Identification of a Single Amino Acid in GluN1 That Is Critical for Glycine-Primed Internalization of NMDA Receptors

Mol Brain. 2013 Aug 13;6:36. doi: 10.1186/1756-6606-6-36.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Protein Complex beta Subunits / metabolism
  • Animals
  • Binding Sites
  • Endocytosis*
  • Glycine / metabolism*
  • HEK293 Cells
  • Humans
  • Ion Channel Gating
  • Mutant Proteins / metabolism
  • Mutation / genetics
  • Rats
  • Receptors, N-Methyl-D-Aspartate / chemistry*
  • Receptors, N-Methyl-D-Aspartate / genetics
  • Receptors, N-Methyl-D-Aspartate / metabolism*
  • Recombinant Proteins / metabolism
  • Structure-Activity Relationship

Substances

  • Adaptor Protein Complex beta Subunits
  • Mutant Proteins
  • NMDA receptor A1
  • Receptors, N-Methyl-D-Aspartate
  • Recombinant Proteins
  • Glycine