Fig. 2: Cation permeability of TPC2 is independently regulated.

a Effect of TPC2-A1-N (N; 30 µM), TPC2-A1-P (P; 30 µM) or a combination of the two (N+P) on Na⁺ levels of individual SBFI-loaded HEK cells stably expressing TPC2^L11A/L12A. Each trace is the normalised fluorescence ratio response of a single cell imaged from a typical field of view. The thicker trace is the average of the population. b Pooled data (mean ± sem) quantifying the rate of Na⁺ influx from 4-8 experiments in response to the indicated concentration of agonists. n.s. not significant (One-way ANOVA followed by Dunnett’s post hoc test). c, d Comparison of TPC2-A1-N and TPC2-A1-P responses on cytosolic Na⁺ and Ca²⁺ in TPC2^L11A/L12A-expressing cells loaded with SBFI and Fura-2, respectively. Data are mean ± sem from 4 to 10 experiments. e–h Effect of sequential agonist additions on currents from HEK cells stably expressing TPC2^L11A/L12A under bi-ionic conditions. Macropatches were stimulated with 10 µM TPC2-A1-N and 10 µM TPC2-A1-P (e) or 1 µM PI(3,5)P₂ and 100 nM NAADP (g) in the indicated order. Pooled data (mean ± sem) quantifying the inward Ca²⁺ currents at −100 mV and outward Na⁺ currents at +100 mV from 3 to 6 experiments before (basal) and after agonist addition are shown in f and h. *p < 0.05, ***p = 0.0003, ****p < 0.0001, n.s. not significant (Paired t-test, two-tailed); *p = 0.04, **p = 0.002, n.s. not significant (Unpaired t-test, two-tailed, in red). i–l Effect of agonist additions on currents from HEK cells stably expressing TPC2 in lysosomes under bi-ionic conditions. Cells were stimulated with 10 µM TPC2-A1-P or 10 µM TPC2-A1-N (i) or 1 µM PI(3,5)P₂ and 100 nM NAADP (k) either alone or in combination. Pooled data (mean ± sem) quantifying the inward Ca²⁺ currents at −60 mV or outward Na⁺ currents at +100 mV from 8 to 13 experiments in response to the agonists are shown in j and l, respectively. *p = 0.02, **p = 0.005 (One-way ANOVA followed by Dunnett’s post hoc test) (j); *p = 0.02, ****p < 0.0001 (One-way ANOVA followed by Dunnett’s post hoc test) (l); n.s. not significant (Unpaired t-test, two-tailed, j and l). m, n Effect of TPC2 agonists on reversal potentials. Pooled data (mean ± sem from 3 to 13 experiments) quantifying the effect of TPC2 agonists on E_rev in HEK cells stably expressing TPC2^L11A/L12A at the cell surface (m) or TPC2 in lysosomes (n). Values were derived from the bi-ionic experiments described in e–l. *p = 0.01, **p = 0.007, ****p < 0.0001, n.s. not significant (One-way ANOVA followed by Tukey’s post hoc test) (m); ***p = 0.0005, ****p < 0.0001, n.s. not significant (One-way ANOVA followed by Dunnett’s post hoc test) (n). Source data are provided as a Source Data file.