The N-Terminal Domains of both NR1 and NR2 Subunits Determine Allosteric Zn²⁺ Inhibition and Glycine Affinity of N-Methyl-d-aspartate Receptors

Abstract

The N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors (iGluRs) is a tetrameric protein composed of homologous NR1 and NR2 subunits, which require the binding of glycine and glutamate, respectively, for efficient channel gating. The extracellular N-terminal domains (NTDs) of iGluR subunits show sequence homology to the bacterial periplasmic leucine/isoleucine/valine binding protein (LIVBP) and have been implicated in iGluR assembly, trafficking, and function. Here, we investigated how deletion of the NR1-and NR2-NTDs affects the expression and function of NMDA receptors. Both proteolytic cleavage of the NR1-NTD from assembled NR1/ NR2 receptors and coexpression of the NTD-deleted NR1 subunit with wild-type or NTD-deleted NR2 subunits resulted in agonist-gated channels that closely resembled wild-type receptors. This indicates that the NTDs of both NMDA receptor subunits are not essential for receptor assembly and function. However, deletion of either the NR1 or the NR2 NTD eliminated high-affinity, allosteric inhibition of agonist-induced currents by Zn 2ϩ and ifenprodil, consistent with the idea that interdomain interactions between these domains are important for allosteric receptor modulation. Furthermore, by replacing the NR2A-NTD with the NR2B NTD, and vice versa, the different glycine affinities of NR1/NR2A and NR1/NR2B receptors were found to be determined by their respective NR2-NTDs. Together, these data show that the NTDs of both the NR1 and NR2 subunits determine allosteric inhibition and glycine potency but are not required for NMDA receptor assembly.

Supralinear potentiation of NR1/NR3A excitatory glycine receptors by Zn 2+ and NR1 antagonist

“…Thus, although conserved mechanisms of agonist-induced channel activation exist within the NMDA receptor family, the differential consequences of glycine binding to NR1/NR2 and NR1/NR3 receptors discriminate NMDA receptor subtypes. Similarly, Zn 2ϩ acts as an allosteric inhibitor of conventional NMDA receptors mediated by the NTDs of the NR1 and NR2 subunits (17,22,23) but displays exclusively potentiating and agonistic effects on NR1/ NR3A receptors via the NR1 LBD. In conclusion, although common gating mechanisms are used in NR1/NR2 and NR1/ NR3A NMDA receptors, the common ligands glycine and Zn 2ϩ have different functional roles.…”

“…Allosteric Zn 2ϩ inhibition of conventional NR1/NR2A and NR1/NR2B receptors is thought to be mediated via the NTD of the NR2 subunit (17,22,23). To examine whether the NTD is also important for Zn 2ϩ modulation of NR1/NR3A receptors, we generated a NR3A subunit lacking the entire NTD ( Fig.…”

“…To examine whether the NTD is also important for Zn 2ϩ modulation of NR1/NR3A receptors, we generated a NR3A subunit lacking the entire NTD ( Fig. S2) and coexpressed it with a similarly NTD-deleted NR1 subunit (23). The resulting NR1 ⌬NTD /NR3A ⌬NTD receptors were found to be fully functional with a slightly lower apparent affinity for glycine than the WT receptor ( Fig.…”

Subtype-dependent N-Methyl-d-aspartate Receptor Amino-terminal Domain Conformations and Modulation by Spermine

“…The GluN1 cleft can regulate channel function as its deletion or cleavage affects allosteric inhibition and potentiation (9,29), and propping open the GluN1 ATD cleft with a thiol-reactive reagent potentiates the receptor (22). However, aside from potentially contributing amino acids to the spermine-binding site (9,10), the structural influence of the GluN1 ATD in spermine modulation is unexamined.…”

Transmembrane Region of N-Methyl-d-aspartate Receptor (NMDAR) Subunit Is Required for Receptor Subunit Assembly

“…Studies using biochemical methods and immunostaining indicate that the C terminus and/or M4 are not involved in the heteromer formation between GluN1 and GluN2 subunits (9,15), whereas the ATD or even the whole N terminus is necessary for NMDAR subunit assembly (15,16). However, still other research indicates that the ATD mainly plays modulatory roles in NMDAR trafficking and function and is not directly involved in receptor assembly (10,17,18).…”