Identification of a single amino acid in GluN1 that is critical for glycine-primed internalization of NMDA receptors

Abstract

Abstract Background NMDA receptors are ligand-gated ion channels with essential roles in glutamatergic synaptic transmission and plasticity in the CNS. As co-receptors for glutamate and glycine, gating of the NMDA receptor/channel pore requires agonist binding to the glycine sites, as well as to the glutamate sites, on the ligand-binding domains of the receptor. In addition to channel gating, glycine has been found to prime NMDA receptors for internalization upon subsequent stimulation of glutamate and glycine sites. Results Here we address the key issue of identifying molecular determinants in the glycine-binding subunit, GluN1, that are essential for priming of NMDA receptors. We found that glycine treatment of wild-type NMDA receptors led to recruitment of the adaptor protein 2 (AP-2), and subsequent internalization after activating the receptors by NMDA plus glycine. However, with a glycine-binding mutant of GluN1 – N710R/Y711R/E712A/A714L – we found that treating with glycine did not promote recruitment of AP-2 nor were glycine-treated receptors internalized when subsequently activated with NMDA plus glycine. Likewise, GluN1 carrying a single point mutation – A714L – did not prime upon glycine treatment. Importantly, both of the mutant receptors were functional, as stimulating with NMDA plus glycine evoked inward currents. Conclusions Thus, we have identified a single amino acid in GluN1 that is critical for priming of NMDA receptors by glycine. Moreover, we have demonstrated the principle that while NMDA receptor gating and priming share a common requirement for glycine binding, the molecular constraints in GluN1 for gating are distinct from those for priming.