Complex binding of L-[3H]glutamate to hippocampal synaptic membranes in the absence of sodium.

Specific binding of L-[3H]glutamate was investigated with a thoroughly washed synaptic membrane preparation from rat hippocampal formation, a region of brain densely innervated by putatively glutamatergic fibers. L-[3H]Glutamate bound rapidly, saturably, and reversibly to these membranes in the absence of Na+. Specific binding was greatest around 38 degrees C and at a slightly acidic pH. Saturation isotherms fit a model of two independent binding sites with dissociation constants of 11 and 570 nM and corresponding densities of 2.5 and 47 pmol/mg protein. All potent amino acid excitants, except N-methyl-D-aspartate and kainate, and several excitatory amino acid antagonists inhibited specific radioligand binding with IC50 values between 10(-7) M and 10(-4) M. In contrast, weak amino acid excitants and an inhibitor of glutamate uptake were nearly inactive. Displacement curves were analyzed with a computer program that assumed the simultaneous contributions of two independent sites at which each compound competitively inhibited the binding of L-[3H]glutamate. According to this analysis, ibotenate and the L- and D-isomers of glutamate and aspartate bind preferentially to the high-affinity site, whereas quisqualate, L-alpha-aminoadipate, and the L- and D-isomers of homocysteate bind preferentially to the low-affinity site. With the notable exception of gamma-D-glutamylglycine, all of the more potent antagonists appear to bind preferentially to the low-affinity site. Both sites exhibit marked stereoselectivity for L-glutamate. D- and L-Homocysteate and most excitatory amino acid antagonists increased specific binding at concentrations below those required to demonstrate inhibition. Some properties of the low-affinity binding site resemble those of junctional glutamate receptors on insect muscle, but neither site appears to correspond to the "N-methyl-D-aspartate receptor" or the "quisqualate receptor."