Fig. 1

Fig. 1

Figure 1. Design, optimization, and characterization of a genetically encoded GRAB_ATP sensor

(A) Schematic drawing depicting the principle of GRAB-based ATP sensors designed using the human P2Y₁ receptor as the scaffold coupled to the circularly permuted enhanced GFP (cpEGFP). Binding of ATP induces a conformational change that increases the fluorescence signal.

(B) Optimization of the N- and C-terminal linkers connecting the hP2Y₁ receptor and the cpEGFP moiety, yielding increasingly responsive ATP sensors. The sensor with the highest response to 100 μM ATP, GRAB_ATP1.0 (ATP1.0), is indicated.

(C) Representative fluorescence images of HEK293T cells expressing the ATP1.0 sensor under the basal condition and in the presence of 100 μM ATP.

(D and E) Summary of ΔF/F₀ measured in ATP1.0-expressing HEK293T cells in the presence of the indicated compounds (each at 10 μM, except for MRS-2500, which was applied at 30 μM), normalized to the peak response measured in ATP; n = 4 independent wells each. ATP, adenosine triphosphate; MRS, MRS-2500; Glu, glutamate; GABA, γ-aminobutyric acid; Gly, glycine; DA, dopamine; NE, norepinephrine; 5-HT, 5-hydroxytryptamine (serotonin); HA, histamine; ACh, acetylcholine; ADP, adenosine diphosphate; AMP, adenosine monophosphate; Ado, adenosine; UDP, uridine diphosphate; UDP-G, UDP-glucose.

(F) Summary of the response kinetics of ATP1.0. Left: the experimental system and representative fluorescence traces of ATP1.0 and co-expressed mCherry-CAAX in HEK293T cell to locally puffed ATP; a line scan was used to measure the fluorescence response, and ATP was puffed with a glass pipette with the duration of ∼0.5 s (see STAR Methods for details). Right: the time constant of on and off kinetics of ATP1.0; n = 22 cells from seven coverslips.

(G) Excitation (Ex) and emission (Em) spectra of the ATP1.0 sensor in the presence of ATP (100 μM) or ATP (100 μM) together with MRS-2500 (300 μM). The isosbestic point at 435 nm is indicated.

(H and I) GFP fluorescence images (left column) and pseudocolor images of the response (right column) measured in HEK293T cells expressing ATP1.0 (top row) or pm-iATPSnFR1.0 (bottom row). (I) shows a summary of the response to 100 μM ATP; n = 40 and n = 30 cells each for ATP1.0 and pm-iATPSnFR1.0, respectively.

(J) The peak fluorescence response measured in HEK293T cells expressing ATP1.0 or pm-iATPSnFR1.0 plotted against the indicated concentrations of ATP; n = 10 and n = 20 cells each, respectively. Inset: the same data, normalized and re-plotted.

(K and L) The fluorescence response (K) and signal-to-noise ratio (L) measured in HEK293T cells expressing ATP1.0 or the FRET-based ecAT3.10 sensor; where indicated, 100 μM ATP was applied; n = 20 cells each. The signal-to-noise ratio is defined as the peak response divided by the SD prior to the application of ATP application.

Scale bars represent 30 μm. Summary data are presented as mean ± SEM. The data in (D) and (E) were analyzed using one-way ANOVA followed by Dunnett’s post hoc test; the data in (I) and (L) were analyzed using Student’s t test; ^∗∗∗p < 0.001; n.s., not significant (p > 0.05). See also Figures S1, S2, and S6.