L‐[3H]Glutamate Binding to Hippocampal Synaptic Membranes: Two Binding Sites Discriminated by Their Differing Affinities for Quisqualate

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Abstract
The excitatory glutamate analogs quisqualate and ibotenate were employed to distinguish multiple binding sites for L-[3H]glutamate on freshly prepared rat hippocampal synaptic membranes. The fraction of bound radioligand and that was displaceable by 5 .mu.M quisqualate was termed GLU A binding. That which persisted in the presence of 5 .mu.M quisqualate, but was displaceable by 100 .mu.M ibotenate, was termed GLU B binding. GLU A binding equilibrated within 5 min and remained unchanged for up to 80 min. GLU B binding appeared to equilibrate at least as rapidly, but incubation with ligand unmasked latent binding sites. Saturation binding curves were best fitted by single exponentials, which yielded Kd values .apprx. 200 nM (GLU A) and 1 .mu.M (GLU B). On the average, GLU B binding sites twice as abundant in these membranes as were GLU A sites. Rapid freezing of the membranes, followed by storage at -26.degree. C and rapid thawing markedly diminished GLU A binding, but nearly tripled GLU B binding. Both sites bound L-glutamate with 10-30 times the affinity of D-glutamate. The GLU A site also bound L-glutamate with .apprx. 10 times the affinity of L-aspartate and discriminated poorly between L- and D-aspartate. The GLU B site bound L-aspartate with an affinity similar to than for L-glutamate, and with an order-of-magnitude greater affinity than D-aspartate. The structural specificities of the GLU A and GLU B binding sites, suggest that these sites may correspond to receptors on hippocampal pyramidal cell dendrites that are activated by iontophoretically applied L-glutamate.