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 Ca²⁺ release or selectively conducts Na⁺ and whether the binding of nicotinic acid adenine dinucleotide phosphate (NAADP) or phosphatidylinositol 3,5-bisphosphate [PI(3,5)P₂] is required for the activity of this ion channel. We show that TPC2 is required for intracellular Ca²⁺ signaling in response to NAADP or to mTOR inhibition by rapamycin. In pulmonary arterial myocytes, rapamycin and NAADP evoked global Ca²⁺ transients that were blocked by depletion of lysosomal Ca²⁺ stores. Preincubation of cells with high concentrations of rapamycin resulted in desensitization and blocked NAADP-evoked Ca²⁺ signals. Moreover, rapamycin and NAADP did not evoke discernable Ca²⁺ transients in myocytes derived from Tpcn2 knockout mice, which showed normal responses to other Ca²⁺-mobilizing signals. In HEK293 cells stably overexpressing human TPC2, shRNA-mediated knockdown of mTOR blocked rapamycin- and NAADP-evoked Ca²⁺ signals. Confocal imaging of a genetically encoded Ca²⁺ indicator fused to TPC2 demonstrated that rapamycin-evoked Ca²⁺ signals localized to lysosomes and were in close proximity to TPC2. Therefore, inactivation of mTOR may activate TPC2 and consequently lysosomal Ca²⁺ release.