Monitoring ER Ca²⁺ by Luminescence with Low Affinity GFP-Aequorin Protein (GAP)

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.