Targeted recombinant aequorins: tools for monitoring [Ca2+] in the various compartments of a living cell.
In the last decade, the study of Ca2+ homeostasis within organelles in living cells has been greatly enhanced by the utilisation of a recombinant Ca(2+)-sensitive photoprotein, aequorin. Aequorin is a Ca2+ sensitive photoprotein of a coelenterate that, in the past, was widely employed to measure Ca2+ concentration in living cells. In fact, the purified protein was widely used to monitor cytoplasmic [Ca2+] changes in invertebrate muscle cells after microinjection. However, due to the time-consuming and traumatic procedure of microinjection, the role of aequorin in the study of Ca2+ homeostasis remained confined to a limited number of cells (giant cells) susceptible to microinjection. Thus, in most instances, it was replaced by the fluorescent indicators developed by Roger Tsien and coworkers. The cloning of aequorin cDNA [Inouye et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82:3154-3158] and the explosive development of molecular biology offered new possibilities in the use of aequorin, as microinjection has been replaced by the simpler technique of cDNA transfection. As a polypeptide, aequorin allows the endogenous production of the photoprotein in cell systems as diverse as bacteria, yeast, slime molds, plants, and mammalian cells. Moreover, it is possible to specifically localise it within the cell by including defined targeting signals in the amino acid sequence. Targeted recombinant aequorins represent to date the most specific means of monitoring [Ca2+] in subcellular organelles. In this review, we will not discuss the procedure of aequorin microinjection and its use as purified protein but we will present the new advances provided by recombinant aequorin in the study of intracellular Ca2+ homeostasis, discussing in greater detail the advantages and disadvantages in the use of this probe.
Brini
M
M
Department of Biochemistry, University of Padova, 35121 Padova, Italy. brini@civ.bio.unipd.it
Pinton
P
P
Pozzan
T
T
Rizzuto
R
R
D000331:Aequorin / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000737:chemistry; D006706:Homeostasis; D006801:Humans; D008928:Mitochondria / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism
D011994:Recombinant Proteins; D000331:Aequorin; D002118:Calcium