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.