title: NMR-sensitive fluorinated and fluorescent intracellular calcium ion indicators with high dissociation constants.
abstract: A new series of high-dissociation constant (KD) Ca2+ indicators has been developed to reduce perturbations due to buffering of transients, to carry out measurements in cells and organelles with high basal Ca2+ concentrations, and to measure cytosolic Ca2+ levels in the presence of perturbations that may significantly increase these levels. A tetrafluorinated derivative of the chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, 1,2-bis(2-amino-5,6-difluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (TF-BAPTA), has a KD of 65 microM and exhibits two fluorine nuclear magnetic resonances, one of which is insensitive to Ca2+ chelation and the second of which shifts by approximately 10 ppm upon Ca2+ binding. TF-BAPTA has pK values of approximately 5.0 and Mg2+ dissociation constants > 50 mM. At a field of 8.5 T, the Ca(2+)-sensitive resonance is in fast-intermediate exchange. Correction factors for the effects of intermediate exchange and for the effect of protonation (pK approximately 5.0) and Mg2+ complexation are discussed. An analogous approach has been used to synthesize 2-[2-(5-carboxyoxazole)]-5-[2-(2-bis(carboxymethyl) amino-5,6-difluorophenoxy)]ethoxy-6-bis(carboxymethyl)aminobenz ofuran (fura F), a structural analogue of fura 2, which exhibits fluorescence characteristics similar to those of fura 2, but has a KD of 20 microM.
authors.affiliation: Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
authors.forename: R E
authors.initials: RE
authors.lastname: London
authors.forename: C K
authors.initials: CK
authors.lastname: Rhee
authors.forename: E
authors.initials: E
authors.lastname: Murphy
authors.forename: S
authors.initials: S
authors.lastname: Gabel
authors.forename: L A
authors.initials: LA
authors.lastname: Levy
mesh_terms: D002118:Calcium / Q000032:analysis* / Q000378:metabolism*; D002614:Chelating Agents / Q000138:chemical synthesis*; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D005456:Fluorescent Dyes / Q000138:chemical synthesis*; D016257:Fura-2 / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D015394:Molecular Structure; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
chemical_list: D002614:Chelating Agents; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; C087511:1,2-bis(2-amino-5,6-difluorophenoxy)ethane-N,N,N',N'-tetraacetic acid; C087512:fura F; D004533:Egtazic Acid; D002118:Calcium; D016257:Fura-2