Evidence for heterogenous glycine domains but conserved multiple states of the excitatory amino acid recognition site of the NMDA receptor: regional binding studies with [3H]glycine and [3H]L-glutamate.
The possible heterogeneity of the agonist and glycine sites of the N-methyl-D-aspartate (NMDA) receptor-complex was examined using receptor binding techniques. Binding of [3H]L-glutamate [( 3H]GLU) and [3H]glycine to synaptic membranes of cerebral and cerebellar cortices, and membranes of a granule cell preparation of rat cerebellum, was characterized. [3H]Glycine always labelled a single population of sites; densities of binding sites (Bmax) in cortical, cerebellar and "granule" membranes were 3.1, 0.87 and 3.6 pmol/mg protein, respectively. Dissociation constants (Kd) in the same three preparations were 0.13, 0.31 and 1.9 microM, respectively. In competition studies, D-cycloserine, but not D-serine and 7-chlorokynurenate, showed varying potency between the membrane preparations, and analysis of variance (ANOVA) revealed a significant interaction between ligands and membrane fractions. Binding of [3H]GLU was saturable and to a single population of sites: Kd 0.5-0.9 microM and Bmax 3.2-3.6 pmol/mg protein. In all three membrane preparations the rank order of potency of NMDA agonists as inhibitors of the binding of [3H]GLU was always L-aspartate greater than L-cysteate greater than L-cysteinesulphinate greater than L-serine-O-sulphate greater than ibotenate greater than L-homocysteate. NMDA, quinolinate and competitive NMDA antagonists were only weak inhibitors of the binding of [3H]GLU and never fully inhibited specific binding. Other subtype-selective excitatory amino acids were very weak or ineffective inhibitors of binding. Binding of NMDA agonists was better described by a two site model whereby the proportion of high affinity sites did not vary significantly across the three membrane preparations.(ABSTRACT TRUNCATED AT 250 WORDS)
University of Melbourne, Clinical Pharmacology and Therapeutics, Austin Hospital, Heidelberg, Victoria, Australia.
R D
RD
O'Shea
D T
DT
Manallack
E L
EL
Conway
L D
LD
Mercer
P M
PM
Beart
D000818:Animals; D001345:Autoradiography; D001667:Binding, Competitive / Q000187:drug effects; D002525:Cerebellar Cortex / Q000033:anatomy & histology / Q000378:metabolism; D002540:Cerebral Cortex / Q000033:anatomy & histology / Q000378:metabolism; D003523:Cycloserine / Q000494:pharmacology; D005971:Glutamates / Q000378:metabolism* / Q000493:pharmacokinetics; D018698:Glutamic Acid; D005998:Glycine / Q000378:metabolism* / Q000493:pharmacokinetics; D066298:In Vitro Techniques; D007736:Kynurenic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D008024:Ligands; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D017459:Receptors, Amino Acid; D011956:Receptors, Cell Surface / Q000378:metabolism*; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012694:Serine / Q000378:metabolism
D005971:Glutamates; D008024:Ligands; D017459:Receptors, Amino Acid; D011956:Receptors, Cell Surface; D016194:Receptors, N-Methyl-D-Aspartate; D018698:Glutamic Acid; D012694:Serine; D003523:Cycloserine; D007736:Kynurenic Acid; C057013:7-chlorokynurenic acid; D005998:Glycine