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

The kinetics of interaction of excitatory amino acid analogues and re puted antagonists with a specific binding site for [³H]l-glutamate, having a K_d of 600 nM, were examined in washed cerebellar membranes incubated at 37°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 simples, mass action kinetics with Hill coefficeents near unity and K_is of 1.1, 9.3 and 23.3 μM, respectively. Quisqualate, ibotenate and cyclopentylglutamate had Hill coefficients less than 0.85 and bound to an high affinity component with K_Hs of 0.4, 0.8 and 1.7 μM, respectively. Neither N-methyl-d-aspartate nor derivatives of kainate, with the exception of α-keto kainate, had K_iS 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 andK_HS varying from 0.5 to 6.6 μM. 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.