NMDA-receptor antagonist requirements in conantokin-G.
A series of variants of the neuroactive 17-residue gamma-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 alpha-helical conformation. Employing the binding of [3H]dizolcipine (MK-801) as an assay for open receptor ion channels in rat brain membranes, which displays inhibition by con-G (IC50 = 0.48 microM), it was found that replacement by an Ala residue of Gla4 led to complete inactivation of the peptide, whereas a similar replacement of Gla3 resulted in a 20-fold decreased potency. Ala substitutions for Gla10 and Gla14 did not substantially affect [3H]MK-801 binding. This same substitution at Gla7 appeared to slightly enhance binding. Ala replacements of non-Gla residues demonstrated that four of them, viz. Glu2, Leu5, Gln9, and Ile12, possessed at least 200-fold decreases in inhibitory potency, whereas similar replacements at Gly1, Leu11, and Arg13 resulted in peptides with 8- to 12-fold increases in the IC50 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 [3H]MK-801 binding with the ability of these peptides to form cation-dependent alpha-helices. Peptides with notably low alpha-helical content in the presence of these cations were lacking at least one, or both, of Gla10 and Gla14. Con-G[Gla3,4,7,10,14E] and con-G[Gla7,10,14E] were the only peptides that remained in a completely random conformation upon metal ion addition.
Blandl
T
T
Department of Chemistry and Biochemistry, University of Notre Dame, IN 4655, USA.
Prorok
M
M
Castellino
F J
FJ
D019943:Amino Acid Substitution; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D020916:Conotoxins; D018691:Excitatory Amino Acid Antagonists / Q000378:metabolism; D007473:Ion Channels / Q000737:chemistry / Q000378:metabolism; D008978:Mollusk Venoms / Q000378:metabolism*; D010456:Peptides, Cyclic / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
D020916:Conotoxins; D018691:Excitatory Amino Acid Antagonists; D007473:Ion Channels; D008978:Mollusk Venoms; D010456:Peptides, Cyclic; D016194:Receptors, N-Methyl-D-Aspartate; C055407:conotoxin GV