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

Calvo, B.; Torres-Vidal, P.; Delrio, A.; Rodriguez, C.; Aulestia, F.; Rojo-Ruiz, J.; Garcia-Sancho, J.; Patel, S.; Alonso, M. T.

doi:10.1101/2023.07.11.547422

bioRxiv · 10.1101/2023.07.11.547422

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

Calvo, B.·Torres-Vidal, P.·Delrio, A.·Rodriguez, C.·Aulestia, F.·Rojo-Ruiz, J.·Garcia-Sancho, J.·Patel, S.·Alonso, M. T.

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Abstract

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 endoplasmic reticulum Ca2+ pumps. We find that the Ca2+ mobilizing messenger IP3 which typically targets the endoplasmic reticulum evokes robust luminal responses in wild type cells, but not in IP3 receptor 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. Super-resolution microscopy analysis confirmed the presence of IP3 receptors within the endo-lysosomal system, both in live and fixed cells. Our data reveal a physiologically-relevant, IP3-sensitive store of Ca2+ within the endo-lysosomal system.

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Calvo, B., Torres-Vidal, P., Delrio, A., Rodriguez, C., Aulestia, F., Rojo-Ruiz, J., Garcia-Sancho, J., Patel, S., Alonso, M. T.. 2023-07-12. Direct measurements of luminal Ca2+ with endo-lysosomal GFP-aequorin (ELGA) reveal functional IP3 receptors. https://doi.org/10.1101/2023.07.11.547422

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