A high signal-to-noise Ca2+ 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 μM Ca 2+ to 2.5 ms at 1 μM 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 μM Ca 2+ to 2.5 ms at 1 μM Ca 2+ …
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 μM Ca 2+ to 2.5 ms at 1 μM Ca 2+. Dissociation kinetics (τ∼ 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 Ca 2+ in living cells. Mutational analysis, together with previous structural information, suggests the residues that may alter the fluorescence of GFP.
nature.com