Genetically Encoded Bright Ca2+ Probe Applicable for Dynamic Ca2+ Imaging of Dendritic Spines

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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 ∼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.