Amino acid determinants for NMDA receptor inhibition by conantokin-T

Several derivatives of conantokin-T (con-T), a naturally occurring, gamma - carboxyglutamate (Gla)-containing peptide with NMDA receptor (NMDAR) antago nist properties, were synthesized and evaluated for their ability to displa ce [H-3]MK-801 from adult rat forebrain membranes. Analyses of progressive C-terminal truncation analogs of the parent 21-mer revealed gradual losses in activity with decreased chain length. In this series, con-T[1-8] was ide ntified as the shortest variant capable of manifesting inhibitory activity (< 1% of the parent peptide). Ala substitution studies of individual residu es identified Gly1, Gla3, Met8 and Leu12 as important for activity, while G lu2, Gla4 and Tyr5 were shown to be essential in this regard. The effect of side-chain length and charge in the N-terminal region was probed by single amino acid replacements. No correlation was observed between potencies and circular dichroism-derived helical contents of the con-T derivatives. Furt her elaboration of structure-function relationships in con-T was effected t hrough the design and synthesis of helically constrained and destabilized a nalogs. The results of the current study were compared with those of a prev ious investigation on con-G, a related conantokin. Substantial differences in activity requirements were noted between the peptides, particularly in t he C-terminal regions. Chimeras of con-T and con-G were generated and revea led virtually no interchangeability of residues between these two peptides. Finally, single amino acid substitutions that resulted in analogs with enh anced inhibitory properties were combined to yield superior conantokin-base d NMDAR inhibitors.