Direct measurements of luminal Ca ²⁺ with endo-lysosomal GFP-aequorin reveal functional IP ₃ receptors

Endo-lysosomes are considered acidic Ca ²⁺ stores but direct measurements of luminal Ca ²⁺ within them are limited. Here we report that the Ca ²⁺ -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 Ca ²⁺ dynamics in this compartment. We show that Ca ²⁺ uptake is ATP-dependent and sensitive to blockers of endoplasmic reticulum Ca ²⁺ pumps. We find that the Ca ²⁺ mobilizing messenger IP ₃ which typically targets the endoplasmic reticulum evokes robust luminal responses in wild type cells, but not in IP ₃ receptor knock-out cells. Responses were comparable to those evoked by activation of the endo-lysosomal ion channel TRPML1. Stimulation with IP ₃ -forming agonists also mobilized the store in intact cells. Super-resolution microscopy analysis confirmed the presence of IP ₃ receptors within the endo-lysosomal system, both in live and fixed cells. Our data reveal a physiologically-relevant, IP ₃ -sensitive store of Ca ²⁺ within the endo-lysosomal system.