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) antagonist properties, were synthesized and evaluated for their ability to displace [(3)H]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 identified as the shortest variant capable of manifesting inhibitory activity (< 1% of the parent peptide). Ala substitution studies of individual residues identified Gly1, Gla3, Met8 and Leu12 as important for activity, while Glu2, 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. Further elaboration of structure-function relationships in con-T was effected through the design and synthesis of helically constrained and destabilized analogs. The results of the current study were compared with those of a previous investigation on con-G, a related conantokin. Substantial differences in activity requirements were noted between the peptides, particularly in the C-terminal regions. Chimeras of con-T and con-G were generated and revealed virtually no interchangeability of residues between these two peptides. Finally, single amino acid substitutions that resulted in analogs with enhanced inhibitory properties were combined to yield superior conantokin-based NMDAR inhibitors.