DOI: 10.1073/pnas.96.5.2135. Dynamic and quantitative Ca <sup>2+</sup> measurements using improved cameleons

Dynamic and quantitative Ca 2+ measurements using improved cameleons

10.1073/pnas.96.5.2135

Crossref journal-article

Proceedings of the National Academy of Sciences

Proceedings of the National Academy of Sciences (341)

Abstract

Cameleons are genetically-encoded fluorescent indicators for Ca 2+ based on green fluorescent protein variants and calmodulin (CaM). Because cameleons can be targeted genetically and imaged by one- or two-photon excitation microscopy, they offer great promise for monitoring Ca 2+ in whole organisms, tissues, organelles, and submicroscopic environments in which measurements were previously impossible. However, the original cameleons suffered from significant pH interference, and their Ca 2+ -buffering and cross-reactivity with endogenous CaM signaling pathways was uncharacterized. We have now greatly reduced the pH-sensitivity of the cameleons by introducing mutations V68L and Q69K into the acceptor yellow green fluorescent protein. The resulting new cameleons permit Ca 2+ measurements despite significant cytosolic acidification. When Ca 2+ is elevated, the CaM and CaM-binding peptide fused together in a cameleon predominantly interact with each other rather than with free CaM and CaM-dependent enzymes. Therefore, if cameleons are overexpressed, the primary effect is likely to be the unavoidable increase in Ca 2+ buffering rather than specific perturbation of CaM-dependent signaling.

Bibliography

Miyawaki, A., Griesbeck, O., Heim, R., & Tsien, R. Y. (1999). Dynamic and quantitative Ca 2+ measurements using improved cameleons. Proceedings of the National Academy of Sciences, 96(5), 2135â2140.

Authors 4

Atsushi Miyawaki (first)
Oliver Griesbeck (additional)
Roger Heim (additional)
Roger Y. Tsien (additional)

References 30 Referenced 693

10.1146/annurev.ne.12.030189.001303
10.1074/jbc.270.17.9896
10.1038/42264
10.1093/protein/7.1.109
10.1126/science.1585175
10.1128/jvi.70.7.4646-4654.1996
10.1016/S0021-9258(20)80699-1
10.1073/pnas.95.12.6803
10.1016/S0960-9822(02)00450-5
F L Graham, L Prevec Methods Mol Biol 7, 109–128 (1991). / Methods Mol Biol by Graham F L (1991)
10.1002/j.1460-2075.1990.tb07564.x
10.1016/0003-2697(79)90115-5
10.1021/bi00166a017
10.1126/science.273.5280.1392
10.1146/annurev.biochem.67.1.509
10.1016/0166-2236(92)90191-A
10.1152/jn.1994.72.6.2563
10.1038/41048
10.1111/j.1751-1097.1982.tb02651.x
10.1016/0896-6273(93)90277-X
10.1016/S0960-9822(02)00609-7
10.1002/j.1460-2075.1987.tb02738.x
10.1210/endo.133.1.8319584
10.1016/S0021-9258(19)70576-6
10.1093/oxfordjournals.jbchem.a134019
10.1074/jbc.272.20.13270
Kerr R. A. Baird G. Schafer W. R. & Tsien R. Y. (1998) Abstracts of the Fifth International Congress of Neuroethology S4.
10.1016/S0896-6273(00)81237-4
Fan G. Y. Fujisaki H. Miyawaki A. Tsay R.-K. Tsien R. Y. & Ellisman M. H. (1999) Biophys. J. in press.
10.1016/S0076-6879(89)72017-6

Dates

Type	When
Created	23 years, 1 month ago (July 26, 2002, 10:39 a.m.)
Deposited	3 years, 4 months ago (April 13, 2022, 5:45 p.m.)
Indexed	1 month ago (Aug. 5, 2025, 8:18 a.m.)
Issued	26 years, 6 months ago (March 2, 1999)
Published	26 years, 6 months ago (March 2, 1999)
Published Online	26 years, 6 months ago (March 2, 1999)
Published Print	26 years, 6 months ago (March 2, 1999)

Funders 0

None

BibTeX

@article{Miyawaki_1999, title={Dynamic and quantitative Ca 2+ measurements using improved cameleons}, volume={96}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.96.5.2135}, DOI={10.1073/pnas.96.5.2135}, number={5}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Miyawaki, Atsushi and Griesbeck, Oliver and Heim, Roger and Tsien, Roger Y.}, year={1999}, month=mar, pages={2135–2140} }

