Direct measurements of luminal Ca²⁺ with endo-lysosomal GFP-aequorin reveal functional IP₃ receptors

Endo-lysosomes are considered acidic Ca²⁺ stores, but direct measurements of luminal Ca²⁺ within them are limited. Here, we report that the Ca²⁺-sensitive luminescent protein aequorin does not reconstitute with its cofactor at highly acidic pH but that a significant fraction of the probe is functional within a mildly acidic compartment when targeted to the endo-lysosomal system. We leveraged this probe (ELGA) to report Ca²⁺ dynamics in this compartment. We show that Ca²⁺ uptake is ATP-dependent and sensitive to blockers of ER Ca²⁺ pumps. We find that the Ca²⁺ mobilizing messenger IP₃ evokes robust luminal responses in wild-type cells, but not in IP₃R knockout cells. Responses were comparable to those evoked by activation of the endo-lysosomal ion channels TPCs and TRPMLs. Stimulation with IP₃-forming agonists also mobilized the store in intact cells. Super-resolution microscopy analysis was consistent with the presence of IP₃Rs within the endo-lysosomal system. Our data reveal a physiologically relevant, IP₃-sensitive store of Ca²⁺ within the endo-lysosomal system.