Photoprotein-mediated measurement of calcium ion concentration in mitochondria of living cells

This chapter discusses the expression of the transfected complementary DNA (cDNA), the intracellular localization of the recombinant photoprotein, the reconstitution of the active, Ca2+ measuring holoprotein, the measurement of aequorin light emission, and the conversion of luminescence data into estimates of [Ca2+]. It describes the development of a new methodology for measuring [Ca2+]m in living cells. By this approach the chapter demonstrates that the in vivo local domains of [Ca2+]c, sufficiently high to induce fast mitochondrial Ca2+ accumulation, are transiently generated close to plasmalemmal or intracellular Ca2+ channels. Two appealing applications appear to be an important goal for the future: single-cell imaging and the generation of transgenic animals. As to the first, the low-light emission of photoprotein represents a major problem, and work needs to be done, both in the development of suitable apparatus designed for low-light imaging and in the enhancement of the levels of aequorin expression. As to transgenic animals, there is the fascinating possibility of studying mitochondrial Ca2+ homeostasis in situ (e.g., in brain slices). © 1995, Elsevier Inc.