A high signal-to-noise ca2+ probe composed of a single green fluorescent protein

J. Nakai, M. Ohkura, K. Imoto

Research output: Contribution to journalArticlepeer-review

935 Citations (Scopus)

Abstract

Recently, several groups have developed green fluorescent protein (GFP)-based Ca2+ 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 Ca2+ probe composed of a single GFP (named G-CaMP). G-CaMP showed an apparent Kd for Ca2+ of 235 nM. Association kinetics of Ca2+ binding were faster at higher Ca2+ concentrations, with time constants decreasing from 230 ms at 0.2 μM Ca2+ to 2.5 ms at 1 μM Ca2+. Dissociation kinetics (τ -200 ms) are independent of Ca2+ 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.

Original languageEnglish
Pages (from-to)137-141
Number of pages5
JournalNature Biotechnology
Volume19
Issue number2
DOIs
Publication statusPublished - 2001
Externally publishedYes

Keywords

  • Calcium
  • Calmodulin
  • Green fluorescent protein
  • Myosin light chain kinase
  • Photoisomerization
  • Protein-protein interaction

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology
  • Molecular Medicine
  • Biomedical Engineering

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