Antagonistic action on NMDA/GluN2B mediated currents of two peptides that were conantokin-G structure-based designed

Edwin A Reyes-Guzman,Esperanza Recio-Pinto,Nohora Vega-Castro,Edgar A Reyes-Montaño

doi:10.1186/s12868-017-0361-4

Edwin A Reyes-Guzman, Esperanza Recio-Pinto + Show 2 more

Open Access

https://doi.org/10.1186/s12868-017-0361-4

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Abstract

BackgroundThe GluN2B subunit of the N-methyl-d-aspartate receptor (NMDAr) modulates many physiological processes including learning, memory, and pain. Excessive increase in NMDAr/GluN2B activity has been associated with various disorders such neuropathic pain and neuronal death following hypoxia. Thus there is an interest in identifying NMDAr antagonists that interact specifically with the GluN2B subunit. Recently based on structural analysis between the GluN2B subunit and conantokin-G, a toxin that interacts selectively with the GluN2B subunit, we designed various peptides that are predicted to act as NMDAr antagonists by interacting with the GluN2B subunit. In this study we tested this prediction for two of these peptides EAR16 and EAR18.ResultsThe effects of EAR16 and EAR18 in NMDA-evoked currents were measured in cultured rat embryonic hippocampal neurons and in HEK-293 cells expressing recombinant NMDAr comprised of GluN1a–GluN2A or GluN1a–GluN2B subunits. In hippocampal neurons, EAR16 and EAR18 reduced the NMDA-evoked calcium currents in a dose-dependent and reversible manner with comparable IC50 (half maximal inhibitory concentration) values of 241 and 176 µM, respectively. At 500 µM, EAR16 blocked more strongly the NMDA-evoked currents mediated by the GluN1a–GluN2B (84%) than those mediated by the GluN1a–GluN2A (50%) subunits. At 500 µM, EAR18 blocked to a similar extent the NMDA-evoked currents mediated by the GluN1a–GluN2B (62%) and the GluN1a–GluN2A (55%) subunits.ConclusionsThe newly designed EAR16 and EAR18 peptides were shown to block in reversible manner NMDA-evoked currents, and EAR16 showed a stronger selectivity for GluN2B than for GluN2A.

Highlights

The GluN2B subunit of the N-methyl-d-aspartate receptor (NMDAr) modulates many physiological pro‐ cesses including learning, memory, and pain
When NMDA stimulations were separated by a 4 min interval the magnitude of the NMDA-evoked Ca2+ currents was similar between consecutive stimulations (Fig. 1a), as reflected by the average behavior shown by the black squares in Figs. 1b, c and 2a, b
We observed that Ro 25-6981 blocked 70% of the NMDA-evoked currents (Fig. 1c, bottom); under our experimental conditions about 70% of the NMDA-evoked currents in hippocampal neurons are mediated by activation of GluN2B/NMDAr

Summary

Introduction

The GluN2B subunit of the N-methyl-d-aspartate receptor (NMDAr) modulates many physiological pro‐ cesses including learning, memory, and pain. Excessive increase in NMDAr/GluN2B activity has been associated with various disorders such neuropathic pain and neuronal death following hypoxia. Based on structural analysis between the GluN2B subunit and conantokin-G, a toxin that interacts selectively with the GluN2B subunit, we designed various peptides that are predicted to act as NMDAr antagonists by interacting with the GluN2B subunit. N-methyl-d-aspartate receptors (NMDAr) are inotropic glutamate receptors (iGluRs) that contribute to multiple neuronal functions such as [1,2,3]. An increase in the expression of GluN1, GluN2A, and GluN2B subunits in the hippocampus contribute to neurological problems that develop following traumatic brain injury [12]. Increase in the activity/ expression of GluN2B has been postulated to contribute to neuronal damage following stroke, as well as to the development of diseases such as Parkinson, Huntington, Alzheimer and chronic neuropathic pain [11]

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