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A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein

Nature Biotechnology volume 19, pages 137–141 (2001)Cite this article

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.

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Figure 1: Schematic representation of GFP-based Ca2+ probes.
Figure 2: Expression of GFP-based Ca2+ probes in HEK-293 cells.
Figure 3: In vitro characterization of G-CaMP.
Figure 4: Expression of G-CaMP in myotubes.

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Acknowledgements

We thank Masayuki Mori for the rat calmodulin cDNA and Michiyo Murata and Mitsutoshi Ono for technical assistance. We also thank Toshihiko Nagamura and Tatsuo Nakagawa of Unisoku Co., Ltd. for technical assistance. The work was supported by grants from the Ministry of Education, Science, Sports and Culture, by “the Research for the Future Program” of the JSPS, and by the JSPS Research Fellowships for Young Scientists.

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  1. Department of Information Physiology, National Institute for Physiological Sciences, Myodaiji, 444-8585, Okazaki, Japan

    Junichi Nakai, Masamichi Ohkura & Keiji Imoto

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  1. Junichi Nakai
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  2. Masamichi Ohkura
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Correspondence to Junichi Nakai.

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Nakai, J., Ohkura, M. & Imoto, K. A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein. Nat Biotechnol 19, 137–141 (2001). https://doi.org/10.1038/84397

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