Direct measurements of luminal Ca2+with endo-lysosomal GFP-aequorin (ELGA) reveal functional IP3receptors

Abstract

ABSTRACTEndo-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 endoplasmic reticulum Ca2+pumps. We find that the Ca2+mobilizing messenger IP3which typically targets the endoplasmic reticulum evokes robust luminal responses in wild type cells, but not in IP3receptor knock-out cells. Responses were comparable to those evoked by activation of the endo-lysosomal ion channel TRPML1. Stimulation with IP3-forming agonists also mobilized the store in intact cells. Our data reveal a physiologically-relevant, IP3-sensitive store of Ca2+within the endo-lysosomal system.