title: A new low-Ca²⁺ affinity GAP indicator to monitor high Ca²⁺ in organelles by luminescence.
abstract: We have recently described a new class of genetically encoded Ca(2+) indicators composed of two jellyfish proteins, a variant of green fluorescent protein (GFP) and the calcium binding protein apoaequorin, named GAP (Rodriguez-García et al., 2014). GAP is a unique dual-mode Ca(2+) indicator, able to function either as a fluorescent or a luminescent probe, depending on whether the photoprotein aequorin is in its apo-state or reconstituted with its cofactor coelenterazine. We describe here a novel application of GAP as a low affinity bioluminescent indicator, suitable for measurements of [Ca(2+)] in ER or in Golgi apparatus. We used the low affinity variant, GAP1, which carries mutations in two EF-hands of aequorin, reconstituted with coelenterazine n. In comparison to previous bioluminescent aequorin fusions, the decay rate of GAP1 was decreased 8 fold and the affinity for Ca(2+) was lowered one order of magnitude. This improvement allows long-term measurements in high Ca(2+) environments avoiding fast aequorin consumption. GAP1 was targeted to the ER of various cell types, where it monitored resting Ca(2+) concentrations in the range from 400 to 600 μM. ER could be emptied of calcium by stimulation with ATP, carbachol or histamine in intact cells, and by challenge with inositol tris-phosphate in permeabilized cells. GAP1 was also targeted to the Golgi apparatus where it was able to precisely monitor long-term calcium dynamics. GAP1 provides a novel and robust indicator applicable to bioluminescent high-throughput quantitative assays.
authors.affiliation: Instituto de Biología y Genética Molecular (IBGM), Universidad de Valladolid and Consejo Superior de Investigaciones Científicas, Sanz y Forés 3, 47003 Valladolid, Spain.
authors.forename: Macarena
authors.initials: M
authors.lastname: Rodríguez-Prados
authors.affiliation: Instituto de Biología y Genética Molecular (IBGM), Universidad de Valladolid and Consejo Superior de Investigaciones Científicas, Sanz y Forés 3, 47003 Valladolid, Spain.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Rojo-Ruiz
authors.affiliation: Instituto de Biología y Genética Molecular (IBGM), Universidad de Valladolid and Consejo Superior de Investigaciones Científicas, Sanz y Forés 3, 47003 Valladolid, Spain.
authors.forename: Francisco Javier
authors.initials: FJ
authors.lastname: Aulestia
authors.affiliation: Instituto de Biología y Genética Molecular (IBGM), Universidad de Valladolid and Consejo Superior de Investigaciones Científicas, Sanz y Forés 3, 47003 Valladolid, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: García-Sancho
authors.affiliation: Instituto de Biología y Genética Molecular (IBGM), Universidad de Valladolid and Consejo Superior de Investigaciones Científicas, Sanz y Forés 3, 47003 Valladolid, Spain. Electronic address: talonso@ibgm.uva.es.
authors.forename: María Teresa
authors.initials: MT
authors.lastname: Alonso
mesh_terms: D000331:Aequorin / Q000378:metabolism; D001059:Apoproteins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D049449:Luminescence; D049408:Luminescent Agents / Q000494:pharmacology*; D015388:Organelles / Q000187:drug effects* / Q000378:metabolism; D011719:Pyrazines / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D001059:Apoproteins; D007093:Imidazoles; D049408:Luminescent Agents; D011719:Pyrazines; D011994:Recombinant Proteins; C060014:apoaequorin; D049452:Green Fluorescent Proteins; C017144:coelenterazine; D000331:Aequorin; D002118:Calcium
keywords: Aequorin; Bioluminescence; Calcium; Endoplasmic reticulum; GAP; GFP; Genetically encoded calcium indicator; Golgi apparatus; Low affinity