Measuring [Ca2+] in the endoplasmic reticulum with aequorin.
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.
Alvarez
J
J
Departamento de Bioquímica y Biología Molecular y Fisiología, Facultad de Medicina, Instituto de Biología y Genética Molecular, Universidad de Valladolid, Ramón y Cajal, 7, E-47005, Valladolid, Spain. jalvarez@ibgm.uva.es
Montero
M
M
D000331:Aequorin / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008163:Luminescent Measurements; D010957:Plasmids / Q000138:chemical synthesis
D000331:Aequorin; D002118:Calcium