Fig. 4: Unveiling P2X3 receptor ICD architecture in apo and open states via engineered disulfide bonds and VCF analysis in the absence of ATP. | Nature Communications

Fig. 4: Unveiling P2X3 receptor ICD architecture in apo and open states via engineered disulfide bonds and VCF analysis in the absence of ATP.

From: Finely ordered intracellular ___domain harbors an allosteric site to modulate physiopathological function of P2X3 receptors

Fig. 4

A Location of engineered disulfide bonds within the 1α3β ___domain of P2X3-ICD. B The S15C-F358C intersubunit disulfide bond formation identified using non-reducing gel analysis. The experiment was repeated thrice with similar results. C, D Representative current traces (C) and pooled data (D) recorded from transfected cells containing either the mutant or WT P2X3 receptors. H2O2 (0.3%) and dithiothreitol (DTT, 10 mM) were employed to facilitate and disrupt the disulfide bond, respectively. The ratio compares the current generated by ATP (10 µM) following DTT treatment to H2O2 treatment. Every measurement is represented by the scatter of the open circles. Data are expressed as mean ± S.E.M. n = 4 (rP2X3F358C), 5 (rP2X3S15C), 6 (rP2X3WT) or 10 (rP2X3 S15C/F358C), **P < 0.0001 vs. rP2X3WT, one-way ANOVA with Dunnett’s multiple comparisons test. E ATP concentration-response curves for rP2X3WT and rP2X3S15C/F358C after the treatment of H2O2 or DTT. Data are expressed as mean ± S.E.M. n = 5 (0.03, 0.1, 0.3, 1, 3 and 10 μM) independent cells for rP2X3WT-H2O2, n = 4 (0.03, 0.1, 0.3 and 10 μM), or 3 (1 μM) independent cells for rP2X3WT-DTT, n = 3 (0.03, 0.1, and 0.3 μM), or 5 (1, 3 and 10 μM) independent cells for rP2X3S15C/F358C-H2O2, n = 4 (0.03, 0.1, 0.3, 1 and 3 μM), or 3 (10 μM) independent cells for rP2X3S15C/F358C-DTT. F A schematic showing how residue 356’s ANAP emission peak shifts as a result of chemically modifying rP2X3V16C in the absence of ATP. GI Effects of MTSEA (1 mM)-treatment on ANAP fluorescence. Pooled data (G) and representative emission peak shifts of ANAP from the cells expressing rP2X3R356ANAP (H) and rP2X3V16C/R356ANAP (I). Each pair of connected open circles in the scatterplot signifies a single measurement in the before-and-after comparison. n = 16 (rP2X3R356ANAP) or 26 (rP2X3V16C/R356ANAP), *P = 0.0214 and **P = 0.0041, two-way ANOVA with Sidak’s multiple comparisons test.

Back to article page