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

The endoplasmic reticulum (ER) is the main cellular reservoir of Ca, able to accumulate high amounts of calcium close to the millimolar range and to release it upon cell activation. Monitoring of Ca 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. Here we describe a robust and quantitative method based on the Ca indicator of the GFP-Aequorin Protein (GAP) family, targeted to the ER lumen. A low Ca affinity version of GAP, GAP1, carrying mutations in two EF-hands of aequorin, reconstituted with coelenterazine n has a reduced affinity for Ca such that it conforms with the [Ca] values found in the ER and it slows the consumption of the probe by Ca. This feature is advantageous because it avoids fast aequorin consumption allowing long-term (longer than 1 h) ER Ca measurements. GAP1 targeted to the ER allows monitoring of resting [Ca] and Ca dynamics in intact cells stimulated with IP-produced agonists. In addition, GAP1 can record Ca mobilization in permeabilized cells challenged with IP. We also provide a detailed calibration procedure which allows to accurately convert the luminescence signal into [Ca].