Calcium- and calmodulin-dependent inhibition of NMDA receptor currents.

Calcium ions (Ca2+) reduce NMDA receptor currents through several distinct mechanisms. Among these, calmodulin (CaM)-dependent inhibition (CDI) accomplishes rapid, reversible, and incomplete reduction of the NMDA receptor currents in response to elevations in intracellular Ca2+. Quantitative and mechanistic descriptions of CDI of NMDA receptor-mediated signals have been marred by variability originating, in part, from differences in the conditions and metrics used to evaluate this process across laboratories. Recent ratiometric approaches to measure the magnitude and kinetics of NMDA receptor CDI have facilitated rapid insights into this phenomenon. Notably, the kinetics and magnitude of NMDA receptor CDI depend on the degree of saturation of its CaM binding sites, which represent the bona fide calcium sensor for this type of inhibition, the kinetics and magnitude of the Ca2+ signal, which depends on the biophysical properties of the NMDA receptor or of adjacent Ca2+ sources, and on the relative distribution of Ca2+ sources and CaM molecules. Given that all these factors vary widely during development, across cell types, and with physiological and pathological states, it is important to understand how NMDA receptor CDI develops and how it contributes to signaling in the central nervous system. Here, we review briefly these recent advances and highlight remaining questions about the structural and kinetic mechanisms of NMDA receptor CDI. Given that pathologies can arise from several sources, including mutations in the NMDA receptor and in CaM, understanding how CaM responds to intracellular Ca2+ signals to initiate conformational changes in NMDA receptors, and mapping the structural domains responsible will help to envision novel therapeutic strategies to neuropsychiatric diseases, which presently have limited available treatments.

Cross-subunit interactions that stabilize open states mediate gating in NMDA receptors.

NMDA receptors are excitatory channels with critical functions in the physiology of central synapses. Their activation reaction proceeds as a series of kinetically distinguishable, reversible steps, whose structural bases are currently under investigation. Very likely, the earliest steps include glutamate binding to glycine-bound receptors and subsequent constriction of the ligand-binding domain. Later, three short linkers transduce this movement to open the gate by mechanical pulling on transmembrane helices. Here, we used molecular and kinetic simulations and double-mutant cycle analyses to show that a direct chemical interaction between GluN1-I642 (on M3 helix) and GluN2A-L550 (on L1-M1 linker) stabilizes receptors after they have opened and thus represents one of the structural changes that occur late in the activation reaction. This native interaction extends the current decay, and its absence causes deficits in charge transfer by GluN1-I642L, a pathogenic human variant.

GluD receptors are functional ion channels.

In this article, we review contemporary evidence that GluD receptors are functional ion channels whose depolarizing currents contribute to their biological functions, akin to all other members of the ionotropic glutamate receptor (iGluR) family.

Membrane Stretch Gates NMDA Receptors.

NMDARs are ionotropic glutamate receptors widely expressed in the CNS, where they mediate phenomena as diverse as neurotransmission, information processing, synaptogenesis, and cellular toxicity. They function as glutamate-gated Ca2+-permeable channels, which require glycine as coagonist, and can be modulated by many diffusible ligands and cellular cues, including mechanical stimuli. Previously, we found that, in cultured astrocytes, shear stress initiates NMDAR-mediated Ca2+ entry in the absence of added agonists, suggesting that more than being mechanosensitive, NMDARs may be mechanically activated. Here, we used controlled expression of rat recombinant receptors and noninvasive on-cell single-channel current recordings to show that mild membrane stretch can substitute for the neurotransmitter glutamate in gating NMDAR currents. Notably, stretch-activated currents maintained the hallmark features of the glutamate-gated currents, including glycine-requirement, large unitary conductance, high Ca2+ permeability, and voltage-dependent Mg2+ blockade. Further, we found that the stretch-gated current required the receptor's intracellular domain. Our results are consistent with the hypothesis that mechanical forces can gate endogenous NMDAR currents even in the absence of synaptic glutamate release, which has important implications for understanding mechanotransduction and the physiological and pathologic effects of mechanical forces on cells of the CNS.SIGNIFICANCE STATEMENT We show that, in addition to enhancing currents elicited with low agonist concentrations, membrane stretch can gate NMDARs in the absence of the neurotransmitter glutamate. Stretch-gated currents have the principal hallmarks of the glutamate-gated currents, including requirement for glycine, large Na+ conductance, high Ca2+ permeability, and voltage-dependent Mg2+ block. Therefore, results suggest that mechanical forces can initiate cellular processes presently attributed to glutamatergic neurotransmission, such as synaptic plasticity and cytotoxicity. Given the ubiquitous presence of mechanical forces in the CNS, this discovery identifies NMDARs as possibly important mechanotransducers during development and across the lifespan, and during pathologic processes, such as those associated with traumatic brain injuries, shaken infant syndrome, and chronic traumatic encephalopathy.

