Intracellular targeting of the photoprotein aequorin: a new approach for measuring, in living cells, Ca2+ concentrations in defined cellular compartments.
We here present a novel method, based on the targeting of the photoprotein aequorin, for measuring the concentration of Ca2+ ions in defined cellular compartments of intact cells. In this contribution we will discuss the application to mitochondria. A chimaeric cDNA was constructed by fusing in frame the aequorin cDNA with that for a mitochondrial protein. The cDNA encoded a "mitochondrially-targeted" aequorin, composed of a typical mitochondrial targeting signal at the N-terminus and the photoprotein at the C-terminus. The cDNA, inserted in the expression vector pMT2, was co-transfected into bovine endothelial and HeLa cells together with the selectable plasmid pSV2-neo and stable transfectants, selected for high aequorin production, were analyzed. In subcellular fractionations, aequorin was shown to be localized in mitochondria; in intact cells, the first direct measurement of mitochondrial free Ca2+, [Ca2+]m, were obtained, which showed that [Ca2+]m is low at rest (< 0.5 microM), but rapidly increases to the micromolar range upon cell stimulation [1]. These data indicate that mitochondria "sense" very accurately the cytosolic Ca2+ concentration ([Ca2+]i), and after cell stimulation [Ca2+]m rises to values capable of activating the Ca(2+)-sensitive mitochondrial dehydrogenases.
Rizzuto
R
R
Dept. Biomedical Sciences, University of Padova, Italy.
Brini
M
M
Pozzan
T
T
D000331:Aequorin / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001709:Biotechnology; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002451:Cell Compartmentation; D002460:Cell Line; D004247:DNA / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008928:Mitochondria / Q000378:metabolism; D014162:Transfection
D000331:Aequorin; D004247:DNA; D002118:Calcium