Figure 1. TRMPL1 mediates lysosomal Ca²⁺ efflux in PS1 KO cells.

(A) Lysosomal Ca²⁺ is reduced (WT: n=336; PS1KO n=338) and cytosolic Ca²⁺ is elevated in PS1KO cells (WT: n=148; PS1KO n=150). (B) Cells were treated with 5μM ionomycin followed by GPN (300 μM) to measure lysosomal Ca²⁺ levels Less Ca²⁺ is released from PS1KO (n=18) cells WT cells treated for 24 hrs with 2μg/ml U18666a (n=33) release less lysosomal Ca²⁺. (C) Compared to WT (n=67), PS1KO cells (n=93) released minimal Ca²⁺ after 1 μM thapsigargin followed by NAADP-AM (100 nM) (arrowhead). Ca²⁺ release was significantly lowered in WT cells treated with U18666a (n=25). (D) Addition of 5mM NH₄Cl increased cytosolic Ca²⁺ in PS1KO (n=64) but not WT cells (n=125). (E) 20μM ML-SA1 (black arrowhead) elevated cytosolic Ca²⁺ levels in PS1KO (n=151) but not WT cells (n=206) and this elevation was diminished by Ned-19 (0.5μM, 24hrs; n=167) (Gray arrowheads indicate ionomycin addition). (F) 72 hr shRNA TPC2KD had no discernible effect on lysosomal or cytosolic Ca²⁺ levels in PS1KO cells (n>90). 0.5μM Ned-19 (24hrs) restored lysosomal and cytosolic Ca²⁺ levels even when TPC2 was knocked down (G) 72hr knockdown of TRPML1 significantly elevated lysosomal Ca²⁺ and decreased cytosolic Ca²⁺ in PS1KO cells (H) Anti-TRMPL1 antibody (16h, 5μg/ml) inhibited ML-SA1 mediated lysosomal Ca²⁺ release in PS1KO cells (n=60). (I) Lysosomal Ca²⁺ levels determined using GPN are almost normalized in PS1KO cells after 10μM YM201636 for 1hr via reduced Ca²⁺ flux through TRPML1 (n=100). (J) ConA followed by ML-SA1 induced a rise in cytosolic Ca²⁺ in WT (n=33) but not MLIV fibroblasts (n=29) imaged in a Ca²⁺ free buffer Osmotic lysis of lysosomes with GPN prior to ML-SA1 abolished Ca²⁺ release in WT (n=11) and MLIV cells (n=17). ***p<0.0001. Error bars: ±S.E.M