Monitoring ER Ca2+ by Luminescence with Low Affinity GFP-Aequorin Protein (GAP).
PMID 39395103 · Methods in molecular biology (Clifton, N.J.) · 2025 · DOI 10.1074/jbc.272.44.27694
Authors: Macarena Rodriguez-Prados; Jonathan Rojo-Ruiz; Belen Calvo; Javier Garcia-Sancho; Maria Teresa Alonso
Abstract
The endoplasmic reticulum (ER) is the main cellular reservoir of Ca2+, able to accumulate high amounts of calcium close to the millimolar range and to release it upon cell activation. Monitoring of Ca2+ 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 Ca2+. Here we describe a robust and quantitative method based on the Ca2+ indicator of the GFP-Aequorin Protein (GAP) family, targeted to the ER lumen. A low Ca2+ affinity version of GAP, GAP1, carrying mutations in two EF-hands of aequorin, reconstituted with coelenterazine n has a reduced affinity for Ca2+ such that it conforms with the [Ca2+] values found in the ER and it slows the consumption of the probe by Ca2+. This feature is advantageous because it avoids fast aequorin consumption allowing long-term (longer than 1 h) ER Ca2+ measurements. GAP1 targeted to the ER allows monitoring of resting [Ca2+]ER and Ca2+ dynamics in intact cells stimulated with IP3-produced agonists. In addition, GAP1 can record Ca2+ mobilization in permeabilized cells challenged with IP3. We also provide a detailed calibration procedure which allows to accurately convert the luminescence signal into [Ca2+]ER.
Aequorin; Endoplasmic Reticulum; Calcium; Humans; Green Fluorescent Proteins; Luminescent Measurements; Calcium Signaling; Animals
Cite
Macarena Rodriguez-Prados; Jonathan Rojo-Ruiz; Belen Calvo; Javier Garcia-Sancho; Maria Teresa Alonso. Monitoring ER Ca2+ by Luminescence with Low Affinity GFP-Aequorin Protein (GAP). Methods in molecular biology (Clifton, N.J.) (2025). PMID 39395103. DOI 10.1074/jbc.272.44.27694.