We here present a novel method for measuring the concentration of Ca2+ ions in defined subcompartments of living cells. The method is based on the recombinant expression of the Ca2+-sensitive photoprotein aequorin, modified in order to include specific targeting signals. The article will focus on the description of the 'mitochondrial' aequorin as the prototype of this class of indicators. A chimeric cDNA was constructed, which encoded a mitochondrial targeting signal fused to the photoprotein. The cDNA was cloned in the expression vector pMT2 and co-transfected into mammalian cells together with the selectable marker pSV2neo. After selection, stable clones were isolated and analyzed for aequorin production. The high producers were further tested. Biochemical analysis showed that the targeted photoprotein was indeed localized in mitochondria. This allowed the first specific measurement of mitochondrial Ca2+ concentration ([Ca2+](m)) in living cells, that showed that [Ca2+](m) is low at rest (< 0.5 μmoles/L), but rapidly rises to the micromolar range upon cell stimulation.
A new approach to cellular Ca2+ determination by the photoprotein aequorine
Brini M.;Rizzuto R.
1993
Abstract
We here present a novel method for measuring the concentration of Ca2+ ions in defined subcompartments of living cells. The method is based on the recombinant expression of the Ca2+-sensitive photoprotein aequorin, modified in order to include specific targeting signals. The article will focus on the description of the 'mitochondrial' aequorin as the prototype of this class of indicators. A chimeric cDNA was constructed, which encoded a mitochondrial targeting signal fused to the photoprotein. The cDNA was cloned in the expression vector pMT2 and co-transfected into mammalian cells together with the selectable marker pSV2neo. After selection, stable clones were isolated and analyzed for aequorin production. The high producers were further tested. Biochemical analysis showed that the targeted photoprotein was indeed localized in mitochondria. This allowed the first specific measurement of mitochondrial Ca2+ concentration ([Ca2+](m)) in living cells, that showed that [Ca2+](m) is low at rest (< 0.5 μmoles/L), but rapidly rises to the micromolar range upon cell stimulation.
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