Specific binding of [3H]+/- 2-amino-7-phosphono heptanoic acid to rat brain membranes in vitro.

The specific binding of [3H]+/- 2-amino-7-phosphono heptanoic acid (3H-APH), a potent N-methyl-D-aspartate (NMDA) antagonist, to extensively washed, previously frozen crude mitochondrial fractions of rat brain is described. Binding was optimal at physiological pH and temperature and, in Triscitrate buffer, attained equilibrium within 60 minutes. Scatchard analysis of the equilibrium data for forebrain revealed a single, non-interacting population of binding sites (BMapp = 15 picomoles/mg protein; KDapp = 3.6 uM; Hill coefficient = 0.92, r = 0.99; N = 5). Specific binding of the ligand was readily reversible by unlabeled APH and was absent in peripheral tissues including heart, lung, kidney, liver, spleen and striate muscle and in heat treated brain sonicates. An 8-fold variation in the amount of ligand bound to brain membranes prepared from different regions was observed with binding being greatest in the hippocampal formation and least in the midbrain. Kainic acid, NMDA and aspartic acid exhibited negligible affinity for the [3H]-APH site; in contrast, quisqualic acid, ibotenic acid, glutamatic acid, homocysteic acid and 2-amino-4-phosphono butyric acid were moderately potent displacers. The results indicate that [3H]-APH labels a quisqualate preferring site in vitro. Unlike the receptor labeled by [3H]-glutamate however, [3H]-APH binding was attenuated in the presence of chloride ions suggesting that this ligand may label a subpopulation of excitatory amino acid receptors.