Conantokin-Br from Conus brettinghami and selectivity determinants for the NR2D subunit of the NMDA receptor

Abstract

Conantokins are venom peptides from marine cone snails that are NMDA receptor antagonists. Here, we report the characterization of a 24 AA conantokin from Conus brettinghami Coomans , H. E. , Moolenbeek , R. G. and Wils , E. ( 1982 ) Basteria 46 ( 1/4 ), 3 - 67 , conantokin-Br (con-Br), the first conantokin that does not have the conserved glutamate residue at position 2. Molecular modeling studies suggest that con-Br has a helical structure between residues 2-13. In contrast to other characterized conantokins, con-Br has a high potency for NMDA receptors with NR2D subunits. To identify determinants for NR2D potency, we synthesized chimeras of con-Br and conantokin-R (con-R); the latter has a approximately 30-fold lower potency for the NR2D subtype. The characterization of two reciprocal chimeras (con-Br/R and con-R/Br), comprising the first 9-10 N-terminal AAs of each conantokin followed by the corresponding C-terminal AAs of the other conantokin demonstrates that determinants for NR2D selectivity are at the N-terminal region. Additional analogues comprising 1-3 amino acid substitutions from each peptide into the homologous region of the other led to the identification of a key determinant; a Tyr residue in position 5 increases potency for NR2D, while Val at this locus causes a decrease. The systematic definition of key determinants in the conantokin peptides for NMDA receptor subtype selectivity is an essential component in the development of conantokin peptides that are highly selective for each specific NMDA receptor subtype.

Figure 1

Forms of Conus sulcatus and Conus brettinghami, the species that produces conantokin-Br. The shells on the extreme left and right are Conus sulcatus; the two middle shells are generally called Conus sulcatus brettinghami; two varieties are shown. As is discussed in the text, we regard Conus brettinghami as a distinct species, not as a subspecies or form of C. sulcatus. The shell on the extreme left is a variant of the typical form. Venom ducts of several specimens of Conus sulcatus brettinghami from Manila Bay were pooled — this was the source of the cDNA clone that yielded the conantokin sequence. The specimens resembled the shell that is second from left.

Figure 2

Con-Br peptide precursor and predicted mature toxin sequences, compared to previously characterized conantokin precursors. In the mature toxin region, Glu residues either known or predicted to be post-translationally modified to Gla are indicated by γ.

Figure 3

NMDA receptor current traces in the presence and absence of con-Br. (A) Left: NR2B current elicited by agonist pulse before and after buffer control. Right: Baseline NR2B response followed by current elicited following a 10 min application of 1µM con-Br. (B) Effects of buffer control (left) and 1µM con-Br (right) on NR2D.

Figure 4

NR2B and NR2D discrimination of con-Br, con-R, and chimeras. (A) Concentration-response curves for con-Br on NR1-3b/NR2B and NR1-3b/NR2D, showing weak discrimination between the two subtypes. (B) Con-R concentration-response curves for NR2B and NR2D, showing high selectivity toward NR2B. (C) Br/R concentration-response curves for NR2B and NR2D. (D) R/Br concentration-response curves for NR2B and NR2D. R/Br and Br/R sequences are shown in Table 3.

Figure 5

Relative inhibitory potencies of con-Br and con-R variants on NR1-3b/NR2B and NR1-3b/NR2D. Con-R [Br 7–9] and con-Br [R 7–10] have greatly reduced potency on NR2B and NR2D (A–B). Con-R [Br 5–6] and con-R [V5Y] retain their potency on NR2B and discriminate less strongly against NR2D (C, E). Con-Br [R 5–6] and con-Br [Y5V] have reduced potency on both subtypes and discriminate strongly against NR2D (D, F). Sequences of con-Br and con-R variants are shown in Table 3.

Figure 6

Molecular modeling of con-Br and con-R/Br. Ribbon representation of con-Br (A) and con-R/Br chimera (B). Superimposion of five averaged structures generated with frequency of 250 ps from 1.25 ns MD simulation run. C - RMS deviation for conantokins con-Br and the con-R/Br chimera calculated during the MD simulation for each residue (all atoms were included). RMS deviation was also calculated for chosen helical region, which included residues 2–13 for both peptides.