title: Using targeted variants of aequorin to measure Ca2+ levels in intracellular organelles.
abstract: 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.
authors.affiliation: Department of Biomedical Sciences, University of Padua and CNR Neuroscience Institute, 35131 Padua, Italy.
authors.forename: Veronica
authors.initials: V
authors.lastname: Granatiero
authors.forename: Maria
authors.initials: M
authors.lastname: Patron
authors.forename: Anna
authors.initials: A
authors.lastname: Tosatto
authors.forename: Giulia
authors.initials: G
authors.lastname: Merli
authors.forename: Rosario
authors.initials: R
authors.lastname: Rizzuto
mesh_terms: D000331:Aequorin / Q000302:isolation & purification; D000818:Animals; D002118:Calcium / Q000032:analysis*; D015870:Gene Expression; D037402:Hydrozoa; D007093:Imidazoles / Q000378:metabolism; D015388:Organelles / Q000737:chemistry*; D017785:Photons; D011485:Protein Binding; D021381:Protein Transport; D011719:Pyrazines / Q000378:metabolism; D011994:Recombinant Proteins
chemical_list: D007093:Imidazoles; D011719:Pyrazines; D011994:Recombinant Proteins; C017144:coelenterazine; D000331:Aequorin; D002118:Calcium