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+-bound Mag-indo1 are identical; (ii) two successive equilibria occur for increasing Ca2+ concentrations; and (iii) the value of the dissociation constant of the first one, as determined by using a probe dilution protocol, amounts to 780 nM. In order to investigate the fluorescence level of Mag-indo1 trapped in cell organelles, fluorescence spectra of Mag-indo1-loaded fibroblasts were recorded before and after a digitonin permeabilization. Their resolution into cation-bound, protein-bound, and free Mag-indo1 characteristic spectra allowed measurement of the fluorescence intensities of these species. 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 compartments for 70% of the cells. We found a concentration value of 500 nM for compartmentalized Ca2+ and concluded that the Mag-indo1 binding to Ca2+ is likely to affect drastically the Mg2+ concentration measurements in cells. Moreover, we showed that the amount variation of protein-bound Mag-indo1 also affects Mg2+ measurements when using the two-wavelength ratio method. (C) 2001 Academic Press.