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Figure 1. Access to the K249C hP2X1R mutant is reduced by PPADS binding and was the starting point for RosettaLigand docking.
a, representative blots of MTSEA-biotinylation of hP2X1 receptor WT and the K249C mutant. Oocytes were treated with apyrase (Apy, 15 units/ml) or apyrase + PPADS (100 μm). Data show expression of the P2X1 in the total samples, however, biotinylation of the WT receptor was below the limit of detection. The K249C mutant receptor was biotinylated and this was reduced by PPADS treatment. b, PPADS docked into both, apo and open state hP2X1R models (for clarity only the open state receptor is shown). Individual subunits are colored in blue, pink, and gray. Docking was focused on the extracellular region centered at Lys-249, the sampled space is indicated by a transparent sphere. Representative docked ligands of the top ranking clusters for open and apo states are shown as sticks. c, zoom into b, representative docking solutions of top ranking clusters for hP2X1R apo state models (bottom) and hP2X1R open state models (top). These clusters were further analyzed for agreement/disagreement with the experimental data presented in this work.
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Figure 2. Effect of PPADS on accessibility changes of the cysteine mutants in the hP2X1 receptor.
a, the closed state homology model of hP2X1 receptor is shown in cartoon, with three subunits in blue, pink, and gray, respectively. The black ring is centered on residue 249 with a radius the length of PPADS, the circle can be divided by the line into two parts (around the ATP-binding sites shown as a smaller circle, and opposite to the ATP-binding site). Cysteine-mutated residues are shown as spheres (red, decreased accessibility in the presence of PPADS, yellow, increased accessibility, gray, no significant change). b, representative blots of MTSEA-biotinylation of hP2X1 receptor mutants. Oocytes were treated with apyrase (Apy, 15 units/ml) or apyrase + PPADS (100 μm). c, densitometric analysis using ImageJ showing any change in MTSEA-biotinylation level at each individual cysteine mutant in the presence of PPADS compared with the apyrase control (n = 3), data are shown as mean ± S.D., (n > 3) (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
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Figure 3. Effect of individual cysteine mutants on PPADS sensitivity of the hP2X1 receptor.
a, the closed state homology model of hP2X1R zoomed in on the extracellular loop showing the location of individual cysteine mutants, blue spheres indicate decreased accessibility but no change in PPADS potency, red spheres indicate decreased accessibility and PPADS potency, yellow spheres indicate increased accessibility and decreased PPADS potency, purple sphere indicates decreased accessibility and increased PPADS potency. The black ring is centered on residue 249 with a radius of the length of PPADS. b, representative traces of inhibition of ATP-evoked currents in the absence (control) and presence of PPADS. Different concentrations of PPADS were applied 5 min before the co-application with ATP (EC90). Orange bar indicates application of PPADS and black bar indicates application of ATP. c, PPADS concentration-dependent inhibition curves of the hP2X1R mutants V74C, K249C, and D170C. The WT receptor response is shown with as a black line (n = 3). d, comparison of PPADS potency (pIC50) between WT and mutant hP2X1Rs. Statistical analysis was performed by one-way analysis of variance, data are shown as mean ± S.D., n = 3 (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
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Figure 4. PPADS quenches MTS-TAMRA fluorescence and highlights cysteine mutants lining the antagonist-binding site.
a, graph showing percentage of MTS-TAMRA fluorescence remaining after addition of different PPADS concentrations. The baseline fluorescence of 1 μm MTS-TAMRA was measured via Flexstation, then different concentrations of PPADS were added after 20 s and any change in fluorescence was monitored, data are shown as mean ± S.D. (n = 3). b, example traces of fluorometry recordings from oocytes expressing hP2X1R single cysteine point mutants labeled with MTS-TAMRA. Oocytes were perfused with ND96, then 10 μm PPADS was applied for 30 s by perfusion (bar above trace) and any changes in fluorescent output were measured. Changes in fluorescence were quantified as the percentage change in fluorescent output compared with baseline level measured before PPADS application. Scale bars apply to all traces. c, graph showing the average change in fluorescence for the mutants tested. A 5% decrease in fluorescence is shown as a dotted line, data are shown as mean ± S.D. (n ≥ 5). The effects of PPADS on cysteine accessibility and antagonist sensitivity were: V74C, E181C, and D320C (light blue) PPADS decreased in MTSEA-biotin and there was no change in PPADS sensitivity, at K70C, K190C, and K249C (red) MTSEA-biotin labeling was reduced following PPADS treatment and PPADS sensitivity was also reduced, at D170C (purple) PPADS decreased MTSEA-biotin access and sensitivity was increased, at K138C (yellow) PPADS increased MTSEA-biotin access and decreased PPADS sensitivity. d, hP2X1R homology model showing the positions of the introduced single cysteine residue mutations. Blue labeling indicates cysteine mutants with a PPADS induced decrease in MTSEA-biotinylation but no effect on PPADS sensitivity, red is for those with a decrease in MTSEA-biotinylation following PPADS treatment and a decrease in PPADS sensitivity, and yellow corresponds to residues where MTSEA-biotinylation was increased by PPADS and also showed a decrease in PPADS sensitivity. e, hP2X1R homology model surface representation with K249C linked MTS-TAMRA (modeled manually). Compared with d, the receptor is slightly rotated to the left for clearer visualization of MTS-TAMRA (shown as spheres).
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Figure 5. PPADS docking pose in best agreement with experimental data.
a, cartoon representation of open state hP2XR1 model with subunits shown in red, blue, and white, respectively. The docked PPADS pose from the largest, top ranked cluster that is best explaining data for Lys-70, Asp-170, Lys-190, and Lys-249 is shown as spheres. b, zoom into a, detailing key charge interactions between PPADS and hP2XR that are discussed in the text.
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Figure 6. Comparison of proposed PPADS and TNP-ATP binding modes.
a, proposed PPADS binding mode for P2X1R. b, for comparison the X-ray structure of chicken P2X7 receptor with TNP-ATP bound is shown. Equivalent lysine residues involved in salt bridges to sulfonate groups of PPADS (a) and phosphate groups of TNP-ATP are labeled. c, visualization of electrostatic potential for the P2X1R model used in a. The electrostatic potential was calculated using the Protein Data Bank 2PQR. d, as in c, but PPADS was omitted.
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Figure 7. Comparison of the effects of PPADS and ATP on MTSEA-biotinylation at cysteine mutants.
Upper panel shows representative blots of the MTSEA-biotinylation levels of mutant P2X1Rs in the presence of PPADS, apyrase control, or ATP. The graph shows the effects of PPADS (gray shading) or ATP (open boxes) on the relative level of biotinylation compared with the control (in the presence of apyrase to break down any endogenous ATP). Data are shown as mean ± S.D. (n = 3), *, p < 0.05.
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Figure 8. Positive charge at the base of the cysteine-rich head region imparts PPADS sensitivity to the rP2X4R.
a, representative traces showing application of ATP and ATP + 1 μm PPADS (filled circles) for rP2X4R, the X4-CRH1 chimera and hP2X1R for rP2X4(2+) and rP2X4(4+) mutant receptors. b, PPADS inhibition curves. All inhibition was measured at an EC90 concentration of ATP. Dotted lines correspond to previously published mean data from Farmer et al. (19). The black bar corresponds to the period of agonist application (3 s) and PPADS was pre-superfused over the oocyte and co-applied with ATP (filled circles). Traces have been normalized to peak current to allow for comparison. Data are plotted as mean ± S.D. n = 3–4.
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