Measuring [Ca2+] in the endoplasmic reticulum with aequorin.

Abstract

The photoprotein aequorin was the first probe used to measure specifically the [Ca(2+)] inside the lumen of the endoplasmic reticulum ([Ca(2+)](ER)) of intact cells and it provides values for the steady-state [Ca(2+)](ER), around 500 microM, that closely match those obtained now by other procedures. Aequorin-based methods to measure [Ca(2+)](ER) offer several advantages: (i) targeting of the probe is extremely precise; (ii) the use of low Ca(2+)-affinity aequorin allows covering a large dynamic range of [Ca(2+)], from 10(-5) to 10(-3)M; (iii) aequorin is nearly insensitive to changes in Mg(2+) or pH, has a high signal-to-noise ratio and calibration of the results in [Ca(2+)] 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 Ca(2+) of the ER for 1-2h, a maneuver that may result in deleterious effects in some cells; (iii) because of the high rate of aequorin consumption at steady-state [Ca(2+)](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 [Ca(2+)](ER) will depend of the correct balance between these properties in a particular problem.

• Authors: J Alvarez, M Montero
• Journal: Cell calcium (Cell Calcium) 2002 Nov-Dec Vol. 32 Issue 5-6 Pg. 251-60 ISSN: 0143-4160 [Print] Netherlands
• PMID: 12543087 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Review)

Chemical References

• Aequorin
• Calcium

Topics

• Aequorin (analysis, genetics, metabolism)
• Animals
• Calcium (analysis, metabolism)
• Endoplasmic Reticulum (chemistry, drug effects, genetics, metabolism)
• Humans
• Luminescent Measurements
• Plasmids (chemical synthesis)