RT info:eu-repo/semantics/article
T1 A new low-Ca2+affinity GAP indicator to monitor high Ca2+inorganelles by luminescence
A1 Rodríguez Prados, Macarena
A1 Rojo Ruiz, Jonathan
A1 Aulestia Araya, Francisco Javier
A1 García-Sancho Martín, Francisco Javier
A1 Alonso Alonso, María Teresa
K1 Bioluminiscencia
K1 Bioluminescence
AB 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.
PB Elsevier
YR 2015
FD 2015
LK http://uvadoc.uva.es/handle/10324/35198
UL http://uvadoc.uva.es/handle/10324/35198
LA eng
NO Cell Calcium Volume 58, Issue 6, 2015, Pages 558-564
NO Producción Científica
DS UVaDOC
RD 27-nov-2024