Calcium-sensitive photoproteins.
The recombinant Ca2+ sensitive photoprotein aequorin was the first probe used to measure specifically the Ca2+ concentration, [Ca2+], 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 Ca2+-affinity aequorins allows covering a large dynamic range of [Ca2+], from 10(-7) to 10(-3)M; (iii) aequorin has a low Ca2+ buffering effect and it is nearly insensitive to changes in Mg2+ or pH; (iv) it has a high signal-to-noise ratio; (v) calibration of the results in [Ca2+] 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 1h; (iii) in the case of aequorin targeted to high Ca2+ 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 Ca2+-response curve, the calibrated [Ca2+] 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.
Brini
Marisa
M
Department of Biochemistry, University of Padova, Viale G. Colombo 3, 35131 Padova, Italy. arisa.brini@unipd.it
D000331:Aequorin / Q000502:physiology*; D000465:Algorithms; D002118:Calcium / Q000502:physiology*; D003600:Cytosol / Q000737:chemistry; D007093:Imidazoles / Q000737:chemistry / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D011719:Pyrazines / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection
D007093:Imidazoles; D008164:Luminescent Proteins; D011719:Pyrazines; D011993:Recombinant Fusion Proteins; C017144:coelenterazine; D000331:Aequorin; D002118:Calcium