Bioluminescent Ca2+ Indicators
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In the last two decades, the study of Ca2+
homeostasis in living cells received a great impulse by the explosive development of genetically encoded Ca2+
-indicators. The cloning of the Ca2+
-sensitive photoprotein aequorin and of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria
has been enormously advantageous for the biologists.
As polypeptides, aequorin and GFP allow their endogenous production in cell system as diverse as bacteria, yeast, slime moulds,
plants and mammalian cells. Moreover, it is possible to specifically localize them within the cell by including defined targeting
signals in the amino acid sequence.
These two proteins have been extensively engineerized to obtain several recombinant probes for different biological parameters,
among which Ca2+
concentration reporters are probably the most relevant. In this review, we will not treat the GFP-based Ca2+
probes, but we will present the applications offered by aequorin in the study of intracellular Ca2+
homeostasis, discussing also the new generation of bioluminescent probes that couple the Ca2+
sensitivity of aequorin to GFP fluorescence emission. In these probes, aequorin Ca2+
-dependent photon emission delivers energy to the GFP acceptor in a bioluminescence resonance energy transfer (BRET): this
process enhances the stability and the high signal-to noise ratio of the probes and permits real-time measurements of subcellular
Ca2+
changes in single cell imaging experiments. Very recently, the development of transgenic animals expressing GFP–aequorin
bi-functional probes has also permitted the video-imaging of Ca2+
concentrations changes in live animals.
