Conantokins derived from the Asprella clade impart conRl-B, an N-methyl d-aspartate receptor antagonist with a unique selectivity profile for NR2B subunits

Abstract

Using molecular phylogeny has accelerated the discovery of peptidic ligands targeted to ion channels and receptors. One clade of venomous cone snails, Asprella, appears to be significantly enriched in conantokins, antagonists of N-methyl d-aspartate receptors (NMDARs). Here, we describe the characterization of two novel conantokins from Conus rolani, including conantokin conRl-B that has shown an unprecedented selectivity for blocking NMDARs that contain NR2B subunits. ConRl-B shares only some sequence similarity with the most studied NR2B selective conantokin, conG. The divergence between conRl-B and conG in the second inter-Gla loop was used to design analogues for structure-activity studies; the presence of Pro10 was found to be key to the high potency of conRl-B for NR2B, whereas the ε-amino group of Lys8 contributed to discrimination in blocking NR2B- and NR2A-containing NMDARs. In contrast to previous findings for Tyr5 substitutions in other conantokins, conRl-B[L5Y] showed potencies on the four NR2 NMDA receptor subtypes that were similar to those of the native conRl-B. When delivered into the brain, conRl-B was active in suppressing seizures in the model of epilepsy in mice, consistent with NR2B-containing NMDA receptors being potential targets for antiepileptic drugs. Circular dichroism experiments confirmed that the helical conformation of conRl-B is stabilized by divalent metal ions. Given the clinical applications of NMDA antagonists, conRl-B provides a potentially important pharmacological tool for understanding the differential roles of NMDA receptor subtypes in the nervous system. This work shows the effectiveness of coupling molecular phylogeny, chemical synthesis, and pharmacology for discovering new bioactive natural products.

Figure 1

Predicted amino acid sequences of ConRl-B and conRl-C Predicted translated sequences from genomic DNA are shown for the pre/propeptide (upper panel, A) and mature toxin regions (lower panel, C) of conRl-B and ConRl-C, aligned to the sequences of conRl-A and conG for comparison. Shading indicates residues conserved among the four sequences. Two potential mature sequences predicted for conRl-B (C). The proline that may undergo post-translational modification to hydroxyproline is highlighted in bold. O denotes hydroxyproline; γ denotes gamma-carboxyglutamate, and # denotes C-terminal amidation.

Figure 2

NMDA receptor subtype selectivity of conRl-B. (A) Current traces from Xenopus oocytes expressing heterologous NR1-2b/NR2B and NR1-2b/NR2D, respectively. ConRl-B blocks most of the agonist-elicited current in oocytes expressing NR1-2b/NR2B (left) but only weakly blocks NR1-2b/NR2D (right). (B) Concentration response curves for conRl-B tested against the four NR2 NMDA receptor subtypes. Data points represent normalized peak current ± SEM from a minimum of 3 oocytes. (C) Normalized current responses of NR1-2a/NR2 and NR1-4b/NR3 subunit combinations, in response to 10 µM conRl-B.

Figure 3

Sequences of native ConRl-B and its analogs. Shaded boxed region indicates region of peptide that primary sequence analysis suggests is important for the selectivity profile of conRl-B. ^a γ denotes gamma-carboxyglutamic acid; ^b # denotes C-terminal amidation; ^c O denotes 4-trans-hydroxyproline; X denotes L-norleucine.

Figure 4

Concentration response curves of ConRl-B analogs on NR2B/NR1-2b, compared to native ConRl-B. Potency is decreased by O10A and O10-, but not by K8Nle. Sequences of ConRl-B and variants are shown in Figure 3. Each data point represents the average peak current, normalized to baseline from a minimum of three oocytes. Error bars represent SEM.

Figure 5

Circular dichroism spectra of ConRl-B. Spectra were recorded with (or) without 2mM CaCl₂ containing 10 mM HEPES buffer at pH 7.0 and shown is an average spectra obtained from five independent scans (n=5). The dual minima at 208 and 222nm, in the presence of calcium, suggest that ConRl-B adopts helical conformation. Estimated percentage of helicity of peptide in the absence of calcium is 10% and in the presence of calcium is 59%.