title: Genetically encoded bright Ca2+ probe applicable for dynamic Ca2+ imaging of dendritic spines.
abstract: G-CaMP is a Ca2+ probe based on a single green fluorescent protein (GFP). G-CaMP shows a large fluorescence increase upon Ca2+ binding, but its fluorescence is dim and pH sensitive, similar to other single GFP-based probes. Here we report an improved G-CaMP, named G-CaMP1.6, which enables easier detection of intracellular Ca2+ signals. G-CaMP1.6 was approximately 40 times more fluorescent than G-CaMP, mainly due to an increase in quantum yield. Furthermore, compared with G-CaMP, G-CaMP1.6 had not only a lower pH sensitivity but also a higher selectivity for divalent cations having an ionic radius similar to Ca2+. Ca2+ sensitivity of G-CaMP1.6 (Kd = 146 nM, Hill coefficient = 3.8, Fmax/Fmin = 4.9) was slightly shifted toward higher affinity compared with that of G-CaMP. When expressed in mammalian cells, G-CaMP1.6 showed large fluorescence changes with drug applications. Notably, local Ca2+ changes in such tiny structures as dendritic spines of neurons were successfully observed with G-CaMP1.6, this being the first observation using a GFP-based probe. Additional mutations in Ca2+-binding sites of G-CaMP1.6 shifted the affinity for Ca2+ and reduced the Ca2+-buffering effect. G-CaMP1.6-CaM(E140K), which has a mutation in the Ca2+ binding site, is an improved probe with its increased brightness and reduced Ca2+-buffering capacity.
authors.affiliation: Department of Information Physiology, and Department of Cell Physiology, National Institute for Physiological Sciences, and School of Life Science, Graduate University for Advanced Studies, Okazaki, Aichi 444-8585, Japan.
authors.forename: Masamichi
authors.initials: M
authors.lastname: Ohkura
authors.forename: Masanori
authors.initials: M
authors.lastname: Matsuzaki
authors.forename: Haruo
authors.initials: H
authors.lastname: Kasai
authors.forename: Keiji
authors.initials: K
authors.lastname: Imoto
authors.forename: Junichi
authors.initials: J
authors.lastname: Nakai
mesh_terms: D000818:Animals; D002118:Calcium / Q000032:analysis* / Q000378:metabolism*; D002460:Cell Line; D049229:Dendritic Spines / Q000235:genetics* / Q000378:metabolism*; D020832:EF Hand Motifs; D014644:Genetic Variation / Q000235:genetics; D006624:Hippocampus / Q000378:metabolism; D006801:Humans; D051381:Rats; D012680:Sensitivity and Specificity; D013696:Temperature
chemical_list: D002118:Calcium