Monitoring ER Ca 2+ by Luminescence with Low Affinity GFP-Aequorin Protein (GAP).

Autor: Rodriguez-Prados M; Instituto de Biomedicina y Genética Molecular de Valladolid (IBGM), Unidad de Excelencia, Universidad de Valladolid y Consejo Superior de Investigaciones Científicas (CSIC), Valladolid, Spain.; Universidad Alfonso X el Sabio, Madrid, Spain., Rojo-Ruiz J; Instituto de Biomedicina y Genética Molecular de Valladolid (IBGM), Unidad de Excelencia, Universidad de Valladolid y Consejo Superior de Investigaciones Científicas (CSIC), Valladolid, Spain., Calvo B; Instituto de Biomedicina y Genética Molecular de Valladolid (IBGM), Unidad de Excelencia, Universidad de Valladolid y Consejo Superior de Investigaciones Científicas (CSIC), Valladolid, Spain., Garcia-Sancho J; Instituto de Biomedicina y Genética Molecular de Valladolid (IBGM), Unidad de Excelencia, Universidad de Valladolid y Consejo Superior de Investigaciones Científicas (CSIC), Valladolid, Spain., Alonso MT; Instituto de Biomedicina y Genética Molecular de Valladolid (IBGM), Unidad de Excelencia, Universidad de Valladolid y Consejo Superior de Investigaciones Científicas (CSIC), Valladolid, Spain. talonso@uva.es.
Jazyk: angličtina
Zdroj: Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2025; Vol. 2861, pp. 141-153.
DOI: 10.1007/978-1-0716-4164-4_11
Abstrakt: The endoplasmic reticulum (ER) is the main cellular reservoir of Ca 2+ , able to accumulate high amounts of calcium close to the millimolar range and to release it upon cell activation. Monitoring of Ca 2+ dynamics within the ER lumen is best achieved using genetically encoded and targeted reporters. Luminescent probes based on the photoprotein aequorin have provided significant insight to measure subcellular Ca 2+ . Here we describe a robust and quantitative method based on the Ca 2+ indicator of the GFP-Aequorin Protein (GAP) family, targeted to the ER lumen. A low Ca 2+ affinity version of GAP, GAP1, carrying mutations in two EF-hands of aequorin, reconstituted with coelenterazine n has a reduced affinity for Ca 2+ such that it conforms with the [Ca 2+ ] values found in the ER and it slows the consumption of the probe by Ca 2+ . This feature is advantageous because it avoids fast aequorin consumption allowing long-term (longer than 1 h) ER Ca 2+ measurements. GAP1 targeted to the ER allows monitoring of resting [Ca 2+ ] ER and Ca 2+ dynamics in intact cells stimulated with IP 3 -produced agonists. In addition, GAP1 can record Ca 2+ mobilization in permeabilized cells challenged with IP 3 . We also provide a detailed calibration procedure which allows to accurately convert the luminescence signal into [Ca 2+ ] ER .
(© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
Databáze: MEDLINE
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