Extended Data Fig. 4: The GRABACh3.0 sensor produces negligible downstream signaling.
From: An optimized acetylcholine sensor for monitoring in vivo cholinergic activity
a–c, HEK293T cells expressing either a GFP-tagged M3R construct or ACh3.0 are loaded with the red Ca2+ dye Cal590 (a), and the change in Cal590 fluorescence is measured in response to various concentrations of ACh (b). The Ca2+ influx is calculated as the integration of Cal590 fluorescent signal (ΔF/F0) to ACh application. The group summary data for Ca2+ influx measured in response to 0.1 μM ACh are shown in panel c; n = 21 and 15 cells for GFP-M3R and ACh3.0, respectively, p=1.06E-7. d, Left, cartoon illustrating the experimental design of the luciferase complementation assay, in which cells expressed M3R-SmBit or ACh2.0/3.0-SmBit together with LgBit-mGq. Middle, the luminescence signal measured in non-transfected HEK293T cells (NT), cells expressing ACh2.0/ACh3.0-SmBit, or cells expressing M3R-SmBit in response to application of the indicated concentrations of ACh, normalized to the signal measured in control buffer-treated cells (n = 6 wells for NT; n = 6 wells for M3R; n = 3 wells for ACh2.0; n = 6 wells for ACh3.0, with >100 cells in each well). Right, group summary of the luminescence signal measured in response to 100 μM ACh (n = 6 wells for NT; n = 6 wells for M3R; n = 3 wells for ACh2.0; n = 6 wells for ACh3.0, with >100 cells in each well; p=7.11E-7 between NT and M3R; p=9.95E-7 between M3R and ACh2.0; p=0.003 between ACh2.0 and ACh3.0). e, Similar to (d), except the luminescence signal is measured in HEK293T cells expressing M3R-SmBit or cells expressing both M3R-SmBit and ACh3.0. The group summary at the right shows the luminescence signal in response to 100 μM ACh; n = 5-8 wells per group, with each group averaging >100 cells, p=7.95E-5 between NT and M3R; p=0.33 between M3R and M3R+ACh3.0. f, Schematic cartoon depicting two-photon imaging of transgenic flies in response to odorant stimulation. Ca2+ influx is measured by expressing jRCaMP1a either alone or together with ACh3.0 in the Kenyon cells (KC) of the mushroom body. g, Representative fluorescence traces (left) and group summary (right) of jRCaMP1a fluorescence measured in response to odorant application in flies expressing jRCaMP1a either alone or together with ACh3.0; n = 10 flies per group, p=0.49. All data are shown as mean value ± SEM, with the error bars or shaded regions indicating SEM. Scale bar represents 10 μm. Two-sides Student’s t test performed in (c), (d), (e) and (g); ***p<0.001 and n.s., not significant.
