Article Figures & Data
Figures
- Figure 1.
P2X2-GCaMP6s (PG6) fusion is functional and displays concentration-dependent ATP-evoked fluorescence changes. A, Cartoon illustrating the biosensor and the change in GCaMP6s fluorescence during P2X2 activation. B, C, Representative traces (B) and normalized dose-response curves (C) of ATP evoked current measured by whole-cell recording in HEK cells expressing WT P2X2 receptors or PG6 fusion. D, Representative images showing fluorescence changes (λexc/em 485/540) of a HEK cell expressing PG6 after ATP application (10 μm). Images show pseudo-colored dF after F0 subtraction. Scale bar: 20 μm. E, Example traces generated from a plate reader of changes in dF/F values triggered by increasing concentration of ATP in HEK cells expressing PG6. F, PG6 time constant of activation (τON) as a function of ATP concentration. PG6 was expressed in HEK cells and fluorescence evoked by increasing ATP concentration was acquired by video microscopy at 100 Hz. ON-time constants were calculated from N = 3 independent experiments and n > 6 cells per experiments. Inset shows representative fluorescence change evoked by 1 μm (black trace) and 10 μm (red trace) ATP. G, H, Representative traces from a plate reader of changes in dF/F values triggered by 10 μm ATP. HEK cells expressed either PG6 in presence or absence of extracellular calcium (G), or PG6 and PKG6 (H). I, Representative traces from a plate reader of changes in dF/F values triggered by increasing concentration of ATP in HEK cells expressing the cytosolic GCaMP6s. J, Normalized concentration-response curves for ATP evoked changes in F/Fmax at the PG6 and PKG6 fusions and cytosolic GCaMP6s. Curves were generated from N > 4 independent experiments. Data are expressed as mean ± SEM in all panels. For panels E, G, H, I, representative data from n = 3 wells per condition.
- Figure 2.
PG6 fluorescence signal is mainly triggered by calcium influx through the pore of the channel. A, B, Representative traces of normalized fluorescence changes evoked by ATP (10 μm, black), HBSS (gray), or NMDA/glycine (50/10 μm, blue) in HEK cells expressing NMDA receptor subunits GluN1 and GluN2A in combination with either PG6 (A) or PKG6 (B). Data were generated from a plate reader with n = 3 wells per condition. C, D, Representative traces of normalized fluorescence changes evoked by ATP (10 μm, black), HBSS (gray), or capsaicin (100 nm, green) in HEK cells expressing TRPV1 in combination either with PG6 (C) or PKG6 (D). Data were generated from a plate reader with n = 3 wells per condition. E, Plots of area under the fluorescence curve obtained from data shown in panels A, B. Values were normalized to response evoked by 10 μm ATP in PG6-expressing cells. F, Plot of area under the fluorescence curve obtained from data shown in panels C, D. Values were normalized to response evoked by 10 μm ATP in PG6-expressing cells. In all panels, data are expressed as mean ± SEM of N ≥ 3 independent experiments; *p < 0.05, **p < 0.005, ***p < 0.001, one-sample t test compared with 100% reference value; #p < 0.005; NS, non-significant, one-way ANOVA with FDR corrected post hoc tests.
- Figure 3.
P2X-GCaMP6s fusion strategy extended to others P2X receptors. A–D, Representative traces of changes in dF/F values triggered by increasing concentration of ATP (A, B, D) or Bz-ATP (C) in HEK cells expressing P2X4-GCaMP6s (A), P2X5-GCaMP6s (B), P2X7-GCaMP6s (C) or P2X6-GCaMP6s (D). Data were generated from a plate reader with n ≥ 3 wells per condition. E, Normalized concentration-response curves for ATP or Bz-ATP evoked changes in F/Fmax at the P2X4-, P2X5-, P2X6-, and P2X7-GCaMP6s fusions. Curves were generated from N = 3 independent experiments. Data are expressed as mean ± SEM in all panels. F, Summary of EC50, nH, and maximal dF/F for P2X4-, P2X5-, P2X6-, and P2X7-GCaMP6s fusions expressed in HEK cells.
- Figure 4.
