EAR-20: A NOVEL PEPTIDE ENHANCING NMDA RECEPTOR FUNCTION

Allosteric modulation of ligand-gated ion channels represents an effective strategy to fine-tune receptor function without directly competing with endogenous ligands. N-methyl-D-aspartate receptors (NMDARs) are glutamatergic ion channels essential for excitatory neurotransmission and synaptic plasticity, and their hypofunction has been implicated in multiple neurological and neurodevelopmental disorders. Consequently, the identification of positive allosteric modulators (PAMs) of NMDARs has emerged as a promising therapeutic approach. In this work, we characterize EAR-20, a peptide derived from structural elements of the marine cone snail toxin conantokin-G, as a novel PAM of NMDARs. Using whole-cell and single-channel patch-clamp electrophysiology in HEK293T cells expressing defined NMDAR subunit combinations, we demonstrate that EAR-20 robustly potentiates NMDAR-mediated currents in a subunit-dependent manner. EAR-20 enhances GluN1–GluN2A and GluN1–GluN2B receptors more than twofold, while exerting a more modest potentiation of tri-heteromeric GluN1–GluN2A–GluN2B receptors. Single-channel recordings reveal that EAR-20 increases channel open probability by prolonging receptor occupancy in the open state and reducing desensitization. Notably, EAR-20 can partially activate NMDARs in the absence of exogenous glutamate and glycine. Molecular docking identifies a binding site at the GluN1–GluN2B interface, with Ser773 in GluN1 being critical for modulation. Importantly, EAR-20 partially rescues the functional deficits of hypofunctional NMDARs carrying patient-derived loss-of-function mutations. Together, these findings establish EAR-20 as a novel subunit-selective NMDAR PAM and highlight rational peptide design as a viable strategy for developing therapeutics targeting NMDAR hypofunction.
Supported by grants PID2020-119932GB-I00 to DS, CEX2021-001159-M, ​J.P. is supported by Ministro de Universidades (Nº FPU22/02071)