Measurements of Ca2+ Concentration with Recombinant Targeted Luminescent Probes

Abstract

In the last two decades the study of Ca(2+) homeostasis in living cells has been enhanced by the explosive development of genetically encoded Ca(2+)-indicators. The cloning of the Ca(2+)-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 Ca(2+) concentration reporters are probably the most relevant. The GFP-based Ca(2+) probes and aequorin are widely employed in the study of intracellular Ca(2+) homeostasis. The new generation of bioluminescent probes that couple the Ca(2+) sensitivity of aequorin to GFP fluorescence emission allows real-time measurements of subcellular Ca(2+) changes in single cell imaging experiments and the video-imaging of Ca(2+) concentrations changes in live transgenic animals that express GFP-aequorin bifunctional probes.