Complex functional phenotypes of NMDA receptor disease variants.

NMDA receptors have essential roles in the physiology of central excitatory synapses and their dysfunction causes severe neuropsychiatric symptoms. Recently, a series of genetic variants have been identified in patients, however, functional information about these variants is sparse and their role in pathogenesis insufficiently known. Here we investigate the mechanism by which two GluN2A variants may be pathogenic. We use molecular dynamics simulation and single-molecule electrophysiology to examine the contribution of GluN2A subunit-residues, P552 and F652, and their pathogenic substitutions, P552R and F652V, affect receptor functions. We found that P552 and F652 interact during the receptors' normal activity cycle; the interaction stabilizes receptors in open conformations and is required for a normal electrical response. Engineering shorter side-chains at these positions (P552A and/or F652V) caused a loss of interaction energy and produced receptors with severe gating, conductance, and permeability deficits. In contrast, the P552R side chain resulted in stronger interaction and produced a distinct, yet still drastically abnormal electrical response. These results identify the dynamic contact between P552 and F652 as a critical step in the NMDA receptor activation, and show that both increased and reduced communication through this interaction cause dysfunction. Results show that subtle differences in NMDA receptor primary structure can generate complex phenotypic alterations whose binary classification is too simplistic to serve as a therapeutic guide.

NMDA receptors: linking physiological output to biophysical operation.

NMDA receptors are preeminent neurotransmitter-gated channels in the CNS, which respond to glutamate in a manner that integrates multiple external and internal cues. They belong to the ionotropic glutamate receptor family and fulfil unique and crucial roles in neuronal development and function. These roles depend on characteristic response kinetics, which reflect the operation of the receptors. Here, we review biologically salient features of the NMDA receptor signal and its mechanistic origins. Knowledge of distinctive NMDA receptor biophysical properties, their structural determinants and physiological roles is necessary to understand the physiological and neurotoxic actions of glutamate and to design effective therapeutics.

Ca2+-Dependent Inactivation of GluN2A and GluN2B NMDA Receptors Occurs by a Common Kinetic Mechanism.

N-Methyl-d-aspartate (NMDA) receptors are Ca2+-permeable channels gated by glutamate and glycine that are essential for central excitatory transmission. Ca2+-dependent inactivation (CDI) is a regulatory feedback mechanism that reduces GluN2A-type NMDA receptor responses in an activity-dependent manner. Although CDI is mediated by calmodulin binding to the constitutive GluN1 subunit, prior studies suggest that GluN2B-type receptors are insensitive to CDI. We examined the mechanism of CDI subtype dependence using electrophysiological recordings of recombinant NMDA receptors expressed in HEK-293 cells. In physiological external Ca2+, we observed robust CDI of whole-cell GluN2A currents (0.42 ± 0.05) but no CDI in GluN2B currents (0.08 ± 0.07). In contrast, when Ca2+ was supplied intracellularly, robust CDI occurred for both GluN2A and GluN2B currents (0.75 ± 0.03 and 0.67 ± 0.02, respectively). To examine how the source of Ca2+ affects CDI, we recorded one-channel Na+ currents to quantify the receptor gating mechanism while simultaneously monitoring ionomycin-induced intracellular Ca2+ elevations with fluorometry. We found that CDI of both GluN2A and GluN2B receptors reflects receptor accumulation in long-lived closed (desensitized) states, suggesting that the observed subtype-dependent differences in macroscopic CDI reflect intrinsic differences in equilibrium open probabilities (Po). We tested this hypothesis by measuring substantial macroscopic CDI, in physiologic conditions, for high Po GluN2B receptors (GluN1A652Y/GluN2B). Together, these results show that Ca2+ flux produces activity-dependent inactivation for both GluN2A and GluN2B receptors and that the extent of CDI varies with channel Po. These results are consistent with CDI as an autoinhibitory feedback mechanism against excessive Ca2+ load during high Po activation.

