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Intrasynaptosomal free Mg2+ concentration measured with the fluorescent indicator mag-fura-2: modulation by Na+
E García-Martín1, F J Martín-Romero, C Gutiérrez-Merino
1Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Extremadura, Badajoz, Spain.
Abstract:
Synaptosomes can be loaded with mag-fura-2 without significant perturbation of their ATP content by incubation for 10 min at 37 degrees C with 10 microM mag-fura-2 acetoxymethyl ester in Hanks'-HEPES buffer (pH 7.45). The intrasynaptosomal free Mg2+ concentration ([Mg2+]i) was found to be dependent on external Mg2+ concentration, increasing from 0.8 to 1.25 mM when the concentration of Mg2+ in the incubation medium increased from 1 to 8 mM. Dissipation of the Na+ gradient across the plasma membrane of synaptosomes by treatment with the Na+ ionophore monensin (0.2 mM) or with veratridine (0.2 mM) and ouabain (0.6 mM) produced a moderate increase of [Mg2+]i, from 1.0 to 1.2-1.3 mM in an incubation medium containing 5 mM Mg2+. Plasma membrane depolarization by incubation of synaptosomes in a medium containing 68 mM KCl and 68 mM NaCl had no effect on [Mg2+]i. Reversal of the Na+ gradient by incubation of synaptosomes in a medium in which external Na+ was replaced by choline increased [Mg2+]i up to 1.6 and 2.2 mM for extrasynaptosomal Mg2+ concentrations of 1 and 8 mM, respectively. We conclude that a Na+/Mg2+ exchange operates in the plasma membrane of synaptosomes. In the presence of Mg2+ in the incubation medium, extrasynaptosomal ATP, but not ADP or adenosine, increased [Mg2+]i from 1.1 +/- 0.1 up to 1.6 +/- 0.1 mM. The nonhydrolyzable ATP analogue adenosine 5'-(beta gamma-imido)triphosphate antagonized the effect of ATP, but had no effect by itself on [Mg2+]i. It is concluded that Mg2+ transport across the plasma membrane of synaptosomes is modulated by the activity of an ecto-ATPase or an ecto-protein kinase.
Insights
Synaptosomes regulate intracellular magnesium ([Mg2+]i) via a Na+/Mg2+ exchange in their plasma membrane. Extracellular ATP also influences Mg2+ levels, suggesting modulation by ecto-enzymes.
Area of Science:
- Neuroscience
- Cell Physiology
- Biochemistry
Background:
- Magnesium ions (Mg2+) are crucial intracellular cations involved in numerous cellular processes.
- Understanding the regulation of intrasynaptosomal free Mg2+ concentration ([Mg2+]i) is vital for synaptic function.
- Synaptosomes, isolated nerve terminals, provide a model system to study ion transport mechanisms.
Purpose of the Study:
- To investigate the mechanisms regulating intrasynaptosomal free Mg2+ concentration ([Mg2+]i) in synaptosomes.
- To identify potential transporters and modulators involved in Mg2+ homeostasis across the synaptosomal plasma membrane.
Main Methods:
- Synaptosomes were loaded with mag-fura-2, a fluorescent indicator for Mg2+.
- Intrasynaptosomal Mg2+ was measured under varying extracellular Mg2+ concentrations.
- Ion gradients were manipulated using ionophores (monensin), ion channel activators (veratridine), ouabain, and ionic substitutions (choline for Na+).
- The effect of extracellular ATP and its analogues on [Mg2+]i was assessed.
Main Results:
- Intrasynaptosomal Mg2+ concentration ([Mg2+]i) is dependent on extracellular Mg2+ levels.
- Dissipating the Na+ gradient moderately increased [Mg2+]i.
- Reversing the Na+ gradient significantly increased [Mg2+]i, indicating a Na+/Mg2+ exchange mechanism.
- Extracellular ATP, but not ADP or adenosine, increased [Mg2+]i, an effect antagonized by a non-hydrolyzable ATP analogue.
Conclusions:
- A Na+/Mg2+ exchange system operates in the plasma membrane of synaptosomes.
- Mg2+ transport across the synaptosomal plasma membrane is modulated by extracellular ATP, likely through ecto-ATPase or ecto-protein kinase activity.