Abstract
BackgroundNMDA 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.ResultsHere 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.ConclusionsThus, 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.
Highlights
NMDA receptors (NMDARs) constitute a major subtype of glutamate receptor and play important roles in numerous physiological and pathophysiological processes in the CNS [1]
Glycine-primed internalization of wild-type NMDARs With wild-type NMDARs, we found that after treating cells with glycine (100 μM; 5 min) the amplitude of NMDAR-mediated currents – evoked by test applications of NMDA (50 μM) plus glycine (1 μM) – was reduced significantly as compared with cells not treated with glycine (Figure 1A and B)
To determine whether glycine stimulation recruits adaptor protein 2 (AP-2) to recombinant NMDARs, we examined the association of GluN1/GluN2A or GluN1/ GluN2B receptors with the adaptin β2 subunit of endogenous AP-2 in the HEK cells
Summary
NMDA receptors (NMDARs) constitute a major subtype of glutamate receptor and play important roles in numerous physiological and pathophysiological processes in the CNS [1]. The number of cell-surface NMDARs is critically regulated by endocytosis [4] which is either constitutive or regulated, i.e. induced by stimulation. Both constitutive and regulated NMDAR endocytosis are dynamin-dependent [5,6]. 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
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