Characterization of Two [3H]Glutamate Binding Sites in Rat Hippocampal Membranes

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Abstract
The specific binding of l‐[3H]glutamate was investigated in the presence and the absence of sodium ions in freshly prepared membranes from rat hippocampus. Sodium ions were found to have a biphasic effect; low concentrations induced a marked inhibition of the binding (in the range 0.5‐5.0 mM), whereas higher concentrations resulted in a dose‐dependent stimulation of binding (in the range 10‐ 150 mM). These results permit the discrimination of two binding sites in hippocampal membranes. Both Na+‐independent and Na+‐dependent binding sites were saturable, exhibiting dissociation constants at 30°C of 750 nM and 2.4 μM, respectively, with Hill coefficients not significantly different from unity, and maximal number of sites of 6.5 and 75 pmol/mg protein, respectively. [3H]Glutamate binding to both sites reached equilibrium between 5 and 10 min and was reversible. The relative potencies of a wide range of compounds, with known pharmacological activities, to inhibit [3H]glutamate binding were very different for the Na+‐independent and Na+‐ dependent binding and suggested that the former sites were related to postsynaptic glutamate receptors, whereas the latter were related to high‐affinity uptake sites. This conclusion was also supported by the considerable variation in the regional distribution of the Na+‐dependent binding site, which paralleled that of the high‐affinity glutamate uptake; the Na+‐independent binding exhibited less regional variation.