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      A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein.

      Nature biotechnology

      Transfection, metabolism, analysis, Recombinant Proteins, chemistry, Peptide Fragments, genetics, Myosins, Myosin-Light-Chain Kinase, Mutagenesis, Site-Directed, Molecular Sequence Data, Luminescent Proteins, Kinetics, Kidney, pharmacology, Ionomycin, Indicators and Reagents, Humans, Green Fluorescent Proteins, Edetic Acid, Chickens, Cell Line, Carbachol, Calcium, Animals, Amino Acid Sequence, Adenosine Triphosphate

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          Abstract

          Recently, several groups have developed green fluorescent protein (GFP)-based Ca(2+) 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 Ca(2+) probe composed of a single GFP (named G-CaMP). G-CaMP showed an apparent K(d) for Ca(2+) of 235 nM. Association kinetics of Ca(2+) binding were faster at higher Ca(2+) concentrations, with time constants decreasing from 230 ms at 0.2 microM Ca(2+) to 2.5 ms at 1 microM Ca(2+). Dissociation kinetics (tau approximately 200 ms) are independent of Ca(2+) 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.

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          Journal
          10.1038/84397
          11175727

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