Intracellular targeting of the photoprotein aequorin: A new approach for measuring, in living cells, Ca(2+) concentrations in defined cellular compartments.

We here present a novel method, based on the targeting of the photoprotein aequorin, for measuring the concentration of Ca(2+) 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 Ca(2+), [Ca(2+)](m), were obtained, which showed that [Ca(2+)](m) is low at rest (<0.5 μM), but rapidly increases to the micromolar range upon cell stimulation [1]. These data indicate that mitochondria "sense" very accurately the cytosolic Ca(2+) concentration ([Ca(2+)](i)), and after cell stimulation [Ca(2+)](m) rises to values capable of activating the Ca(2+)-sensitive mitochondrial dehydrogenases.