Expression and functional analysis of ATP sensors in hippocampal neurons. Neurons were transduced with a lentivirus expressing PG6 or PKG6 under the control of the CamKIIα promoter. A, Transduced neurons were stained using anti-GFP (green) and MAP2 (red) antibodies and nucleus was stained with DAPI (blue). PG6 is localized in the whole dendritic tree as well as in varicosities. Scale bar: 20 μm. B, Fluorescence changes generated from a plate reader and triggered by increasing concentration of ATP in hippocampal neurons expressing PG6. Data are expressed as mean ± SEM of N = 2 independent experiments and three wells per point. C, Normalized concentration-response curves for ATP evoked fluorescence in neurons expressing PG6. Curves were generated from the data presented in B and are expressed as mean ± SEM. D, Representative images of ATP-evoked fluorescence. Intensity of fluorescence was color coded. Scale bar: 20 μm. E, Representative fluorescence recording of KCl-evoked activation of PG6 (black trace) and PKG6 (red trace) in neurons; 3 μm ATP was first applied as above followed by 25 and 50 mm KCl. Fluorescence was acquired as above. KCl-evoked depolarization induced ATP release which in turn activated PG6. In neuron expressing PKG6, only a small and transient fluorescence signal was evoked by depolarization. For PG6 and PKG6, N = 2 independent experiments, n > 12 neurons. F, Plot of area under the curves obtained from panel E, F. One-way ANOVA with Tukey’s multiple comparisons test, **p < 0.01, ***p < 0.001, ****p < 0.0001.
- Figure 5.
Functional characterization of high sensitivity ATP sensors. A, ATP potency at different single residue mutated PG6. Dose-response curves for ATP were performed in HEK cells transfected with each mutant. Normalized curves were generated from N = 4 independent experiments. B, Dose-response curves for each mutant were normalized to that of PG6, showing that some mutations have significant lower response to saturating dose of ATP. N = 4 experiments. C, Table of the main characteristics for each mutant. D, Example traces generated from a plate reader of changes in dF/F values triggered by 300 nm ATP in HEK cells expressing PG6, PNG6 (N333A mutant), and PPG6 (P339A mutant); n = 3 wells. E, Epifluorescence images showing fluorescence changes (λexc/em 485/540) of HEK cells expressing PG6-P2A-Scarlet, PNG6-P2A-Scarlet, and PPG6-P2A-Scarlet during 300 nm ATP application. Scale bar: 20 μm. F, Sensing ATP release from HEK cell during hypo-osmotic stimulation. Comparison of fluorescence signals recorded in a plate reader from cells expressing PG6 or PNG6 during a 160 mOsm application. Data are normalized to the fluorescence evoked by a 10 μm ATP application; 20 U/ml apyrase strongly inhibited hypotonicity-evoked fluorescence in cells expressing the high sensitive ATP sensor; n = 3 wells. G, Estimation of the maximal concentration of ATP release during hypotonic challenge. Maximal fluorescent values measured from cells expressing PG6 or PNG6 and reported on their respective dose-response fitted curve. H, Group data quantification of ATP release during 240 and 160 mOsm hypotonic challenge of cells expressing PG6, PNG6, and PPG6. N = 6 independent experiments. Data from all panels are mean ± SEM.
- Figure 6.
Comparison of the red-shifted ATP sensors. A, Pharmacological characterization of P2X2- and P2X2-N333A-RCaMP2. Normalized ATP dose responses curves were performed using a plate reader on HEK cells expressing either P2X2-, P2X2-N333A-, or P2X2-K69A-RCaMP2. B, Representative traces of changes in dF/F values triggered after 1 μm ATP application in HEK cells expressing P2X2-N333A-RCaMP2 or P2X2-N333A-jRGECO1a. Note that the jRGECO1a-evoked fluorescence is significantly brighter than RCaMP2. Data were generated from a plate reader with n ≥ 3 wells per condition. C, Comparison of hypotonicity-evoked fluorescence in HEK cells expressing either P2X2-N333A-RCaMP2 or P2X2-N333A-jRGECO1a. Data were normalized to the fluorescence evoked by a 10 μm ATP application. D, Normalized concentration-response curves for ATP for P2X2-N333A-RCaMP2 or P2X2-N333A-jRGECO1a. Curves were generated from N = 3 independent experiments. E, Quantification of ATP release during a 160 mOsm hypotonic challenge of cells expressing P2X2-N333A-RCaMP2 and P2X2-N333A-jRGECO1a. N = 4 independent experiments. Data from all panels are mean ± SEM. F, Summary of EC50, nH, and maximal dF/F for P2X2 red shifted biosensors.
Movies
- Movie 1.
Representative movie of ATP responses in hippocampal neurons expressing PG6, related to Figure 4D. Different ATP concentrations were applied for 10 s at, at least, 2-min interval. Baseline fluorescence (F0) was calculated by average of the 20 initial frames. Images were pseudo-colored by F/F0 ratio and exported as JPEG and played at 31.25 fps. The timer indicates real recording time. Indications of ATP concentration are displayed during 1 min before application (in white) and during the 10-s perfusion (in yellow). Scale bar: 5 μm.
