The fidelity of NMDA receptors (NMDARs) to integrate pre- and post-synaptic activity requires a match between agonist binding and ion channel opening. To address how agonist binding is transduced into pore opening in NMDARs, we manipulated the coupling between the ligand binding domain (LBD) and the ion channel by inserting residues in a linker between them. We find that a single residue insertion dramatically attenuates the ability of NMDARs to convert a glutamate transient into a functional response. This is largely due to a decreased likelihood for the channel to open and remain open. Computational and thermodynamic analyses suggest that insertions prevent the agonist-bound LBD from effectively pulling on pore lining elements, thereby destabilizing pore opening. Further, this pulling energy is more prominent in the GluN2 subunit. We conclude that an efficient NMDAR-mediated synaptic response relies on a mechanical coupling between the LBD and the ion channel.

Glutamate-gated ion channels embedded within the neuronal membrane are the primary mediators of fast excitatory synaptic transmission in the CNS. The ion channel of these glutamate receptors contains a pore-lining transmembrane M3 helix surrounded by peripheral M1 and M4 helices. In the NMDA receptor subtype, opening of the ion channel pore, mediated by displacement of the M3 helices away from the central pore axis, occurs in a highly concerted fashion, but the associated temporal movements of the peripheral helices are unknown. To address the gating dynamics of the peripheral helices, we constrained the relative movements of the linkers that connect these helices to the ligand-binding domain using engineered cross-links, either within (intra-GluN1 or GluN2A) or between subunits. Constraining the peripheral linkers in any manner dramatically curtailed channel opening, highlighting the requirement for rearrangements of these peripheral structural elements for efficient gating to occur. However, the magnitude of this gating effect depended on the specific subunit being constrained, with the most dramatic effects occurring when the constraint was between subunits. Based on kinetic and thermodynamic analysis, our results suggest an asynchrony in the displacement of the peripheral linkers during the conformational and energetic changes leading to pore opening. Initially there are large-scale rearrangements occurring between the four subunits. Subsequently, rearrangements occur within individual subunits, mainly GluN2A, leading up to or in concert with pore opening. Thus, the conformational changes induced by agonist binding in NMDA receptors converge asynchronously to permit pore opening.

Most fast excitatory synaptic transmission in the nervous system is mediated by glutamate acting through ionotropic glutamate receptors (iGluRs). iGluRs (AMPA, kainate, and NMDA receptor subtypes) are tetrameric assemblies, formed as a dimer of dimers. Still, the mechanism underlying tetramerization – the necessary step for the formation of functional receptors that can be inserted into the plasma membrane – is unknown. All eukaryotic compared to prokaryotic iGluR subunits have an additional transmembrane segment, the M4 segment, which positions the physiologically critical C-terminal domain on the cytoplasmic side of the membrane. AMPA receptor (AMPAR) subunits lacking M4 do not express on the plasma membrane. Here, we show that these constructs are retained in the endoplasmic reticulum, the major cellular compartment mediating protein oligomerization. Using approaches to assay the native oligomeric state of AMPAR subunits, we find that subunits lacking M4 or containing single amino-acid substitutions along an ‘interacting’ face of the M4 helix that block surface expression, no longer tetramerize in either homo- or heteromeric assemblies. In contrast, subunit dimerization appears to be largely intact. These experiments define the M4 segment as a unique functional unit in AMPARs that is required for the critical dimer to tetramer transition.

AMPA and NMDA receptors are glutamate-gated ion channels that mediate fast excitatory synaptic transmission throughout the nervous system. In the continual presence of glutamate, AMPA and NMDA receptors containing the GluN2A or GluN2B subunit enter into a nonconducting, desensitized state that can impact synaptic responses and glutamate-mediated excitotoxicity. The process of desensitization is dramatically different between subtypes, but the basis for these differences is unknown. We generated an extensive sequence alignment of ionotropic glutamate receptors (iGluRs) from diverse animal phyla and identified a highly conserved motif, which we termed the "hydrophobic box," located at the extracellular interface of transmembrane helices. A single position in the hydrophobic box differed between mammalian AMPA and NMDA receptors. Surprisingly, we find that an NMDAR-to-AMPAR exchange mutation at this position in the rat GluN2A or GluN2B subunit had a dramatic and highly specific effect on NMDAR desensitization, making it AMPARlike. In contrast, a reverse exchange mutation in AMPARs had minimal effects on desensitization. These experiments highlight differences in desensitization between iGluR subtypes and the highly specific contribution of the GluN2 subunit to this process.

Background:In contrast to prokaryotic ionotropic glutamate receptors (GluRs), eukaryotic GluRs have an additional transmembrane segment, M4, that has unknown functional significance. Results: Interaction of a specific face of the M4 segment with other transmembrane segments is necessary for AMPAR surface expression. Conclusion:The M4 segment is required for AMPAR surface expression. Significance: This work suggests a mechanism regulating AMPAR biogenesis.