NMDA‐receptor antagonist requirements in conantokin‐G

A series of variants of the neuroactive 17‐residue γ‐carboxyglutamate‐(Gla)‐containing polypeptide, conantokin‐G (con‐G), were synthesized with the intention of determining those features that were important for its N ‐methyl‐ d ‐aspartate (NMDA) receptor‐targeted antagonist activity and for adoption of its divalent cation‐dependent α‐helical conformation. Employing the binding of [ 3 H]dizolcipine (MK‐801) as an assay for open receptor ion channels in rat brain membranes, which displays inhibition by con‐G (IC 50 =0.48 μM), it was found that replacement by an Ala residue of Gla 4 led to complete inactivation of the peptide, whereas a similar replacement of Gla 3 resulted in a 20‐fold decreased potency. Ala substitutions for Gla 10 and Gla 14 did not substantially affect [ 3 H]MK‐801 binding. This same substitution at Gla 7 appeared to slightly enhance binding. Ala replacements of non‐Gla residues demonstrated that four of them, viz. Glu 2 , Leu 5 , Gln 9 , and Ile 12 , possessed at least 200‐fold decreases in inhibitory potency, whereas similar replacements at Gly 1 , Leu 11 , and Arg 13 resulted in peptides with 8‐ to 12‐fold increases in the IC 50 values. The remaining amino acid residues tested in the single Ala replacement series showed no significant changes in the inhibitory characteristics of wild‐type con‐G. Additional studies with carboxyl‐terminal truncated peptides revealed that the carboxyl‐terminal 4 amino acids were unimportant for this activity. There was no strict correlation of inhibition of [ 3 H]MK‐801 binding with the ability of these peptides to form cation‐dependent α‐helices. Peptides with notably low α‐helical content in the presence of these cations were lacking at least one, or both, of Gla 10 and Gla 14 . Con‐G[Gla 3,4,7,10,14 E] and con‐G[Gla 7,10,14 E] were the only peptides that remained in a completely random conformation upon metal ion addition.