Figure 3.

Stimulation of P2X7 receptors promotes endolysosome alkalinization and an enhanced P2X4 receptor-dependent Ca²⁺ signal. (A) Representative images of cells co-expressing rP2X4 and rP2X7 with endolysosomes labeled with the pH-sensitive Oregon Green 488 10 kD Dextran (DexOG) and pH-insensitive DexTR either with or without treatment with 30 µM BzATP for 0.5 h. (B) The fluorescence ratio of DexOG to DexTR per lysosome was increased in response to BzATP addition. The mean and SEM of this ratio for control and BzATP treated cells is shown for three independent experiments (557–2050 lysosomes per experiment). (C) Colocalization between LAMP1-GECO- and DexTR-positive compartments. (D) Changes in mean cellular LAMP1-GECO fluorescence during application of 30 µM BzATP in HBS is shown for cells expressing rP2X4 and rP2X7 receptors individually or together, or rP2X7 with the rP2X4K6A mutant, as indicated. The mean cellular fluorescence ± SEM was obtained from 50 cells per condition from a single experiment, and, for each cell, the response was normalized to the maximal response (F_max) obtained by a final addition of ionomycin plus 5 mM Ca²⁺. (E) The mean peak amplitudes of the BzATP-evoked responses normalized to F_max, from three separate experiments, are plotted for all of the receptor combinations. Results are the mean ± SEM. * p < 0.05, and *** p < 0.001; one-way ANOVA followed by Tukey’s analysis. All scale bars, 10 µm.