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.
Authors
- Belén Calvo (ORCID: https://orcid.org/0000-0003-1415-1363)
- David I. Yule (ORCID: https://orcid.org/0000-0002-6743-0668)
- Patricia Torres-vidal (ORCID: https://orcid.org/0009-0005-9616-8258)
- Marı́a Teresa Alonso (ORCID: https://orcid.org/0000-0002-9176-3788)
- Christian Grimm (ORCID: https://orcid.org/0000-0002-0177-5559)
- Marco Keller (ORCID: https://orcid.org/0000-0003-4792-3980)
- Francisco J. Aulestia (ORCID: https://orcid.org/0000-0002-7841-812X)
- Viola Oorschot (ORCID: https://orcid.org/0000-0002-8122-8271)
- Vera Y. Moiseenkova‐Bell (ORCID: https://orcid.org/0000-0002-0589-4053)
- Bridget M. McVeigh (ORCID: https://orcid.org/0000-0003-4200-6016)
- Javier García‐Sancho (ORCID: https://orcid.org/0000-0003-4573-7930)
- Alba Delrio-Lorenzo (ORCID: https://orcid.org/0000-0002-3176-3380)
- Sandip Patel (ORCID: https://orcid.org/0000-0001-7247-2013)
- Carla Rodríguez-Crespo (ORCID: https://orcid.org/0009-0000-1174-6658)
- Beatriz Callejo (ORCID: https://orcid.org/0009-0003-4592-0994)
Institutions
- University of Oxford (GB)
- MRC Laboratory for Molecular Cell Biology (GB)
- Fraunhofer Institute for Translational Medicine and Pharmacology (DE)
- Translational Therapeutics (United States) (US)
- European Molecular Biology Laboratory (DE)
- Instituto de Biomedicina y Genética Molecular de Valladolid (ES)
- University College London (GB)
- University of Rochester (US)
- Ludwig-Maximilians-Universität München (DE)
Publication Details
- Journal
- The Journal of Cell Biology
- Published
- 2025-11-07
- DOI
- https://doi.org/10.1083/jcb.202410094
- Citations
- 4
- Primary Topic
- Calcium signaling and nucleotide metabolism
- Type
- article
- Field-Weighted Citation Impact
- 4.47