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

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.

Hydroxyproline-induced Helical Disruption in Conantokin Rl-B Affects Subunit-selective Antagonistic Activities toward Ion Channels of N-Methyl-d-aspartate Receptors

“…Thus, it is not surprising that conRl-B lacks helical character in the apo-form. However, in the presence of divalent cations, negative charge repulsions are eliminated and the ␣-helix forms (30,50,61). The further effect of Hyp 10 that is present in a central primary location of conRl-B on overall helix propensity of conRl-B is of interest, especially with regard to whether it destabilizes the global helix or is more simply a small region within the overall helix.…”

“…Cell Cultures of Dissociated Primary Neurons-Primary mouse cortical cell cultures were prepared from embryonic day 18 for WT, GluN2A Ϫ/Ϫ , and GluN2B Ϫ/Ϫ mice as described (50). Mice heterozygous for the GluN2A (GluN2A ϩ/Ϫ ) or the GluN2B (GluN2B ϩ/Ϫ ) allele were bred separately, and the embryos were harvested and genotyped for the GluN2A Ϫ/Ϫ or GluN2B Ϫ/Ϫ alleles, which were used for neuron culture.…”

Conantokin-G Attenuates Detrimental Effects of NMDAR Hyperactivity in an Ischemic Rat Model of Stroke

“…Since our con-G administration method was i.t., a dose of 2 μM con-G was employed to establish the neuroprotective model. Furthermore, from our in vitro studies, we have observed that 2–5 μM conantokins could antagonize NMDA-evoked current and intracellular calcium influx in dissociated rat hippocampal or mouse cortical neurons [ 29 ]. The intrathecal catheter (Braintree Scientific, Braintree, MA) was inserted into the spinal column of the rat at the junction of the skull and 1 st vertebra while the rat was anesthetized with 100 μg ketamine/10 μg xylazine/gr weight and held in place on a stereotaxic frame.…”

Discerning the Role of the Hydroxyproline Residue in the Structure of Conantokin Rl-B and Its Role in GluN2B Subunit-Selective Antagonistic Activity toward N-Methyl-d-Aspartate Receptors

“…The nonextensively studied GluN2D subunit is thought to be mostly located in the extrasynaptic spaces . Previously, we have shown that mature (>DIV 13) WT, GluN2A –/– , and GluN2B –/– mice cortical neurons express the GluN2D subunit . The Xenopus oocyte system that supports heterologous expression of NMDAR subunits is excellent for discerning pharmacological properties of inhibitors without interference from other glutamate receptors or ion channels.…”