Spatial Coupling Tunes NMDA Receptor Responses via Ca2+ Diffusion.

In the CNS, NMDA receptors generate large and highly regulated Ca2+ signals, which are critical for synaptic development and plasticity. They are highly clustered at postsynaptic sites and along dendritic arbors, but whether this spatial arrangement affects their output is unknown. Synaptic NMDA receptor currents are subject to Ca2+-dependent inactivation (CDI), a type of activity-dependent inhibition that requires intracellular Ca2+ and calmodulin (CaM). We asked whether Ca2+ influx through a single NMDA receptor influences the activity of nearby NMDA receptors, as a possible coupling mechanism. Using cell-attached unitary current recordings from GluN1-2a/GluN2A receptors expressed in human HEK293 cells and from NMDA receptors native to hippocampal neurons from male and female rats, we recorded unitary currents from multichannel patches and used a coupled Markov model to determine the extent of signal coupling (κ). In the absence of extracellular Ca2+, we observed no cooperativity (κ < 0.1), whereas in 1.8 mm external Ca2+, both recombinant and native channels showed substantial negative cooperativity (κ = 0.27). Intracellular Ca2+ chelation or overexpression of a Ca2+-insensitive CaM mutant, reduced coupling, which is consistent with CDI representing the coupling mechanism. In contrast, cooperativity increased substantially (κ = 0.68) when overexpressing the postsynaptic scaffolding protein PSD-95, which increased receptor clustering. Together, these new results demonstrate that NMDA receptor currents are negatively coupled through CDI, and the degree of coupling can be tuned by the distance between receptors. Therefore, channel clustering can influence the activity-dependent reduction in NMDA receptor currents.SIGNIFICANCE STATEMENT At central synapses, NMDA receptors are a major class of excitatory glutamate-gated channels and a source of activity-dependent Ca2+ influx. In turn, fluxed Ca2+ ions bind to calmodulin-primed receptors and reduce further entry, through an autoinhibitory mechanism known as Ca2+ -dependent inactivation (CDI). Here, we show that the diffusion of fluxed Ca2+ between active channels situated within submicroscopic distances amplified receptor inactivation. Thus, calmodulin-mediated gating modulation, an evolutionarily conserved regulatory mechanism, endows synapses with sensitivity to both the temporal sequence and spatial distribution of Ca2+ signals. Perturbations in this mechanism, which coordinates the activity of NMDA receptors within a cluster, may cause signaling alterations that contribute to neuropsychiatric conditions.

Hydroxynorketamine Blocks N-Methyl-d-Aspartate Receptor Currents by Binding to Closed Receptors.

Ketamine, a dissociative anesthetic, is experiencing a clinical resurgence as a fast-acting antidepressant. In the central nervous system, ketamine acts primarily by blocking NMDA receptor currents. Although it is generally safe in a clinical setting, it can be addictive, and several of its derivatives are being investigated as preferable alternatives. 2R,6R-Hydroxynorketamine (HNK), a ketamine metabolite, reproduces some of the therapeutic effects of ketamine and appears to lack abuse liability. Here, we report a systematic investigation of the effects of HNK on macroscopic responses elicited from recombinant NMDA receptors expressed in human embryonic kidney 293 cells. We found that, like ketamine, HNK reduced NMDA receptor currents in a dose-, pH-, and voltage-dependent manner. Relative to ketamine, it had 100-fold-lower potency (46 µM at pH 7.2), 10-fold-slower inhibition onset, slower apparent dissociation rate, weaker voltage dependence, and complete competition by magnesium. Notably, HNK inhibition was fully effective when applied to resting receptors. These results revealed unexpected properties of hydroxynorketamine that warrant its further investigation as a possible therapeutic in pathologies associated with NMDA receptor dysfunction. SIGNIFICANCE STATEMENT: NMDA receptors are excitatory ion channels with fundamental roles in synaptic transmission and plasticity, and their dysfunction associates with severe neuropsychiatric disorders. 2R,6R-Hydroxynorketamine, a metabolite of ketamine, mimics some of the neuroactive properties of ketamine and may lack its abuse liability. Results show that 2R,6R-hydroxynorketamine blocks NMDA receptor currents with low affinity and weak voltage dependence and is effective when applied to resting receptors. These properties highlight its effectiveness to a subset of NMDA receptor responses and recommend it for further investigation.

Reprogramming Postnatal Human Epidermal Keratinocytes Toward Functional Neural Crest Fates.

