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    Citas

    Por favor, use este identificador para citar o enlazar este ítem:http://uvadoc.uva.es/handle/10324/35198

    Título
    A new low-Ca2+affinity GAP indicator to monitor high Ca2+inorganelles by luminescence
    Autor
    Rodríguez Prados, Macarena
    Rojo Ruiz, JonatanAutoridad UVA Orcid
    Aulestia Araya, Francisco Javier
    García-Sancho Martín, Francisco JavierAutoridad UVA Orcid
    Alonso Alonso, María TeresaAutoridad UVA
    Año del Documento
    2015
    Editorial
    Elsevier
    Descripción
    Producción Científica
    Documento Fuente
    Cell Calcium Volume 58, Issue 6, 2015, Pages 558-564
    Zusammenfassung
    We have recently described a new class of genetically encoded Ca2+ 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 Ca2+ 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 [Ca2+] 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 Ca2+ was lowered one order of magnitude. This improvement allows long-term measurements in high Ca2+ environments avoiding fast aequorin consumption. GAP1 was targeted to the ER of various cell types, where it monitored resting Ca2+ 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.
    Palabras Clave
    Bioluminiscencia
    Bioluminescence
    Revisión por pares
    SI
    DOI
    10.1016/j.ceca.2015.09.002
    Patrocinador
    Ministerio de Economía, Industria y Competitividad (Project BFU2014-534698P)
    Instituto de Salud Carlos III (RD06/0010/0000 and RD12/0019/0036)
    Version del Editor
    https://www.sciencedirect.com/science/article/pii/S0143416015001463
    Idioma
    eng
    URI
    http://uvadoc.uva.es/handle/10324/35198
    Derechos
    openAccess
    Aparece en las colecciones
    • IBGM - Artículos de revista [78]
    Zur Langanzeige
    Attribution-NonCommercial-NoDerivatives 4.0 InternationalSolange nicht anders angezeigt, wird die Lizenz wie folgt beschrieben: Attribution-NonCommercial-NoDerivatives 4.0 International

    Universidad de Valladolid

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