A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein.
Nat Biotechnol
; 19(2): 137-41, 2001 Feb.
Article
in En
| MEDLINE
| ID: mdl-11175727
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.
Search on Google
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Calcium
/
Luminescent Proteins
Limits:
Animals
/
Humans
Language:
En
Journal:
Nat Biotechnol
Journal subject:
BIOTECNOLOGIA
Year:
2001
Type:
Article
Affiliation country:
Japan