSG, sucrose) were dissolved directly into reading buffer. If experimental data indicated a more sensitive response than this range, the concentration was adjusted accordingly. Ligand concentrations for mutants were the same as for the corresponding wild-type OR. Each plate contained a negative control of GCaMP6s transfected alone and exposed to eugenol for MhOR5 and VUAA1 for Orco experiments. Additionally, each plate included the corresponding wild-type OR with its cognate ligand—MhOR5 and MhOR1 with eugenol, Orco with VUAA1, and Orco–AgOR28 with acetophenone—as a positive control to account for plate-to-plate variation in transfection efficiency and cell count. A control of DMSO alone was also tested to ensure no activity effects were due to the solvent. Each concentration of ligand was applied to four technical replicates, which were averaged and considered a single biological replicate.

The baseline fluorescence (F) was calculated as the average fluorescence of the 30 s before odour was added to the plate. Within each well, ΔF was calculated as the difference between the average of the last 10 s of fluorescence and the baseline F. ΔF/F was then calculated as the ΔF divided by the baseline fluorescence (F). Finally, the ΔF/F for each concentration was normalized to the maximum ΔF/F value of the corresponding positive control present on each plate: MhOR5 and MhOR1 with eugenol, Orco with VUAA1, and Orco–AgOR28 with acetophenone to account for inevitable variations in transfection efficiency and cell counts across different plates. The normalized ΔF/F averaged across all experiments for a given condition is the value used to construct the dose–response curves in all plots (Figs. ​(Figs.1b,1b, 2e–g, Extended Data Figs. ​Figs.2d,2d, ​d,9a–c,9a–c, 10c, 11b). All wild-type curves come from the same plates as the experimental data in the same plot. Baseline values for wild-type and mutant channels were found by normalizing each F value by the negative GCaMP6s-only control on the same plate (Extended Data Figs. ​Figs.1c,1c, 9a, e).

For all experiments, GraphPad Prism 8 was used to fit the dose–responses curves to the Hill equation from which the EC₅₀ of the curve was extracted. Three metrics were used to characterize the dose–response curve for each ligand: activity index, log(EC₅₀) and max ΔF/F. For conditions where EC₅₀ was too high for the dose–response curve to reach saturation and therefore could not be fitted to a Hill equation, a value of −2 was assigned to the EC₅₀, which is more than an order of magnitude higher than the highest concentration used. Max ΔF/F is the maximum response achieved at the highest concentration. Activity index is defined as the negative product of log(EC₅₀) and max ΔF/F, as follows:

 Activity index = −log(EC₅₀) × max ΔF/F