Recombinant aequorin as reporter of changes in intracellular calcium in mammalian cells

Abstract

Publisher Summary The photoprotein aequorin, from the coelenterate jellyfish Aequorea victoria , is a bioluminescent complex formed from the 21-kDa apoaequorin, the luminophore cofactor coelenterazine, and molecular oxygen. Apoaequorin is a 189-amino acid polypeptide chain containing three calcium-binding sites (EF-hand structures). After the binding of calcium to these sites, aequorin undergoes a conformational change, converting into an oxygenase. Aequorin has been used as a reporter of changes in intracellular calcium concentration in mammalian cells or Xenopus oocytes. In these experiments, loading of cells with aequorin has been involved microinjection of purified protein. Aequorin offers a highly sensitive method with which to assess changes in the level of intracellular calcium that offers significant time savings over the use of calcium-sensitive fluorescent dyes such as Fura-2 or Fluo-3. The detection of changes in intracellular calcium concentration using expressed aequorin does not require loading of the cells with dye, the extensive washing steps involved in the dye-loading procedure, the leaching of dye from the cells, or fluorescence quenching associated with the use of these dyes.