Abstract

Conantokins are ~20-amino acid peptides present in predatory marine snail venoms that function as allosteric antagonists of ion channels of the N-methyl-d-aspartate receptor (NMDAR). These peptides possess a high percentage of post-/co-translationally modified amino acids, particularly γ-carboxyglutamate (Gla). Appropriately spaced Gla residues allow binding of functional divalent cations, which induces end-to-end α-helices in many conantokins. A smaller number of these peptides additionally contain 4-hydroxyproline (Hyp). Hyp should prevent adoption of the metal ion-induced full α-helix, with unknown functional consequences. To address this disparity, as well as the role of Hyp in conantokins, we have solved the high resolution three-dimensional solution structure of a Gla/Hyp-containing 18-residue conantokin, conRl-B, by high field NMR spectroscopy. We show that Hyp(10) disrupts only a small region of the α-helix of the Mn(2+)·peptide complex, which displays cation-induced α-helices on each terminus of the peptide. The function of conRl-B was examined by measuring its inhibition of NMDA/Gly-mediated current through NMDAR ion channels in mouse cortical neurons. The conRl-B displays high inhibitory selectivity for subclasses of NMDARs that contain the functionally important GluN2B subunit. Replacement of Hyp(10) with N(8)Q results in a Mg(2+)-complexed end-to-end α-helix, accompanied by attenuation of NMDAR inhibitory activity. However, replacement of Hyp(10) with Pro(10) allowed the resulting peptide to retain its inhibitory property but diminished its GluN2B specificity. Thus, these modified amino acids, in specific peptide backbones, play critical roles in their subunit-selective inhibition of NMDAR ion channels, a finding that can be employed to design NMDAR antagonists that function at ion channels of distinct NMDAR subclasses.

Highlights

  • N-methyl-D-aspartate receptor (NMDAR) subunit plasticity governs its function in health and disease

  • Cation-induced ␣-Helicity Observed by Circular Dichroism (CD)—CD experiments were designed to reveal the maximal helicity adopted by various conantokins in the presence of saturating Mg2ϩ

  • In the conantokin family of peptides, the presence of generated ␥-carboxyglutamate (Gla) side chains at the i,i ϩ 3, i ϩ 7, and i ϩ 11 positions is conducive to their binding to divalent ions, and the ability of cations to span Gla 3– 4 residues apart is central to the induction of cation-dependent ␣-helical states [58, 59]

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Summary

Introduction

NMDAR subunit plasticity governs its function in health and disease. Results: Substitutions of key amino acids in conantokins change their selectivity for NMDAR subunits. Conantokins are ϳ20-amino acid peptides present in predatory marine snail venoms that function as allosteric antagonists of ion channels of the N-methyl-D-aspartate receptor (NMDAR). These peptides possess a high percentage of post-/ co-translationally modified amino acids, ␥-carboxyglutamate (Gla). Replacement of Hyp with Pro allowed the resulting peptide to retain its inhibitory property but diminished its GluN2B specificity These modified amino acids, in specific peptide backbones, play critical roles in their subunit-selective inhibition of NMDAR ion channels, a finding that can be employed to design NMDAR antagonists that function at ion channels of distinct NMDAR subclasses

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