Intrasynaptosomal Free Mg 2+ Concentration Measured with the Fluorescent Indicator Mag‐Fura‐2: Modulation by Na + Gradient and by Extrasynaptosomal ATP

Synaptosomes can be loaded with mag‐fura‐2 without significant perturbation of their ATP content by incubation for 10 min at 37°C with 10 µ M mag‐fura‐2 acetoxymethyl ester in Hanks'‐HEPES buffer (pH 7.45). The intrasynaptosomal free Mg 2+ concentration ([Mg 2+ ] i ) was found to be dependent on external Mg 2+ concentration, increasing from 0.8 to 1.25 m M when the concentration of Mg 2+ in the incubation medium increased from 1 to 8 m M . Dissipation of the Na + gradient across the plasma membrane of synaptosomes by treatment with the Na + ionophore monensin (0.2 m M ) or with veratridine (0.2 m M ) and ouabain (0.6 m M ) produced a moderate increase of [Mg 2+ ] i , from 1.0 to 1.2–1.3 m M in an incubation medium containing 5 m M Mg 2+ . Plasma membrane depolarization by incubation of synaptosomes in a medium containing 68 m M KCl and 68 m M NaCl had no effect on [Mg 2+ ] i . Reversal of the Na + gradient by incubation of synaptosomes in a medium in which external Na + was replaced by choline increased [Mg 2+ ] i up to 1.6 and 2.2 m M for extrasynaptosomal Mg 2+ concentrations of 1 and 8 m M , respectively. We conclude that a Na + /Mg 2+ exchange operates in the plasma membrane of synaptosomes. In the presence of Mg 2+ in the incubation medium, extrasynaptosomal ATP, but not ADP or adenosine, increased [Mg 2+ ] i from 1.1 ± 0.1 up to 1.6 ± 0.1 m M . The nonhydrolyzable ATP analogue adenosine 5′‐(βγ‐imido)triphosphate antagonized the effect of ATP, but had no effect by itself on [Mg 2+ ] i . It is concluded that Mg 2+ transport across the plasma membrane of synaptosomes is modulated by the activity of an ecto‐ATPase or an ecto‐protein kinase.