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

A new series of high-dissociation constant (KD) Ca2+ indicators has be en developed to reduce perturbations due to buffering of transients, t o carry out measurements in cells and organelles with high basal Ca3concentrations, and to measure cytosolic Ca2+ levels in the presence o f perturbations that may significantly increase these levels. A tetraf luorinated derivative of the chelator ,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 exhi bits two fluorine nuclear magnetic resonances, one of which is insensi tive 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 M g2+ dissociation constants >50 mM. At a field of 8.5 T, the Ca2+-sensi tive resonance is in fast-intermediate exchange. Correction factors fo r the effects of intermediate exchange and for the effect of protonati on (pK similar to 5.0) and Mg2+ complexation are discussed. An analogo us approach has been used to synthesize -[2-(5-carboxyoxazole)]-5-[2-( 2-bis(carboxymethyl) henoxy)]ethoxy-6-bis(carboxymethyl)aminobenzofura n (fura Fl, a structural analogue of fura 2, which exhibits fluorescen ce characteristics similar to those of fura 2, but has a K(D)of 20 mu M.