EAR-20 peptide, a novel NMDA receptor positive allosteric modulator

Roberto García-Díaz^1,2,3*Aida Castellanos⁴Federico Miguez-Cabello⁴Javier Picañol⁴Anna Priscil·la Pérez-González⁴Esther Gratacòs-Batlle⁵Nohora Vega²Xavier Altafaj^4,6Edwin A Reyes-Guzmán⁷Edgar A Reyes-Montaño^2*Xavier Gasull^4,6David Soto^4,6*

• ¹Neurophysiology Laboratory, Department of Biomedicine, Faculty of Medicine and Health Sciences, Institute of Neurosciences, University of Barcelona,, 08036, University of Barcelona, Barcelona, Spain
• ²Grupo de Investigación en Proteínas, Departamento de Química, Universidad Nacional de Colombia, Cra 30 No 45‑03 Edificio 451 Lab 201‑1, Bogotá, Colombia., Universidad Nacional de Colombia, Bogotá, Colombia
• ³Facultad de Ingeniería, Programa de Ingeniería Biomédica, Universidad Militar Nueva Granada, kilómetro 2 Cajicá-Zipaquirá, Colombia., Universidad Militar Nueva Granada, Bogotá, Colombia
• ⁴Faculty of Medicine and Health Sciences, Institute of Neurosciences, University of Barcelona, 08036 Barcelona, Spain., University of Barcelona, Barcelona, Spain
• ⁵Department of Fundamental and Clinical Nursing. Faculty of Nursing. University of Barcelona. L'Hospitalet del Llobregat, 08907, Spain., University of Barcelona, Barcelona, Spain
• ⁶Institut d'Investigacions Biomediques August Pi i Sunyer, Barcelona, Spain
• ⁷Doctorado en Ciencia Aplicada, Universidad Antonio Nariño, Bogotá, Colombia., Universidad Antonio Narino, Bogotá, Colombia

Allosteric modulation of ligand-gated ion channels provides a powerful mechanism to fine-tune their activity without competing with endogenous ligands. In the case of NMDA receptors (NMDARs), which are critical for excitatory neurotransmission and synaptic plasticity, allosteric modulators represent potential therapeutic tools, particularly in conditions involving NMDAR hypofunction. Here, we characterize EAR-20, a 17-amino-acid peptide derived from the marine cone snail toxin Conantokin-G, as a novel positive allosteric modulator (PAM) of NMDARs. Using molecular docking, whole-cell and single-channel patch-clamp electrophysiology, and recordings in cultured hippocampal neurons, we show that EAR-20 enhances receptor function by increasing channel open probability and reducing desensitization, and can even activate NMDARs in the absence of exogenous glutamate and glycine, albeit to a lower extent. EAR-20 decreased desensitization, potentiating GluN1-GluN2A and GluN1-GluN2B receptors more than twofold, modestly enhanced (~25%) GluN1-GluN2A-GluN2B tri-heteromers, and increased NMDAR-mediated currents in primary hippocampal neurons. Molecular docking identified a binding site at the GluN1-GluN2B interface, with Ser773 in GluN1 being critical for the modulatory effect. Importantly, EAR-20 partially rescued hypofunctional NMDARs carrying patient-derived loss-of-function mutations. Together, these findings identify EAR-20 as a novel subunit-dependent PAM with the potential to inspire the development of small molecules targeting the same binding site, offering proof of concept for therapeutic strategies to treat neurological and neurodevelopmental disorders.