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

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...

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Bibliographic Details
Published in:Nature biotechnology 2001-02, Vol.19 (2), p.137
Main Authors: Nakai, J, Ohkura, M, Imoto, K
Format: Article
Language:English
Subjects:
Adenosine Triphosphate - pharmacology
Amino Acid Sequence
Animals
Calcium - analysis
Calcium - metabolism
Carbachol - pharmacology
Cell Line
Chickens
Edetic Acid - pharmacology
Green Fluorescent Proteins
Humans
Indicators and Reagents
Ionomycin - pharmacology
Kidney
Kinetics
Luminescent Proteins - analysis
Luminescent Proteins - genetics
Luminescent Proteins - metabolism
Molecular Sequence Data
Mutagenesis, Site-Directed
Myosin-Light-Chain Kinase - genetics
Myosin-Light-Chain Kinase - metabolism
Myosins - chemistry
Myosins - genetics
Peptide Fragments - chemistry
Peptide Fragments - metabolism
Recombinant Proteins - analysis
Recombinant Proteins - metabolism
Transfection
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Summary: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.
ISSN:1087-0156
DOI:10.1038/84397