Measuring [Ca2+] in the endoplasmic reticulum with aequorin

Abstract

The photoprotein aequorin was the first probe used to measure specifically the [Ca2+] inside the lumen of the endoplasmic reticulum ([Ca2+]ER) of intact cells and it provides values for the steady-state [Ca2+]ER, around 500 M, that closely match those obtained now by other procedures. Aequorin-based methods to measure [Ca2+]ER offer several advantages: (i) targeting of the probe is extremely precise; (ii) the use of low Ca2+-affinity aequorin allows covering a large dynamic range of [Ca2+], from 10−5 to 10−3 M; (iii) aequorin is nearly insensitive to changes in Mg2+ or pH, has a high signal-to-noise ratio and calibration of the results in [Ca2+] is made straightforward using a simple algorithm; and (iv) the equipment required for luminescence measurements in cell populations is simple and low-cost. On the negative side, this technique has also some disadvantages: (i) the relatively low amount of emitted light makes difficult performing single-cell imaging studies; (ii) reconstitution of aequorin with coelenterazine requires previous complete depletion of Ca2+ of the ER for 1–2 h, a maneuver that may result in deleterious effects in some cells; (iii) because of the high rate of aequorin consumption at steady-state [Ca2+]ER, only relatively brief experiments can be performed; and (iv) expression of ER-targeted aequorin requires previous transfection or infection to introduce the appropriate DNA construct, or alternatively the use of stable cell clones. Choosing aequorin or other techniques to measure [Ca2+]ER will depend of the correct balance between these properties in a particular problem.