Figure 2.

Activation of plasma membrane P2X7 receptors increases endolysosome size and trafficking in a P2X4 receptor-dependent manner. (A) In NRK cells expressing rP2X4-EGFP and rP2X7 receptors, endolysosomes were labeled with DexTR prior to treatment with 30 µM BzATP for 0.5 h at 37 °C. (B) A histogram of the percentage of cells with two or more enlarged DexTR positive lysosomes (≥1.8 µm) for control and BzATP-stimulated conditions. Stimulation with BzATP was carried out in normal DMEM (2 mM Ca²⁺) or zero Ca²⁺ DMEM. (C) Representative images of DexTR-labeled lysosomes in cells expressing the human isoform of P2X7 alone, in combination with rP2X4-EGFP, the rP2X4K67A-EGFP mutant, or rP2X4-EGFP alone. Control cells and those treated with 100 µM BzATP in DMEM for 0.5 h at 37 °C are shown. In cells expressing hP2X7 alone, EGFP was co-expressed to identify transfected cells. The frequency distribution of lysosome size for control and BzATP-stimulated cells is shown for cells co-expressing rP2X4 and hP2X7 receptors (D), expressing hP2X7 alone (E), and expressing rP2X4 alone (F). For cells expressing rP2X4 alone, a comparison was also made with cells treated with MgATP (100 µM) for 0.5 h at 37 °C. For statistical analysis, control versus agonist-stimulated was compared for each of the size groups. (G) To investigate lysosome trafficking, cells expressing either rP2X4 alone or co-expressing either wild-type rP2X4 or rP2X4-K67A with hP2X7 were treated with BzATP (100 µM, 0.5 h) and dynasore (80 µM, 0.5 h), fixed, and immunostained with an anti-P2X4 antibody and a FITC-labeled anti-rabbit secondary antibody. The nucleus was stained with DapI. (H) The distribution of rP2X4 was assessed by calculating the perinuclear index, which provides a measure of the proportion of receptors in the vicinity of the nucleus ($\le$5 µm) compared to those with a peripheral distribution ($\ge$5 µm). All results are the mean ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001; one-way ANOVA followed by Tukey’s analysis. All scale bars, 10 µm.