A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein

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Abstract
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 Kd 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.