During development, neural crest (NC) cells are induced by signaling events at the neural plate border of all vertebrate embryos. Initially arising within the central nervous system, NC cells subsequently undergo an epithelial to mesenchymal transition to migrate into the periphery, where they differentiate into diverse cell types. Here we provide evidence that postnatal human epidermal keratinocytes (KC), in response to fibroblast growth factor 2 and insulin like growth factor 1 signals, can be reprogrammed toward a NC fate. Genome-wide transcriptome analyses show that keratinocyte-derived NC cells are similar to those derived from human embryonic stem cells. Moreover, they give rise in vitro and in vivo to NC derivatives such as peripheral neurons, melanocytes, Schwann cells and mesenchymal cells (osteocytes, chondrocytes, adipocytes, and smooth muscle cells). By demonstrating that human keratin-14+ KC can form NC cells, even from clones of single cells, our results have important implications in stem cell biology and regenerative medicine. Stem Cells 2017;35:1402-1415.

Resident Calmodulin Primes NMDA Receptors for Ca2+-Dependent Inactivation.

N-methyl-d-aspartate (NMDA) receptors are glutamate- and glycine-gated channels that flux Na+ and Ca2+ into postsynaptic neurons during synaptic transmission. The resulting intracellular Ca2+ transient is essential to physiological and pathological processes related to synaptic development, plasticity, and apoptosis. It also engages calmodulin (CaM) to reduce subsequent NMDA receptor activity in a process known as Ca2+-dependent inactivation (CDI). Here, we used whole-cell electrophysiology to measure CDI and computational modeling to dissect the sequence of events that underlies it. With these approaches, we estimate that CaM senses NMDA receptor Ca2+ influx at ∼9 nm from the channel pore. Further, when we controlled the frequency of Ca2+ influx through individual channels, we found that a kinetic model where apoCaM associates with channels before their activation best predicts the measured CDI. These results provide, to our knowledge, novel functional evidence for CaM preassociation to NMDA receptors in living cells. This particular mechanism for autoinhibitory feedback reveals strategies and challenges for Ca2+ regulation in neurons during physiological synaptic activity and disease.

Probing the Structural Dynamics of the NMDA Receptor Activation by Coarse-Grained Modeling.

N-Methyl-D-aspartate (NMDA) receptors are glutamate-gated excitatory channels that play essential roles in brain functions. High-resolution structures have been solved for an allosterically inhibited and agonist-bound form of a functional NMDA receptor; however, other key functional states (particularly the active open-channel state) were only resolved at moderate resolutions by cryo-electron microscopy (cryo-EM). To decrypt the mechanism of the NMDA receptor activation, structural modeling is essential to provide presently missing information about structural dynamics. We performed systematic coarse-grained modeling using an elastic network model and related modeling/analysis tools (e.g., normal mode analysis, flexibility and hotspot analysis, cryo-EM flexible fitting, and transition pathway modeling) based on an active-state cryo-EM map. We observed extensive conformational changes that allosterically couple the extracellular regulatory and agonist-binding domains to the pore-forming trans-membrane domain (TMD), and validated these, to our knowledge, new observations against known mutational and functional studies. Our results predict two key modes of collective motions featuring shearing/twisting of the extracellular domains relative to the TMD, reveal subunit-specific flexibility profiles, and identify functional hotspot residues at key domain-domain interfaces. Finally, by examining the conformational transition pathway between the allosterically inhibited form and the active form, we predict a discrete sequence of domain motions, which propagate from the extracellular domains to the TMD. In summary, our results offer rich structural and dynamic information, which is consistent with the literature on structure-function relationships in NMDA receptors, and will guide in-depth studies on the activation dynamics of this important neurotransmitter receptor.

Actions of bupivacaine, a widely used local anesthetic, on NMDA receptor responses.

