Properties of Quisqualate-Sensitive L-[ 3 H]Glutamate Binding Sites in Rat Brain as Determined by Quantitative Autoradiography

Abstract

Quisqualate, a glutamate analogue, displaced L-[ 3 H]glutamate binding in a biphasic manner, corresponding to “high-affinity” and “low-affinity” binding sites. High-affinity quisqualate sites were termed “quisqualate-sensitive L-[ 3 H]glutamate” binding sites. Quisqualate-sen-sitive L-[ 3 H]glutamate binding was regionally distributed, with the highest levels present in the cerebellar molecular layer. This binding was stimulated by millimolar concentrations of chloride and calcium. The stimulatory effects of calcium required the presence of chloride ions, whereas chloride's stimulatory effects did not require calcium. All of the L-[ 3 H]glutamate binding stimulated by chloride/calcium was quisqualate sensitive and only weakly displaced by N -methyl-D-aspartate, L-aspartate, or kainate. At high concentrations (1 m M ), the anion blockers 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid and 4 ,4′-diisothio-cyanatostilbene-2,2′-disulfonic acid both reduced, by 41 and 43%, respectively, the stimulatory effects of chloride. At concentrations of 100 Μ M , kynurenate, L-aspartate, ( RS )-Α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and L-2-amino-4-phosphonobutyric acid (L-APB) failed to displace quisqualate-sensitive L-[ 3 H]-glutamate binding in the cerebellar molecular layer. In the presence of KSCN, however, 100 Μ M AMPA displaced 44% of binding. Quisqualate-sensitive L-[ 3 H]glutamate binding was not sensitive to freezing, and, in contrast to other chloride- and calcium-dependent L-[ 3 H]glutamate binding sites that have been reported, quisqualate-sensitive binding observed by autoradiography was enhanced at 4°C compared with 37°C. Quisqualate-sensitive L-[ 3 H]glutamate binding likely represents binding to the subclass of postsynaptic neuronal glutamate receptors known as quisqualate receptors, rather than binding to previously described APB receptors, chloride-driven sequestration into vesicles, or binding to astrocytic membrane binding sites.