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.

两孔段通道2（TPC2）是一种普遍表达的，溶酶体靶向的离子通道，有助于终止自噬，并在与雷帕霉素（mTOR）的机械靶标结合时受到抑制。TPC2是否介导溶酶体Ca ²⁺释放或选择性地传导Na ⁺以及是否需要烟酸腺嘌呤二核苷酸磷酸（NAADP）或磷脂酰肌醇3,5-双磷酸[PI（3,5）P ₂ ]的结合存在争议。该离子通道的活性。我们显示TPC2是响应NAADP或雷帕霉素对mTOR抑制的细胞内Ca ²⁺信号传导所必需的。在肺动脉心肌细胞中，雷帕霉素和NAADP引起整体Ca ²⁺瞬变被溶酶体Ca ²⁺储存的耗尽所阻止。用高浓度雷帕霉素对细胞进行预孵育会导致脱敏并阻断NAADP引起的Ca ²⁺信号。此外，雷帕霉素和NAADP不会在源自Tpcn2基因敲除小鼠的心肌细胞中引起可辨别的Ca ²⁺瞬变，这显示出对其他Ca ²⁺动员信号的正常反应。在稳定表达人类TPC2的HEK293细胞中，mTOR的shRNA介导的敲低阻断了雷帕霉素和NAADP引起的Ca ²⁺信号。与TPC2融合的遗传编码Ca ²⁺指示剂的共聚焦成像表明雷帕霉素诱发的Ca ²⁺信号定位于溶酶体，并且非常接近TPC2。因此，mTOR的失活可能激活TPC2，从而激活溶酶体Ca ²⁺释放。