Using Targeted Variants of Aequorin to Measure Ca2+ Levels in Intracellular Organelles

Aequorin is a Ca-sensitive photoprotein isolated from the jellyfish Aequorea victoria. It is an ideal probe for measuring Ca concentration ([Ca]) in intracellular organelles because it can be modified to include specific targeting sequences. On the binding of Ca 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] in various subcellular compartments.