NMR-SENSITIVE FLUORINATED AND FLUORESCENT INTRACELLULAR CALCIUM-ION INDICATORS WITH HIGH DISSOCIATION-CONSTANTS

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 Ca3+ 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 K-D of 65 mu M and exhibits two fluorine nuclear magnetic resonances, one of which is insensitive to Ca2+ chelation and the second of which shifts by similar to 10 ppm upon Ca2+ binding. TF-BAPTA has pK values of similar to 5.0 and Mg2+ dissociation constants >50 mM. At a field of 8.5 T, the Ca2+-sensitive resonance is in fast-intermediate exchange. Correction factors for the effects of intermediate exchange and for the effect of protonation (pK similar to 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)aminobenzofuran (fura Fl, a structural analogue of fura 2, which exhibits fluorescence characteristics similar to those of fura 2, but has a K(D)of 20 mu M.