NMDA receptors mediate excitatory neurotransmission in brain and spinal cord and play a pivotal role in the neurological disease state of chronic pain, which is caused by central sensitization. Bupivacaine is the indicated local anesthetic in caudal, epidural, and spinal anesthesia and is widely used clinically to manage acute and chronic pain. In addition to blocking Na(+) channels, bupivacaine affects the activity of many other channels, including NMDA receptors. Importantly, bupivacaine inhibits NMDA receptor-mediated synaptic transmission in the dorsal horn of the spinal cord, an area critically involved in central sensitization. We used recombinant NMDA receptors expressed in HEK293 cells and found that increasing concentrations of bupivacaine decreased channel open probability in GluN2 subunit- and pH-independent manner by increasing the mean duration of closures and decreasing the mean duration of openings. Using kinetic modeling of one-channel currents, we attributed the observed current decrease to two main mechanisms: a voltage-dependent "foot-in-the-door" pore block and an allosteric gating effect. Further, the inhibition was state-independent because it occurred to the same degree whether the drug was applied before or after glutamate stimulation and was mediated by extracellular and intracellular inhibitory sites, via hydrophilic and hydrophobic pathways. These results predict that clinical doses of bupivacaine would decrease the peak and accelerate the decay of synaptic NMDA receptor currents during normal synaptic transmission. These quantitative predictions inform possible applications of bupivacaine as preventative and therapeutic approaches in chronic pain.

Kinetic contributions to gating by interactions unique to N-methyl-D-aspartate (NMDA) receptors.

Among glutamate-gated channels, NMDA receptors produce currents that subside with unusually slow kinetics, and this feature is essential to the physiology of central excitatory synapses. Relative to the homologous AMPA and kainate receptors, NMDA receptors have additional intersubunit contacts in the ligand binding domain that occur at both conserved and non-conserved sites. We examined GluN1/GluN2A single-channel currents with kinetic analyses and modeling to probe these class-specific intersubunit interactions for their role in glutamate binding and receptor gating. We found that substitutions that eliminate such interactions at non-conserved sites reduced stationary gating, accelerated deactivation, and imparted sensitivity to aniracetam, an AMPA receptor-selective positive modulator. Abolishing unique contacts at conserved sites also reduced stationary gating and accelerated deactivation. These results show that contacts specific to NMDA receptors, which brace the heterodimer interface within the ligand binding domain, stabilize actively gating receptor conformations and result in longer bursts and slower deactivations. They support the view that the strength of the heterodimer interface modulates gating in both NMDA and non-NMDA receptors and that unique interactions at this interface are responsible in part for basic differences between the kinetics of NMDA and non-NMDA currents at glutamatergic synapses.

Phosphorylation of Ser1166 on GluN2B by PKA is critical to synaptic NMDA receptor function and Ca2+ signaling in spines.

The NMDA-type glutamate receptor (NMDAR) is essential for synaptogenesis, synaptic plasticity, and higher cognitive function. Emerging evidence indicates that NMDAR Ca(2+) permeability is under the control of cAMP/protein kinase A (PKA) signaling. Whereas the functional impact of PKA on NMDAR-dependent Ca(2+) signaling is well established, the molecular target remains unknown. Here we identify serine residue 1166 (Ser1166) in the carboxy-terminal tail of the NMDAR subunit GluN2B to be a direct molecular and functional target of PKA phosphorylation critical to NMDAR-dependent Ca(2+) permeation and Ca(2+) signaling in spines. Activation of ß-adrenergic and D1/D5-dopamine receptors induces Ser1166 phosphorylation. Loss of this single phosphorylation site abolishes PKA-dependent potentiation of NMDAR Ca(2+) permeation, synaptic currents, and Ca(2+) rises in dendritic spines. We further show that adverse experience in the form of forced swim, but not exposure to fox urine, elicits striking phosphorylation of Ser1166 in vivo, indicating differential impact of different forms of stress. Our data identify a novel molecular and functional target of PKA essential to NMDAR-mediated Ca(2+) signaling at synapses and regulated by the emotional response to stress.

Separate intramolecular targets for protein kinase A control N-methyl-D-aspartate receptor gating and Ca2+ permeability.

Protein kinase A (PKA) enhances synaptic plasticity in the central nervous system by increasing NMDA receptor current amplitude and Ca(2+) flux in an isoform-dependent yet poorly understood manner. PKA phosphorylates multiple residues on GluN1, GluN2A, and GluN2B subunits in vivo, but the functional significance of this multiplicity is unknown. We examined gating and permeation properties of recombinant NMDA receptor isoforms and of receptors with altered C-terminal domain (CTDs) prior to and after pharmacological inhibition of PKA. We found that PKA inhibition decreased GluN1/GluN2B but not GluN1/GluN2A gating; this effect was due to slower rates for receptor activation and resensitization and was mediated exclusively by the GluN2B CTD. In contrast, PKA inhibition reduced NMDA receptor-relative Ca(2+) permeability (PCa/PNa) regardless of the GluN2 isoform and required the GluN1 CTD; this effect was due primarily to decreased unitary Ca(2+) conductance, because neither Na(+) conductance nor Ca(2+)-dependent block was altered substantially. Finally, we show that both the gating and permeation effects can be reproduced by changing the phosphorylation state of a single residue: GluN2B Ser-1166 and GluN1 Ser-897, respectively. We conclude that PKA effects on NMDA receptor gating and Ca(2+) permeability rely on distinct phosphorylation sites located on the CTD of GluN2B and GluN1 subunits. This separate control of NMDA receptor properties by PKA may account for the specific effects of PKA on plasticity during synaptic development and may lead to drugs targeted to alter NMDA receptor gating or Ca(2+) permeability.

