A series of variants of the neuroactive 17-residue Q Q-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 K K-helical conformation. Employing the binding of [ Q H]dizolcipine (MK-801) as an assay for open receptor ion channels in rat brain membranes, which displays inhibition by con-G (IC SH = 0.48 W WM), it was found that replacement by an Ala residue of Gla R led to complete inactivation of the peptide, whereas a similar replacement of Gla Q resulted in a 20-fold decreased potency. Ala substitutions for Gla IH and Gla IR did not substantially affect [ Q H]MK-801 binding. This same substitution at Gla U appeared to slightly enhance binding. Ala replacements of non-Gla residues demonstrated that four of them, viz. Glu P , Leu S , Gln W , and Ile IP , possessed at least 200-fold decreases in inhibitory potency, whereas similar replacements at Gly I , Leu II , and Arg IQ resulted in peptides with 8-to 12-fold increases in the IC SH 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 [ Q H]MK-801 binding with the ability of these peptides to form cationdependent K K-helices. Peptides with notably low K K-helical content in the presence of these cations were lacking at least one, or both, of Gla IH and Gla IR . Con-G[Gla QYRYUYIHYIR E] and con-G[Gla UYIHYIR E] were the only peptides that remained in a completely random conformation upon metal ion addition.z 1998 Federation of European Biochemical Societies.