Mag-indo1 affinity for Ca2+, compartmentalization and binding to proteins:The challenge of measuring Mg2+ concentrations in living cells

A physicochemical study of the MAg-indo1 binding to Ca2+ in solution showed that: (i) the characteristic fluorescence spectra of Ca2+-bound and Mg2+-b ound Mag-indo1 are identical; (ii) two successive equilibria occur for incr easing Ca2+ concentrations; and (iii) the value of the dissociation constan t of the first one, as determined by using a probe dilution protocol, amoun ts to 780 nM. In order to investigate the fluorescence level of Mag-indo1 t rapped in cell organelles, fluorescence spectra of Mag-indo1-loaded fibrobl asts were recorded before and after a digitonin permeabilization. Their res olution into cation-bound, protein-bound, and free Mag-indo1 characteristic spectra allowed measurement of the fluorescence intensities of these speci es. The intensities emitted from whole cells were compared to those emitted from organelles (assumed to be endoplasmic reticulum according to a DiOC(6 ) loading). The cation-bound Mag-indo1 fluorescence resulted partially (20 to 50%) from the cytosol for 30% of the cells, and totally from compartment s for 70% of the cells. We found a concentration value of 500 nM for compar tmentalized Ca2+ and concluded that the Mag-indo1 binding to Ca2+ is likely to affect drastically the Mg2+ concentration measurements in cells. Moreov er, we showed that the amount variation of protein-bound Mag-indo1 also aff ects Mg2+ measurements when using the two-wavelength ratio method. (C) 2001 Academic Press.