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

Abstract

Abstract Lysosomal two-pore channels (TPC) trigger Ca 2+ release from the endoplasmic reticulum (ER). The ensuing ER Ca 2+ depletion activates STIM1-gated store-operated Ca 2+ entry (SOCE) channels that sustain Ca 2+ signals regulating fundamental cellular processes. How TPC channels and STIM1 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 activators 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 Ca 2+ stores 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 TPC2 genetic or pharmacological invalidation, 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.