Two-pore channel-2 controls calmodulin-dependent STIM1 inactivation

Lysosomes and the endoplasmic reticulum (ER) are Ca ²⁺ stores that interact to generate Ca ²⁺ signals regulating fundamental cellular processes. NAADP-sensitive TPC2 channels on lysosomes induce local Ca ²⁺ elevations that sensitize ER Ca ²⁺ release channels, triggering global Ca ²⁺ signals. The ensuing ER Ca ²⁺ depletion activates STIM1-gated Orai1 channels that mediate store-operated Ca ²⁺ entry (SOCE) to sustain long-lasting Ca ²⁺ signals. How TPC2 channels interact with STIM1 to integrate distinct intra and extracellular cues is unclear. Here, we show that TPC2 activation inhibits SOCE by enforcing rapid and persistent Ca ²⁺ -CaM-dependent inactivation of the STIM-Orai activating region (SOAR). The TPC2 agonists NAADP and TPC2-A1-N abrogated SOCE in multiple cell lines and enhanced the slow Ca ²⁺ dependent inactivation (SCDI) of STIM1-gated Orai1 channels. TPC2 engagement triggered lysosomal Ca ²⁺ release and mobilized ER Ca2+ stores independently of inositol trisphosphate receptors but prevented RFP-STIM1 recruitment to the TIRF plane by thapsigargin and disassembled RFP-STIM1 clusters forming after store depletion, preventing and acutely reversing SOCE. These effects persisted in STIM1 mutants truncated after the SOAR and were prevented by genetic or pharmacological invalidation of TPC2, Calmodulin (CaM) inhibition, and cytosolic Ca ²⁺ chelation. We conclude that Ca ²⁺ ions released by TPC2 channels on lysosomes regulate CaM-dependent STIM1 inactivation.