Antagonist Properties of Conus parius Peptides on N-Methyl-D-Aspartate Receptors and Their Effects on CREB Signaling

Shailaja Kunda,John Cheriyan,Francis J Castellino,Rashna D Balsara,Michael Hur

doi:10.1371/journal.pone.0081405

Abstract

Three members of a family of small neurotoxic peptides from the venom of Conus parius, conantokins (Con) Pr1, Pr2, and Pr3, function as antagonists of N-methyl-D-aspartate receptors (NMDAR). We report structural characterizations of these synthetic peptides, and also demonstrate their antagonistic properties toward ion flow through NMDAR ion channels in primary neurons. ConPr1 and ConPr2 displayed moderate increases in α-helicity after addition of Mg2+. Native apo-ConPr3 possessed an α-helical conformation, and the helicity increased only slightly on addition of Mg2+. Additionally, these peptides diminished NMDA/Gly-mediated currents and intracellular Ca2+ (iCa2+) influx in mature rat primary hippocampal neurons. Electrophysiological data showed that these peptides displayed slower antagonistic properties toward the NMDAR than conantokins from other species of cone snails, e.g., ConT and ConG. Furthermore, to demonstrate selectivity of the C. parius-derived conantokins towards specific NMDAR subunits, cortical neurons from GluN2A-/- and GluN2B-/- mice were utilized. Robust inhibition of NMDAR-mediated stimulation in GluN2A-/--derived mouse neurons, as compared to those isolated from GluN2B-/--mouse brains, was observed, suggesting a greater selectivity of these antagonists towards the GluN2B subunit. These C. parius conantokins mildly inhibited NMDAR-induced phosphorylation of CREB at Ser133, suggesting that the peptides modulated iCa2+ entry and, thereby, activation of CREB, a transcription factor that is required for maintaining long-term synaptic activity. Our data mechanistically show that while these peptides effectively antagonize NMDAR-directed current and iCa2+ influx, receptor-coupled CREB signaling is maintained. The consequence of sustained CREB signaling is improved neuronal plasticity and survival during neuropathologies.

Highlights

The conantokins are a diverse array of 17-27-amino acid residue peptides found in the venoms of marine snails of the genus Conus that aid the host in the capture of prey and enable their defense against predators [1]
The C. parius-derived conantokins synthesized in our laboratory were characterized qualitatively for α-helicity by Circular dichroism (CD) spectroscopy, in the absence and presence of Mg2+, and compared to published data [22,28,30,31]
The data revealed that ConPr1 and ConPr3 showed a Mg2+dependent increase of α-helical character, as reflected by the negative increase in the molar ellipticity at 222 nm, to an extent trending toward that of Mg2+/ConG and apo- and divalent cation-loaded ConT (Figure 1A), each of which is known to exist as an end-to-end α-helix [5,29,30,32,33]

Summary

Introduction

The conantokins are a diverse array of 17-27-amino acid residue peptides found in the venoms of marine snails of the genus Conus that aid the host in the capture of prey and enable their defense against predators [1]. Later a variety of conantokins, such as ConT [3] and ConR [4], were discovered and characterized from other hunting snail species. These peptides have multiple conserved gammacarboxyglutamate (Gla) residues that are central to their biological activity, viz., selective inhibition of ion flow through the Glu/Gly co-agonized channels of the N-methyl-D-aspartate receptor (NMDAR) [2]. ConT assumes a high degree of α-helicity in the metal-free (apo) state, whereas ConG adopts a random conformation in absence of divalent ions [6,7]. ConG binds to divalent cations to generate an α-helical structure comparable to apo- or metal-bound ConT [8]

Methods

Results

Conclusion