A high signal-to-noise ca²⁺ probe composed of a single green fluorescent protein

Recently, several groups have developed green fluorescent protein (GFP)-based Ca²⁺ probes. When applied in cells, however, these probes are difficult to use because of a low signal-to-noise ratio. Here we report the development of a high-affinity Ca²⁺ probe composed of a single GFP (named G-CaMP). G-CaMP showed an apparent K_d for Ca²⁺ of 235 nM. Association kinetics of Ca²⁺ binding were faster at higher Ca²⁺ concentrations, with time constants decreasing from 230 ms at 0.2 μM Ca²⁺ to 2.5 ms at 1 μM Ca²⁺. Dissociation kinetics (τ -200 ms) are independent of Ca²⁺ concentrations. In HEK-293 cells and mouse myotubes expressing G-CaMP, large fluorescent changes were observed in response to application of drugs or electrical stimulations. G-CaMP will be a useful tool for visualizing intracellular Ca²⁺ in living cells. Mutational analysis, together with previous structural information, suggests the residues that may alter the fluorescence of GFP.