JSON

{
  "indexed": {
    "date-parts": [
      [
        2025,
        8,
        5
      ]
    ],
    "date-time": "2025-08-05T12:18:41Z",
    "timestamp": 1754396321281
  },
  "reference-count": 30,
  "publisher": "Proceedings of the National Academy of Sciences",
  "issue": "5",
  "content-domain": {
    "domain": [
      "www.pnas.org"
    ],
    "crossmark-restriction": true
  },
  "published-print": {
    "date-parts": [
      [
        1999,
        3,
        2
      ]
    ]
  },
  "abstract": "<jats:p>\n            Cameleons are genetically-encoded fluorescent indicators for Ca\n            <jats:sup>2+</jats:sup>\n            based on green fluorescent protein variants and calmodulin (CaM). Because cameleons can be targeted genetically and imaged by one- or two-photon excitation microscopy, they offer great promise for monitoring Ca\n            <jats:sup>2+</jats:sup>\n            in whole organisms, tissues, organelles, and submicroscopic environments in which measurements were previously impossible. However, the original cameleons suffered from significant pH interference, and their Ca\n            <jats:sup>2+</jats:sup>\n            -buffering and cross-reactivity with endogenous CaM signaling pathways was uncharacterized. We have now greatly reduced the pH-sensitivity of the cameleons by introducing mutations V68L and Q69K into the acceptor yellow green fluorescent protein. The resulting new cameleons permit Ca\n            <jats:sup>2+</jats:sup>\n            measurements despite significant cytosolic acidification. When Ca\n            <jats:sup>2+</jats:sup>\n            is elevated, the CaM and CaM-binding peptide fused together in a cameleon predominantly interact with each other rather than with free CaM and CaM-dependent enzymes. Therefore, if cameleons are overexpressed, the primary effect is likely to be the unavoidable increase in Ca\n            <jats:sup>2+</jats:sup>\n            buffering rather than specific perturbation of CaM-dependent signaling.\n          </jats:p>",
  "DOI": "10.1073/pnas.96.5.2135",
  "type": "journal-article",
  "created": {
    "date-parts": [
      [
        2002,
        7,
        26
      ]
    ],
    "date-time": "2002-07-26T14:39:15Z",
    "timestamp": 1027694355000
  },
  "page": "2135-2140",
  "update-policy": "http://dx.doi.org/10.1073/pnas.cm10313",
  "source": "Crossref",
  "is-referenced-by-count": 693,
  "title": "Dynamic and quantitative Ca\n            <sup>2+</sup>\n            measurements using improved cameleons",
  "prefix": "10.1073",
  "volume": "96",
  "author": [
    {
      "given": "Atsushi",
      "family": "Miyawaki",
      "sequence": "first",
      "affiliation": [
        {
          "name": "Department of Pharmacology and the Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093-0647"
        }
      ]
    },
    {
      "given": "Oliver",
      "family": "Griesbeck",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Department of Pharmacology and the Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093-0647"
        }
      ]
    },
    {
      "given": "Roger",
      "family": "Heim",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Department of Pharmacology and the Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093-0647"
        }
      ]
    },
    {
      "given": "Roger Y.",
      "family": "Tsien",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Department of Pharmacology and the Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093-0647"
        }
      ]
    }
  ],
  "member": "341",
  "published-online": {
    "date-parts": [
      [
        1999,
        3,
        2
      ]
    ]
  },
  "reference": [
    {
      "key": "e_1_3_3_1_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1146/annurev.ne.12.030189.001303"
    },
    {
      "key": "e_1_3_3_2_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1074/jbc.270.17.9896"
    },
    {
      "key": "e_1_3_3_3_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/42264"
    },
    {
      "key": "e_1_3_3_4_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/protein/7.1.109"
    },
    {
      "key": "e_1_3_3_5_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1126/science.1585175"
    },
    {
      "key": "e_1_3_3_6_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/jvi.70.7.4646-4654.1996"
    },
    {
      "key": "e_1_3_3_7_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0021-9258(20)80699-1"
    },
    {
      "key": "e_1_3_3_8_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.95.12.6803"
    },
    {
      "key": "e_1_3_3_9_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0960-9822(02)00450-5"
    },
    {
      "key": "e_1_3_3_10_2",
      "first-page": "109",
      "volume": "7",
      "author": "Graham F L",
      "year": "1991",
      "unstructured": "F L Graham, L Prevec Methods Mol Biol 7, 109\u2013128 (1991).",
      "journal-title": "Methods Mol Biol"
    },
    {
      "key": "e_1_3_3_11_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/j.1460-2075.1990.tb07564.x"
    },
    {
      "key": "e_1_3_3_12_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0003-2697(79)90115-5"
    },
    {
      "key": "e_1_3_3_13_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/bi00166a017"
    },
    {
      "key": "e_1_3_3_14_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1126/science.273.5280.1392"
    },
    {
      "key": "e_1_3_3_15_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1146/annurev.biochem.67.1.509"
    },
    {
      "key": "e_1_3_3_16_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0166-2236(92)90191-A"
    },
    {
      "key": "e_1_3_3_17_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1152/jn.1994.72.6.2563"
    },
    {
      "key": "e_1_3_3_18_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/41048"
    },
    {
      "key": "e_1_3_3_19_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1111/j.1751-1097.1982.tb02651.x"
    },
    {
      "key": "e_1_3_3_20_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0896-6273(93)90277-X"
    },
    {
      "key": "e_1_3_3_21_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0960-9822(02)00609-7"
    },
    {
      "key": "e_1_3_3_22_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/j.1460-2075.1987.tb02738.x"
    },
    {
      "key": "e_1_3_3_23_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1210/endo.133.1.8319584"
    },
    {
      "key": "e_1_3_3_24_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0021-9258(19)70576-6"
    },
    {
      "key": "e_1_3_3_25_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/oxfordjournals.jbchem.a134019"
    },
    {
      "key": "e_1_3_3_26_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1074/jbc.272.20.13270"
    },
    {
      "key": "e_1_3_3_27_2",
      "unstructured": "Kerr R. A. Baird G. Schafer W. R. & Tsien R. Y. (1998) Abstracts of the Fifth International Congress of Neuroethology S4."
    },
    {
      "key": "e_1_3_3_28_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0896-6273(00)81237-4"
    },
    {
      "key": "e_1_3_3_29_2",
      "unstructured": "Fan G. Y. Fujisaki H. Miyawaki A. Tsay R.-K. Tsien R. Y. & Ellisman M. H. (1999) Biophys. J. in press."
    },
    {
      "key": "e_1_3_3_30_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0076-6879(89)72017-6"
    }
  ],
  "container-title": "Proceedings of the National Academy of Sciences",
  "original-title": [],
  "language": "en",
  "link": [
    {
      "URL": "https://pnas.org/doi/pdf/10.1073/pnas.96.5.2135",
      "content-type": "unspecified",
      "content-version": "vor",
      "intended-application": "similarity-checking"
    }
  ],
  "deposited": {
    "date-parts": [
      [
        2022,
        4,
        13
      ]
    ],
    "date-time": "2022-04-13T21:45:44Z",
    "timestamp": 1649886344000
  },
  "score": 1,
  "resource": {
    "primary": {
      "URL": "https://pnas.org/doi/full/10.1073/pnas.96.5.2135"
    }
  },
  "subtitle": [],
  "short-title": [],
  "issued": {
    "date-parts": [
      [
        1999,
        3,
        2
      ]
    ]
  },
  "references-count": 30,
  "journal-issue": {
    "issue": "5",
    "published-print": {
      "date-parts": [
        [
          1999,
          3,
          2
        ]
      ]
    }
  },
  "alternative-id": [
    "10.1073/pnas.96.5.2135"
  ],
  "URL": "http://dx.doi.org/10.1073/pnas.96.5.2135",
  "relation": {},
  "ISSN": [
    "0027-8424",
    "1091-6490"
  ],
  "subject": [],
  "container-title-short": "Proc. Natl. Acad. Sci. U.S.A.",
  "published": {
    "date-parts": [
      [
        1999,
        3,
        2
      ]
    ]
  },
  "assertion": [
    {
      "value": "1998-12-29",
      "order": 1,
      "name": "accepted",
      "label": "Accepted",
      "group": {
        "name": "publication_history",
        "label": "Publication History"
      }
    },
    {
      "value": "1999-03-02",
      "order": 2,
      "name": "published",
      "label": "Published",
      "group": {
        "name": "publication_history",
        "label": "Publication History"
      }
    }
  ]
}