Figure 3: ML-SA1 induces TRPML1-mediated Ca²⁺ release from lysosomes.

(a) ML-SA1 (20 μM) induced rapid increases in GCaMP3 fluorescence (measured as change of GCaMP3 fluorescence ΔF over basal fluorescence F₀; ΔF/F₀) under low (< 10 nM) external Ca²⁺ in CHO cells transfected with GCaMP3-ML1. Subsequent application of GPN (200 μM) induced smaller responses. Maximal responses were induced by ionomycin (1 μM) application. (b) Representative micrographs from panel (a) to show the changes of GCaMP3 fluorescence upon bath application of ML-SA1 and ionomycin to GCaMP3-ML1-transfected CHO cells. Scale bar=5 μm. (c) ML-SA1 failed to induce significant Ca²⁺ increases in CHO cells transfected with GCaMP3-ML1-KK (non-conducting pore mutation). (d) Basal GCaMP3 fluorescence (normalized to the maximal ionomycin-induced fluorescence) of GCaMP3-ML1-KK and GCaMP3-ML1. (e) GPN (200 μM) pretreatment abolished ML-SA1-induced responses in GCaMP3-ML1-expressing CHO cells. (f) Repetitive applications of ML-SA1 (20 μM) induced little or no responses in GCaMP3-ML1-expressing CHO cells. (g) Repetitive applications of GPN (200 μM) induced smaller GCaMP3 responses in GCaMP3-ML1-expressing CHO cells. (h) ML-SA1 failed to further increase GCaMP3 fluorescence in GCaMP3-ML1-expressing CHO cells in the presence of GPN (200 μM). (i) ML-SA1 induced small responses in GCaMP3-ML1-expressing CHO cells that had received an application of GPN. (j) Baf-A1 (500 nM) pretreatment abolished ML-SA1-induced Ca²⁺ response in GCaMP3-ML1-expressing CHO cells. (k) ML-SA1-induced responses in CHO cells transfected with GCaMP3-ML1-KK (non-conducting pore mutation), and GCaMP3-ML1-transfected cells pretreated with Baf-A1, GPN or TG. For panels d and k, the responses were averaged for 40–100 transfected cells from at least three independent experiments; data are presented as the mean±s.e.m. Statistical comparisons were made using analysis of variance: *P<0.05.