Genetically encoded bright Ca²⁺ probe applicable for dynamic Ca²⁺ imaging of dendritic spines

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 (K_d = 146 nM, Hill coefficient = 3.8, F_max/ F_min = 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 Ca ²⁺ binding site, is an improved probe with its increased brightness and reduced Ca²⁺-buffering capacity.