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

Recently, several groups have developed green fluorescent protein (GFP)-bas ed 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 developme nt 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 Ca 2+ binding were faster at higher Ca2+ concentrations, with time constants d ecreasing from 230 ms at 0.2 muM Ca2+ to 2.5 ms at 1 muM Ca2+. Dissociation kinetics (tau similar to 200 ms) are independent of Ca2+ concentrations. I n HEK-293 cells and mouse myotubes expressing G-CaMP, large fluorescent cha nges were observed in response to application of drugs or electrical stimul ations. G-CaMP will be a useful tool for visualizing intracellular Ca2+ in living cells. Mutational analysis, together with previous structural inform ation, suggests the residues that may alter the fluorescence of GFP.