Extended Data Figure 2-1
Calcium dependency of P2X-GCaMP6s. A, Representative traces of normalized fluorescence changes evoked by ATP (10 μm, black), HBSS (gray), or NMDA/glycine (50/10 μm, blue) in HEK cells expressing NMDA receptor subunits GluN1 and GluN2A in combination with cytosolic GCaMP6s. B, Representative traces of normalized fluorescence changes evoked by ATP (10 μm, black), HBSS (gray), or capsaicin (100 nm, green) in HEK cells expressing TRPV1 in combination GCaMP6s. For D, E, data were generated from a plate reader with n = 3 wells per condition. Download Figure 2-1, EPS file.
Extended Data Figure 3-1
A, Characterization of P2X4-GCaMP6s. Normalized ATP dose-response curves for P2X4-GCaMP6s, P2X4-K69A-GCaMP6s and cytosolic GCaMP6s in transfected HEK cells. B, C, Representative traces from a plate reader of changes in dF/F values triggered by 10 μm ATP. HEK cells expressed either P2X4-GCaMP6s in the presence or the absence of extracellular calcium (B), or P2X4-GCaMP6s and P2X4-K69A-GCaMP6s (C). For all panels, data are mean ± SEM of at least N > 3. Download Figure 3-1, EPS file.
Extended Data Figure 4-1
Analysis of PG6 expression in neurons. A, Analysis of cell surface expression of PG6 series by biotinylation. After biotinylation of living neurons, proteins were pulled down using neutravidin conjugated beads; biotinylated and total proteins were identified using an anti-GFP antibody. All PG6s (WT and mutants) are present at the cell surface. B, Localization of PG6 and PKG6 in the synaptic fraction. Representative Western blot analysis of PG6, PKG6, and GCaMP6s in homogenate (H), cytosolic (C), and synaptic (S) fractions. Proteins were revealed using anti-GFP or anti-actin antibodies. PG6 and PKG6 are present in the synaptic fraction and to a lower extend in the homogenate, while cytosolic GCaMP6s is found in all fractions. C, Quantification of N = 6 for PG6 and PKG6, and N = 3 experiments for GCaMP6s. Results are mean ± SEM. D, Representative video-microscopy recording of ATP-evoked fluorescence in neurons expressing PG6 (λem/exc = 485/538, acquisition rate 1 Hz). ATP was applied by gravity for 10 s. Note the apparent desensitization of the peak response for high ATP concentrations while a steady state response developed; 10 μm ionomycin was applied for 5 min at the end of the experiment. N = 2 experiment, n > 10 neurons. E, Neuronal expression of PG6 does not perturb intracellular calcium homeostasis. Potential deregulation of neuronal intracellular calcium homeostasis by the expression of PG6 was evaluated through-c-Fos immunostaining. Resting transduced and untransduced neurons were fixed, and c-Fos, GFP, and NeuN staining were performed. c-Fos was not expressed in either neuronal populations while 50 ng/ml BDNF treatment induced a sustained expression of c-Fos. F, Quantification of the number of neurons expressing c-Fos in resting and BDNF-treated conditions. PG6-positive indicate that only neurons expressing PG6 were quantified. Data are mean ± SEM of N = 2 experiments, n > 170 neurons for each condition. Download Figure 4-1, EPS file.
Extended Data Figure 5-1
Electrophysiological characterization of PG6 high sensitivity mutants. A, B, Representative traces (A) and normalized dose-response curves (B) of ATP-evoked currents measured by whole-cell recording in HEK cells expressing WT PG6, PNG6, and PPG6; data are mean ± SEM of N = 4, 3, and 4 experiments, respectively. Download Figure 5-1, EPS file.
Extended Data Figure 6-1
Sensing ATP release during hypotonic challenges. A, B, HEK cells transfected with either PNG6 (A) or PPG6 (B) were challenged by medium with normal osmolarity (320 mOsm, grey) or medium with osmolarity reduced by 25% (240 mOsm, pink) or 50% (160 mOsm, brown). Variations of fluorescence were recorded with a plate reader. Representative results are mean ± SEM, n = 3. C. ATP dosage in the extracellular media of untransfected HEK cells using a bioluminescent plate reader assay (CellTiter-Glo). Extracellular ATP concentration is measured at 0, 5, 10, 15, 20, 25, and 30 min after HBBS (320 mOsm, grey) or medium with osmolarity reduced by 50% (160 mOsm, black) application. Two-way ANOVA with Bonferroni’s multiple comparison, *p < 0.05, ***p < 0.001, ****p < 0.0001. Download Figure 6-1, EPS file.
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