mTORC1 controls lysosomal Ca2+ release through the two-pore channel TPC2

Two-pore segment channel 2 (TPC2) is a ubiquitously expressed, lysosomally targeted ion channel that aids in terminating autophagy and is inhibited upon its association with mechanistic target of rapamycin (mTOR). It is controversial whether TPC2 mediates lysosomal Ca 2+ release or selectively conducts Na + and whether the binding of nicotinic acid adenine dinucleotide phosphate (NAADP) or phosphatidylinositol 3,5-bisphosphate [PI(3,5)P 2 ] is required for the activity of this ion channel. We show that TPC2 is required for intracellular Ca 2+ signaling in response to NAADP or to mTOR inhibition by rapamycin. In pulmonary arterial myocytes, rapamycin and NAADP evoked global Ca 2+ transients that were blocked by depletion of lysosomal Ca 2+ stores. Preincubation of cells with high concentrations of rapamycin resulted in desensitization and blocked NAADP-evoked Ca 2+ signals. Moreover, rapamycin and NAADP did not evoke discernable Ca 2+ transients in myocytes derived from Tpcn2 knockout mice, which showed normal responses to other Ca 2+ -mobilizing signals. In HEK293 cells stably overexpressing human TPC2, shRNA-mediated knockdown of mTOR blocked rapamycin- and NAADP-evoked Ca 2+ signals. Confocal imaging of a genetically encoded Ca 2+ indicator fused to TPC2 demonstrated that rapamycin-evoked Ca 2+ signals localized to lysosomes and were in close proximity to TPC2. Therefore, inactivation of mTOR may activate TPC2 and consequently lysosomal Ca 2+ release.