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). 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.
Nakai
J
J
Department of Information Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki, 444-8585, Japan. jnakai@nips.ac.jp
Ohkura
M
M
Imoto
K
K
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000032:analysis* / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D002645:Chickens; D004492:Edetic Acid / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents; D015759:Ionomycin / Q000494:pharmacology; D007668:Kidney; D007700:Kinetics; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009219:Myosin-Light-Chain Kinase / Q000235:genetics / Q000378:metabolism; D009218:Myosins / Q000737:chemistry / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D014162:Transfection
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D015759:Ionomycin; D000255:Adenosine Triphosphate; D002217:Carbachol; D004492:Edetic Acid; D009219:Myosin-Light-Chain Kinase; D009218:Myosins; D002118:Calcium