Ionic selectivity of low-affinity ratiometric calcium indicators: mag-Fura-2, Fura-2FF and BTC

Accurate measurement of elevated intracellular calcium levels requires indi cators with low calcium affinity and high selectivity. We examined fluoresc ence spectral properties and ionic specificity of three low-affinity, ratio metric indicators structurally related to Fura-2: mag-fura-2 (furaptra), Fu ra-2FF, and BTC. The indicators differed in respect to their excitation wav elengths, affinity for Ca2+ (K-d similar to 20 mu M, 6 mu M and 12 mu M res pectively) and selectivity over Mg2(+) (K-d similar to 2 mM for mag-fura-2, >10 mM for Fura-2FF and ETC). Among the tested indicators, BTC was limited by a modest dynamic range upon Ca2+ binding, susceptibility to photodamage , and sensitivity to alterations in pH. All three indicators bound other me tal ions including Zn2+, Cd2+ and Gd3+. Interestingly, only in the case of BTC were spectral differences apparent between Ca2+ and other metal ions. F or example, the presence of Zn2+ increased BTC fluorescence 6-fold at the C a2+ isosbestic point, suggesting that this dye may be used as a fluorescent Zn2+ indicator. Fura-2FF has high specificity, wide dynamic range, and low pH sensitivity, and is an optimal low-affinity Ca2+ indicator for most ima ging applications. BTC may be useful if experimental conditions require vis ible wavelength excitation or sensitivity to other metal ions including Zn2 +. (C) 2000 Harcourt Publishers Ltd.