Abstract

Five structural features important for activation of the glycine recognition site on N-methyl-D-aspartate (NMDA) receptors were identified by structure-activity studies of more than 60 glycine analogues in voltage-clamped Xenopus oocytes injected with rat brain mRNA. First, sterically unhindered and ionized carboxyl and amino termini were essential for action at this site. Second, an increase in the interterminal separation by greater than one carbon (e.g., beta-alanine) markedly attenuated activity at this site. Third, activity at the glycine site was stereoselective. The D-isomers of alanine and serine were approximately 20 and 30 times more potent than the L-isomers. Fourth, only small sterically unobtrusive substitutions at the alpha-carbon could be tolerated. alpha-Methyl (D-alanine) and alpha-cyclopropyl (1-amino-cyclopropane carboxylic acid) (ACC) substitutions were effective as agonists but most larger aliphatic and aromatic alpha-carbon substitutions were simply inactive. Glycine, D-alanine, and ACC probably have only a two-point attachment to the receptor. Finally the alpha-carbon substituent of D-serine is envisioned as binding to a third site on the receptor probably via hydrogen bonding of the omega-terminal hydroxyl group. Thus, serine, an hydroxymethyl substitution of glycine, permitted activation of NMDA receptor-mediated currents, whereas isosteric substitutions incapable of hydrogen bonding (e.g., 2-aminobutyric acid) were inactive. Additionally, the position and size of the hydroxyl-containing group is critical for agonist action; D-threonine, DL-homoserine, and hydroxyphenolic substitutions at the alpha-carbon were all inactive. Halogenated analogs of a size comparable to D-serine but capable only of proton acceptance at the omega-terminus (beta-fluoro-D-alanine and beta-chloro-D-alanine) possessed agonist action, whereas an analog capable of only proton donation (1,2-diaminopropionic acid) was inactive. Full concentration-response curves were constructed for those analogs displaying greater than 25% of the effect of glycine when tested at 3 microM. With the exception of (R)-(+)cycloserine and beta-fluoro-D-alanine, all compounds were nearly full agonists and had Hill coefficients not significantly different from unity. The order of relative potency of the active analogs was ACC greater than glycine greater than D-serine greater than D-alanine greater than beta-fluoro-D-alanine greater than (R)-(+)-cycloserine greater than L-serine greater than L-alanine. Molecular modelling of a series of active and inactive analogs with close structural relation to glycine was undertaken. These results were complementary to those data obtained from the electrophysiological investigation.(ABSTRACT TRUNCATED AT 400 WORDS)