Measurements of Ca2+ Concentration with Recombinant Targeted Luminescent Probes

In the last two decades the study of Ca²⁺ homeostasis in living cells has been enhanced 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. As polypeptides, aequorin and GFP allow their endogenous production in cell systems as diverse as bacteria, yeast, slime molds, 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 engineered to obtain several recombinant probes for different biological parameters, among which Ca²⁺ concentration reporters are probably the most relevant. The GFP-based Ca²⁺ probes and aequorin are widely employed in the study of intracellular Ca²⁺ homeostasis. The new generation of bioluminescent probes that couple the Ca²⁺ sensitivity of aequorin to GFP fluorescence emission allows real-time measurements of subcellular Ca²⁺ changes in single cell imaging experiments and the video-imaging of Ca²⁺ concentrations changes in live transgenic animals that express GFP-aequorin bifunctional probes.