Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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


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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others


  1. Inouye, S. et al. Cloning and sequence analysis of cDNA for the luminescent protein aequorin. Proc. Natl. Acad. Sci. USA 82, 3154–3158 (1985).

    Article  CAS  Google Scholar 

  2. Prasher, D.C., Eckenrode, V.K., Ward, W.W., Prendergast, F.G. & Cormier, M.J. Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111, 229–233 (1992).

    Article  CAS  Google Scholar 

  3. Miyawaki, A. et al. Fluorescent indicators for Ca 2+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887 (1997).

    Article  CAS  Google Scholar 

  4. Miyawaki, A., Griesbeck, O., Heim, R. & Tsien, R.Y. Dynamic and quantitative Ca2+ measurements using improved cameleons. Proc. Natl. Acad. Sci. USA 96, 2135–2140 (1999).

    Article  CAS  Google Scholar 

  5. Romoser, V.A., Hinkle, P.M. & Persechini, A. Detection in living cells of Ca2+-dependent changes in the fluorescence emission of an indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence. A new class of fluorescent indicators. J. Biol. Chem. 272, 13270–13274 (1997).

    Article  CAS  Google Scholar 

  6. Persechini, A., Lynch, J.A. & Romoser, V.A. Novel fluorescent indicator proteins for monitoring free intracellular Ca2+. Cell Calcium 22, 209–216 (1997).

    Article  CAS  Google Scholar 

  7. Baird, G.S., Zacharias, D.A. & Tsien, R.Y. Circular permutation and receptor insertion within green fluorescent proteins. Proc. Natl. Acad. Sci. USA 96, 11241–11246 (1999).

    Article  CAS  Google Scholar 

  8. Baubet, V. et al. Chimeric green fluorescent protein–aequorin as bioluminescent Ca2+ reporters at the single-cell level. Proc. Natl. Acad. Sci. USA 97, 7260–7265 (2000).

    Article  CAS  Google Scholar 

  9. Allen, G.J. et al. Cameleon calcium indicator reports cytoplasmic calcium dynamics in Arabidopsis guard cells. Plant J. 19, 735–747 (1999).

    Article  CAS  Google Scholar 

  10. Emmanouilidou, E. et al. Imaging Ca2+ concentration changes at the secretory vesicle surface with a recombinant targeted cameleon. Curr. Biol. 9, 915–918 (1999).

    Article  CAS  Google Scholar 

  11. Fan, G.Y. et al. Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons. Biophys. J. 76, 2412–2420 (1999).

    Article  CAS  Google Scholar 

  12. Jaconi, M. et al. Inositol 1,4,5-trisphosphate directs Ca2+ flow between mitochondria and the endoplasmic/sarcoplasmic reticulum: a role in regulating cardiac autonomic Ca2+ spiking. Mol. Biol. Cell 11, 1845–1858 (2000).

    Article  CAS  Google Scholar 

  13. Foyouzi-Youssefi, R. et al. Bcl-2 decreases the free Ca2+ concentration within the endoplasmic reticulum. Proc. Natl. Acad. Sci. USA 97, 5723–5728 (2000).

    Article  CAS  Google Scholar 

  14. Yu, R. & Hinkle, P.M. Rapid turnover of calcium in the endoplasmic reticulum during signaling: studies with cameleon calcium indicators. J. Biol. Chem. 275, 23648–23653 (2000).

    Article  CAS  Google Scholar 

  15. Kerr, R. et al. Optical imaging of calcium transients in neurons and pharyngeal muscle of C. elegans. Neuron 26, 583–594 (2000).

    Article  CAS  Google Scholar 

  16. Allen, G.J. et al. Alteration of stimulus-specific guard cell calcium oscillations and stomatal closing in Arabidopsis det3 mutant. Science 289, 2338–2342 (2000).

    Article  CAS  Google Scholar 

  17. Rhoads, A.R. & Friedberg, F. Sequence motifs for calmodulin recognition. FASEB J. 11, 331–340 (1997).

    Article  CAS  Google Scholar 

  18. Mori, M. et al. Novel interaction of the voltage-dependent sodium channel (VDSC) with calmodulin: does VDSC acquire calmodulin-mediated Ca2+-sensitivity? Biochemistry 39, 1316–1323 (2000).

    Article  CAS  Google Scholar 

  19. Bischof, G., Serwold, T.F. & Machen, T.E. Does nitric oxide regulate capacitative Ca influx in HEK 293 cells? Cell Calcium 21, 135–142 (1997).

    Article  CAS  Google Scholar 

  20. Yang, F., Moss, L.G. & Phillips, G.N. Jr. The molecular structure of green fluorescent protein. Nat. Biotechnol. 14, 1246–1251 (1996).

    Article  CAS  Google Scholar 

  21. Dickson, R.M., Cubitt, A.B., Tsien, R.Y. & Moerner, W.E. On/off blinking and switching behavior of single molecules of green fluorescent protein. Nature 388, 355–358 (1997).

    Article  CAS  Google Scholar 

  22. Okada, T. et al. Molecular and functional characterization of a novel mouse transient receptor potential protein homologue TRP7. J. Biol. Chem. 274, 27359–27370 (1999).

    Article  CAS  Google Scholar 

  23. Cormack, B.P., Valdivia, R.H. & Falkow, S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38 (1996).

    Article  CAS  Google Scholar 

  24. Nakai, J. et al. Functional nonequality of the cardiac and skeletal ryanodine receptors. Proc. Natl. Acad. Sci. USA 94, 1019–1022 (1997).

    Article  CAS  Google Scholar 

  25. Bers, D.M., Patton, C.W. & Nuccitelli, R. A practical guide to the preparation of Ca2+ buffers. Methods Cell Biol. 40, 3–29 (1994).

    Article  CAS  Google Scholar 

  26. James-Kracke, M.R. Quick and accurate method to convert BCECF fluorescence to pHi: calibration in three different types of cell preparations. J. Cell. Physiol. 151, 596–603 (1992).

    Article  CAS  Google Scholar 

  27. Tanabe, T., Beam, K.G., Powell, J.A. & Numa, S. Restoration of excitation–contraction coupling and slow calcium current in dysgenic muscle by dihydropyridine receptor complementary DNA. Nature 336, 134–139 (1988).

    Article  CAS  Google Scholar 

Download references


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.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Junichi Nakai.

Rights and permissions

Reprints and permissions

About this article

Cite this article

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).

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing