Dynamic and quantitative Ca2+ measurements using improved cameleons.
Cameleons are genetically-encoded fluorescent indicators for Ca2+ 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 Ca2+ 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 Ca2+-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 Ca2+ measurements despite significant cytosolic acidification. When Ca2+ 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 Ca2+ buffering rather than specific perturbation of CaM-dependent signaling.
Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093-0647, USA.
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Miyawaki
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Griesbeck
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Heim
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Tsien
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D000256:Adenoviridae; D019943:Amino Acid Substitution; D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D002147:Calmodulin / Q000032:analysis / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006624:Hippocampus / Q000378:metabolism; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence / Q000379:methods; D014162:Transfection
D002147:Calmodulin; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D006632:Histamine; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D002118:Calcium