RRC ID 29151
Author Nakai J, Ohkura M, Imoto K.
Title A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein.
Journal Nat Biotechnol
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.
Volume 19(2)
Pages 137-41
Published 2001-2-1
DOI 10.1038/84397
PMID 11175727
MeSH 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
IF 36.553
Times Cited 786
DNA material pN1-G-CaMP (RDB06747) pN1-G-CaMP1.3 (RDB06748)