From bedside-to-bench: What disease-associated variants are teaching us about the NMDA receptor.

NMDA receptors (NMDARs) are glutamate-gated ion channels that contribute to nearly all brain processes. Not surprisingly then, genetic variations in the genes encoding NMDAR subunits can be associated with neurodevelopmental, neurological and psychiatric disorders. These disease-associated variants (DAVs) present challenges, such as defining how DAV-induced alterations in receptor function contribute to disease progression and how to treat the affected individual clinically. As a starting point to overcome these challenges, we need to refine our understanding of the complexity of NMDAR structure function. In this regard, DAVs have expanded our knowledge of NMDARs because they do not just target well-known structure-function motifs, but rather give an unbiased view of structural elements that are important to the biology of NMDARs. Indeed, established NMDAR structure-function motifs have been validated by the appearance of disorders in patients where these motifs have been altered, and DAVs have identified novel structural features in NMDARs such as gating triads and hinges in the gating machinery. Still, the majority of DAVs remain unexplored and occur at sites in the protein with unidentified function or alter receptor properties in multiple and unanticipated ways. Detailed mechanistic and structural investigations are required of both established and novel motifs to develop a highly refined pathomechanistic model that accounts for the complex machinery that regulates NMDARs. Such a model would provide a template for rational drug design and a starting point for personalized medicine.

De novo GRIN variants in NMDA receptor M2 channel pore-forming loop are associated with neurological diseases.

N-methyl-D-aspartate receptors (NMDARs) mediate slow excitatory postsynaptic transmission in the central nervous system, thereby exerting a critical role in neuronal development and brain function. Rare genetic variants in the GRIN genes encoding NMDAR subunits segregated with neurological disorders. Here, we summarize the clinical presentations for 18 patients harboring 12 de novo missense variants in GRIN1, GRIN2A, and GRIN2B that alter residues in the M2 re-entrant loop, a region that lines the pore and is intolerant to missense variation. These de novo variants were identified in children with a set of neurological and neuropsychiatric conditions. Evaluation of the receptor cell surface expression, pharmacological properties, and biophysical characteristics show that these variants can have modest changes in agonist potency, proton inhibition, and surface expression. However, voltage-dependent magnesium inhibition is significantly reduced in all variants. The NMDARs hosting a single copy of a mutant subunit showed a dominant reduction in magnesium inhibition for some variants. These variant NMDARs also show reduced calcium permeability and single-channel conductance, as well as altered open probability. The data suggest that M2 missense variants increase NMDAR charge transfer in addition to varied and complex influences on NMDAR functional properties, which may underlie the patients' phenotypes.

Two gates mediate NMDA receptor activity and are under subunit-specific regulation.

Kinetics of NMDA receptor (NMDAR) ion channel opening and closing contribute to their unique role in synaptic signaling. Agonist binding generates free energy to open a canonical gate at the M3 helix bundle crossing. Single channel activity is characterized by clusters, or periods of rapid opening and closing, that are separated by long silent periods. A conserved glycine in the outer most transmembrane helices, the M4 helices, regulates NMDAR function. Here we find that the GluN1 glycine mainly regulates single channel events within a cluster, whereas the GluN2 glycine mainly regulates entry and exit from clusters. Molecular dynamics simulations suggest that, whereas the GluN2 M4 (along with GluN2 pre-M1) regulates the gate at the M3 helix bundle crossing, the GluN1 glycine regulates a 'gate' at the M2 loop. Subsequent functional experiments support this interpretation. Thus, the distinct kinetics of NMDARs are mediated by two gates that are under subunit-specific regulation.

NMDA Receptors Require Multiple Pre-opening Gating Steps for Efficient Synaptic Activity.

NMDA receptors (NMDARs) are glutamate-gated ion channels that mediate fast excitatory synaptic transmission in the nervous system. Applying glutamate to outside-out patches containing a single NMDAR, we find that agonist-bound receptors transition to the open state via two conformations, an "unconstrained pre-active" state that contributes to fast synaptic events and a "constrained pre-active" state that does not. To define how glutamate drives these conformations, we decoupled the ligand-binding domains from specific transmembrane segments for GluN1 and GluN2A. Displacements of the pore-forming M3 segments define the energy of fast opening. However, to enter the unconstrained conformation and contribute to fast signaling, the GluN2 pre-M1 helix must be displaced before the M3 segments move. This pre-M1 displacement is facilitated by the flexibility of the S2-M4 of GluN1 and GluN2A. Thus, outer structures-pre-M1 and S2-M4-work in concert to remove constraints and prime the channel for rapid opening, facilitating fast synaptic transmission.

A Swiss army knife for targeting receptors.

A compound can change the activity of NMDA receptors in some regions of a synapse without affecting those in other regions.

A conserved glycine harboring disease-associated mutations permits NMDA receptor slow deactivation and high Ca2+ permeability.

A variety of de novo and inherited missense mutations associated with neurological disorders are found in the NMDA receptor M4 transmembrane helices, which are peripheral to the pore domain in eukaryotic ionotropic glutamate receptors. Subsets of these mutations affect receptor gating with dramatic effects, including in one instance halting it, occurring at a conserved glycine near the extracellular end of M4. Functional experiments and molecular dynamic simulations of constructs with and without substitutions at this glycine indicate that it acts as a hinge, permitting the intracellular portion of the ion channel to laterally expand. This expansion stabilizes long-lived open states leading to slow deactivation and high Ca2+ permeability. Our studies provide a functional and structural framework for the effect of missense mutations on NMDARs at central synapses and highlight how the M4 segment may represent a pathway for intracellular modulation of NMDA receptor function.

Divergent roles of a peripheral transmembrane segment in AMPA and NMDA receptors.

Ionotropic glutamate receptors (iGluRs), including AMPA receptor (AMPAR) and NMDA receptor (NMDAR) subtypes, are ligand-gated ion channels that mediate signaling at the majority of excitatory synapses in the nervous system. The iGluR pore domain is structurally and evolutionarily related to an inverted two-transmembrane K+ channel. Peripheral to the pore domain in eukaryotic iGluRs is an additional transmembrane helix, the M4 segment, which interacts with the pore domain of a neighboring subunit. In AMPARs, the integrity of the alignment of a specific face of M4 with the adjacent pore domain is essential for receptor oligomerization. In contrast to AMPARs, NMDARs are obligate heterotetramers composed of two GluN1 and typically two GluN2 subunits. Here, to address the function of the M4 segments in NMDARs, we carry out a tryptophan scan of M4 in GluN1 and GluN2A subunits. Unlike AMPARs, the M4 segments in NMDAR subunits makes only a limited contribution to their biogenesis. However, the M4 segments in both NMDAR subunits are critical for receptor activation, with mutations at some positions, most notably at the extreme extracellular end, completely halting the gating process. Furthermore, although the AMPAR M4 makes a minimal contribution to receptor desensitization, the NMDAR M4 segments have robust and subunit-specific effects on desensitization. These findings reveal that the functional roles of the M4 segments in AMPARs and NMDARs have diverged in the course of their evolution and that the M4 segments in NMDARs may act as a transduction pathway for receptor modulation at synapses.