Specific binding of [3H]L-glutamate to cerebellar membranes: evidence for recognition site heterogeneity

The kinetics of interaction of excitatory amino acid analogues and reputed antagonists with a specific binding site for [3H]L-glutamate, having a Kd of 600 nM, were examined in washed cerebellar membranes incubated at 37 degrees C. Displacement curves were analyzed by an iterative computer program for a non-cooperative two-site competitive inhibition model. L-Glutamate, D-glutamate and D-aspartate exhibited simple, mass action kinetics with Hill coefficients near unity and Kis of 1.1, 9.3 and 23.3 microM, respectively. Quisqualate, ibotenate and cyclopentylglutamate had Hill coefficients less than 0.85 and bound to an high affinity component with KHS of 0.4, 0.8 and 1.7 microM, respectively. Neither N-methyl-D-aspartate nor derivatives of kainate, with the exception of a-keto kainate, had KiS less than 0.1 mM. Linear analogues of glutamate with reputed antagonistic properties all exhibited shallow displacement curves with Hill coefficients less than 0.6 and KHS varying from 0.5 to 6.6 microM. Notably, 2-amino-6-phosphonocaproic acid had negligible affinity for the site in contrast to the valeric and pimelic phosphono analogues. The results indicate that [3H]L-glutamate labels a single class of sites that can be resolved into subpopulations by agonists and antagonists and provide additional evidence of excitatory amino acid receptor heterogeneity.