Using targeted variants of aequorin to measure Ca2+ levels in intracellular organelles

Aequorin is a Ca(2+)-sensitive photoprotein isolated from the jellyfish Aequorea victoria. It is an ideal probe for measuring Ca(2+) concentration ([Ca(2+)]) in intracellular organelles because it can be modified to include specific targeting sequences. On the binding of Ca(2+) to three high-affinity sites in aequorin, an irreversible reaction occurs in which the prosthetic group coelenterazine is released and a photon is emitted. This protocol presents procedures for expressing, targeting, and reconstituting aequorin in intact and permeabilized mammalian cells and describes how to use this photoprotein to measure intracellular [Ca(2+)] in various subcellular compartments.