The migration of divalent cations in mitochondria visualized by a fluorescent chelate probe

The use of the fluorescent chelate probe, chlorotetracycline, in mitochondria is described. The probe shows a high fluorescence in the presence of mitochondria which may be ascribed to binding of the probe to membrane-associated Ca(++) and Mg(++). The fluorescence excitation and emission spectra are diagnostic of binding of the probe to Ca(++) in coupled mitochondria and Mg(++) in uncoupled mitochondria. The fluorescence polarization spectra are diagnostic of the cations having a moderately high mobility in the membrane environment. The effects of exogenous EDTA and of endogenous Mn(++) indicate that the probe is primarily visualizing actively accumulated Ca(++) on the inner surface of the inner membrane. By employing the Ca(++) transport inhibitor, Tb(+++), the fluorescence changes associated with metabolic alterations are shown to arise partly from cation transport and partly through alterations in the binding properties of the inner surface of the membrane. Chlorotetracycline is a probe for divalent cations associated with the membrane and is of general utility in the study of cation migrations in cellular and subcellular systems.