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      A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein.

      1 , ,
      Nature biotechnology
      Springer Science and Business Media LLC

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          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.

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          Author and article information

          Journal
          Nat Biotechnol
          Nature biotechnology
          Springer Science and Business Media LLC
          1087-0156
          1087-0156
          Feb 2001
          : 19
          : 2
          Affiliations
          [1 ] Department of Information Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki, 444-8585, Japan. jnakai@nips.ac.jp
          Article
          10.1038/84397
          11175727
          f3178124-6254-4908-968e-2b21eeef567a
          History

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