Abstract
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.