A High Signal-To-Noise Ca(2+) Probe Composed of a Single Green Fluorescent Protein

Nat Biotechnol. 2001 Feb;19(2):137-41. doi: 10.1038/84397.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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

Substances

  • Indicators and Reagents
  • Luminescent Proteins
  • Peptide Fragments
  • Recombinant Proteins
  • Green Fluorescent Proteins
  • Ionomycin
  • Adenosine Triphosphate
  • Carbachol
  • Edetic Acid
  • Myosin-Light-Chain Kinase
  • Myosins
  • Calcium