Estimating the Ca2+ Permeability of NMDA Receptors with Whole-Cell Patch-Clamp Electrophysiology.

In the mammalian central nervous system (CNS), fast excitatory transmission relies primarily on the ionic fluxes generated by ionotropic glutamate receptors (iGluRs). Among iGluRs, NMDA receptors (NMDARs) are unique in their ability to pass large, Ca2+-rich currents. Importantly, their high Ca2+ permeability is essential for normal CNS function and is under physiological control. For this reason, the accurate measurement of NMDA receptor Ca2+ permeability represents a valuable experimental step in evaluating the mechanism by which these receptors contribute to a variety of physiological and pathological conditions. In this chapter, we provide a theoretical and practical overview of the common methods used to estimate the Ca2+ permeability of ion channels as they apply to NMDA receptors. Specifically, we describe the principles and methodology used to calculate relative permeability (PCa/PNa) and fractional permeability (Pf), along with the relationship between these two metrics. With increasing knowledge about the structural dynamics of ion channels and of the ongoing environmental fluctuations in which channels operate in vivo, the ability to quantify the Ca2+ entering cells through specific ion channels remains a tool essential to delineating the molecular mechanisms that support health and cause disease.

Estimating the Ca2+ Block of NMDA Receptors with Single-Channel Electrophysiology.

In vertebrate central neurons, NMDA receptors are glutamate- and glycine-gated ion channels that allow the passage of Na+ and Ca2+ ions into the cell when these neurotransmitters are simultaneously present. The passage of Ca2+ is critical for initiating the cellular processes underlying various forms of synaptic plasticity. These Ca2+ ions can autoregulate the NMDA receptor signal through multiple distinct mechanisms to reduce the total flux of cations. One such mechanism is the ability of Ca2+ ions to exclude the passage of Na+ ions resulting in a reduced unitary current conductance. In contrast to the well-characterized Mg2+ block, this "channel block" mechanism is voltage-independent. In this chapter, we discuss theoretical and experimental considerations for the study of channel block by Ca2+ using single-channel patch-clamp electrophysiology. We focus on two classic methodologies to quantify the dependence of unitary channel conductance on external concentrations of Ca2+ as the basis for quantifying Ca2+ block.

Inhibition of GluN2A-containing N-methyl-D-aspartate receptors by 2-naphthoic acid.

N-Methyl-D-aspartate (NMDA) receptors mediate excitatory synaptic transmission in the central nervous system and play important roles in synaptic development and plasticity, but also mediate glutamate neurotoxicity. Recently, 2-naphthoic acid (NPA) and its derivatives have been identified as allosteric, noncompetitive NMDA receptor inhibitors. The selectivity of NPA derivatives among NMDA receptor subtypes was mapped structurally to the ligand-binding domain, and was proposed to be mediated by residues on the S1 segment. To delineate the kinetic mechanism by which NPA inhibits NMDA receptor activity, we examined its effects on the NMDA receptor gating reaction. Using whole-cell patch clamping on human embryonic kidney 293 cells expressing recombinant NMDA family of glutamate receptor subunits, GluN1/GluN2A, we found that NPA has a 50% inhibitory effect at 1.9 mM. Further, from one-channel current recordings, we found that 4 mM NPA caused a 62% decrease in open probability by decreasing mean open time 2.5-fold and by increasing mean closed time 2-fold. Kinetic modeling suggested that NPA binding stabilized NMDA receptor closed states and increased the energy barriers toward open states, causing NMDA receptors to dwell longer in pre-open states along the activation pathway. The reaction mechanisms we derived provide quantitative insight into the inhibitory mechanism of NPA and help anticipate its effects on GluN1/GluN2A receptors during both physiologic and pathologic activation modalities.