Calcium-sensitive photoproteins

The recombinant Ca(2+) sensitive photoprotein aequorin was the first probe used to measure specifically the Ca(2+) concentration, [Ca(2+)], inside the intracellular organelles of intact cells. Aequorin-based methods offer several advantages: (i) targeting of the probe is extremely precise, thus permitting a selective intracellular distribution; (ii) the use of wild-type and low Ca(2+)-affinity aequorins allows covering a large dynamic range of [Ca(2+)], from 10(-7) to 10(-3) M; (iii) aequorin has a low Ca(2+) buffering effect and it is nearly insensitive to changes in Mg(2+) or pH; (iv) it has a high signal-to-noise ratio; (v) calibration of the results in [Ca(2+)] is made straightforward using a simple algorithm; and (vi) 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 is necessary to generate the functional photoprotein and this procedure requires at least 1 h; (iii) in the case of aequorin targeted to high Ca(2+) compartments, because of the high rate of aequorin consumption at steady-state, only relatively brief experiments can be performed and, because of the steepness of the Ca(2+)-response curve, the calibrated [Ca(2+)] values may not reflect the real mean in cells or compartments with dyshomogeneous behavior; and (iv) expression of targeted aequorins requires previous transfection or infection to introduce the appropriate DNA construct, or alternatively the use of stable cell clones. (C) 2008 Elsevier Inc. All rights reserved.