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

Abstract

The specific binding of L-[3H]glutamate was investigated in the presence and the absence of Na+ in freshly prepared membranes from rat hippocampus. Na+ had 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 2 binding sites in hippocampal membranes. Both Na+-independent and Na+-dependent binding sites were saturable: they exhibit dissociation constants at 30.degree. C of 750 nM and 2.4 .mu.M, respectively, with Hill coefficients not significantly different from unity, and maximal number of sites at 6.5 and 75 pmol/mg protein, respectively. [3H]Glutamate binding to both sites reached equilibrium between 5-10 min and was reversible. The relative potencies of a wide range of compounds with known pharmacological activities were very different in inhibiting [3H]glutamate binding for the Na+-independent and Na+-dependent binding; this 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.