Fig. 2: Effect of Rab7a on TPC2 activity.

a Effect of PI(3,5)P₂ in endolysosomal vesicles coexpressing human TPC2 and Rab7a or mutant variants of Rab7a. Shown are representative current density-voltage relationships from −100 to +100 mV with basal currents in black, 1 µM PI(3,5)P₂ activated currents in red and ATP (1 mM) blocked currents in blue, measured from apilimod-treated, enlarged endolysosomal vesicles expressing either hTPC2^WT, hTPC2^WT + hRAB7a^WT, hTPC2^WT + hRAB7a^Q67L (constitutively active Rab7a) or hTPC2^WT + hRAB7a^T22N (dominant negative Rab7a). b, c Analogous experiments for the lipophilic small molecule agonists of TPC2 TPC2-A1-P and TPC2-A1-N (10 µM, each). d Statistical summary of data comprising average current densities (mean ± SEM) at −100 mV measured in endolysosomal patch-clamp experiments as shown in (a–c). Each dot on the bar graph represents a single current density value measured from one endolysosome (n = 3–9). Data were tested for statistical significance with one-way ANOVA test followed by Tukey’s post-test (*p < 0.0357, ***p < 0.001, ****p < 0.0001). e Representative GCaMP6s traces, with mean value curves highlighted in bold (color coded), each. Bar chart: Maximal change in fluorescence after application of TPC2 agonist TPC2-A1-N (mean ± SEM). Change in GCaMP6s fluorescence (ΔF) was normalized to baseline value (ΔF/F0), each. The baseline value (F0) was acquired by averaging fluorescence from a 30 s recording before addition of compound. One dot corresponds to one experiment with 3–7 transfected cells, each. TPC2-GCaMP6s (n = 12), TPC2-GCaMP6s + Rab7 WT (n = 9), TPC2-GCaMP6s + Rab7 Q67L (n = 10), TPC2-GCaMP6s + Rab7 T22N (n = 12), L265P-TPC2-GCaMP6s + Rab7 WT (n = 8). Statistical significance was determined via one-way ANOVA followed by Bonferroni multiple comparisons test (p (L265P TPC2 + Rab7a vs. WT TPC2) = 0.0356, p (WT TPC2 vs. TPC2 + Rab7a) = 0.0261, p (WT TPC2 vs. TPC2 + QL Rab7a) < 0.0001, p (TPC2 + Rab7a vs. TPC2 + QL Rab7a) = 0.0037, p (TPC2 + Rab7a vs. TPC2 + TN Rab7a) < 0.0001). Source data are provided as Source Data file.