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1.  Direct pharmacological monitoring of the developmental switch in NMDA receptor subunit composition using TCN 213, a GluN2A-selective, glycine-dependent antagonist 
British Journal of Pharmacology  2012;166(3):924-937.
Developmental switches in NMDA receptor subunit expression have been inferred from studies of GluN2 expression levels, changes in kinetics of glutamatergic synaptic currents and sensitivity of NMDA receptor-mediated currents to selective GluN2B antagonists. Here we use TCN 213, a novel GluN2A-selective antagonist to identify the presence of this subunit in functional NMDA receptors in developing cortical neurones.
Two-electrode voltage-clamp (TEVC) recordings were made from Xenopus laevis oocytes to determine the pharmacological activity of TCN 213 at recombinant NMDA receptors. TCN 213 antagonism was studied in cultures of primary cortical neurones, assessing the NMDA receptor dependency of NMDA-induced excitotoxicity and monitoring developmental switches in NMDA receptor subunit composition.
TCN 213 antagonism of GluN1/GluN2A NMDA receptors was dependent on glycine but independent of glutamate concentrations in external recording solutions. Antagonism by TCN 213 was surmountable and gave a Schild plot with unity slope. TCN 213 block of GluN1/GluN2B NMDA receptor-mediated currents was negligible. In cortical neurones, at a early developmental stage predominantly expressing GluN2B-containing NMDA receptors, TCN 213 failed to antagonize NMDA receptor-mediated currents or to prevent GluN2B-dependent, NMDA-induced excitoxicity. In older cultures (DIV 14) or in neurones transfected with GluN2A subunits, TCN 213 antagonized NMDA-evoked currents. Block by TCN 213 of NMDA currents inversely correlated with block by ifenprodil, a selective GluN2B antagonist.
TCN 213 selectively blocked GluN1/GluN2A over GluN1/GluN2B NMDA receptors allowing direct dissection of functional NMDA receptors and pharmacological profiling of developmental changes in native NMDA receptor subunit composition.
PMCID: PMC3417419  PMID: 22022974
NMDA; glutamate; glycine; antagonism; oocyte; two-electrode voltage clamp; electrophysiology; neurotoxicity; development
2.  TCN 201 selectively blocks GluN2A-containing NMDARs in a GluN1 co-agonist dependent but non-competitive manner 
Neuropharmacology  2012;63-540(3-7):441-449.
Antagonists that are sufficiently selective to preferentially block GluN2A-containing N-methyl-d-aspartate receptors (NMDARs) over GluN2B-containing NMDARs are few in number. In this study we describe a pharmacological characterization of 3-chloro-4-fluoro-N-[4-[[2-(phenylcarbonyl)hydrazino]carbonyl]benzyl]benzenesulphonamide (TCN 201), a sulphonamide derivative, that was recently identified from a high-throughput screen as a potential GluN2A-selective antagonist. Using two-electrode voltage-clamp (TEVC) recordings of NMDAR currents from Xenopus laevis oocytes expressing either GluN1/GluN2A or GluN1/GluN2B NMDARs we demonstrate the selective antagonism by TCN 201 of GluN2A-containing NMDARs. The degree of inhibition produced by TCN 201 is dependent on the concentration of the GluN1-site co-agonist, glycine (or d-serine), and is independent of the glutamate concentration. This GluN1 agonist-dependency is similar to that observed for a related GluN2A-selective antagonist, N-(cyclohexylmethyl)-2-[{5-[(phenylmethyl)amino]-1,3,4-thiadiazol-2-yl}thio]acetamide (TCN 213). Schild analysis of TCN 201 antagonism indicates that it acts in a non-competitive manner but its equilibrium constant at GluN1/GluN2A NMDARs indicates TCN 201 is around 30-times more potent than TCN 213. In cortical neurones TCN 201 shows only modest antagonism of NMDAR-mediated currents recorded from young (DIV 9–10) neurones where GluN2B expression predominates. In older cultures (DIV 15–18) or in cultures where GluN2A subunits have been over-expressed TCN 201 gives a strong block that is negatively correlated with the degree of block produced by the GluN2B-selective antagonist, ifenprodil. Nevertheless, while TCN 201 is a potent antagonist it must be borne in mind that its ability to block GluN2A-containing NMDARs is dependent on the GluN1-agonist concentration and is limited by its low solubility.
► TCN 201 is a potent and selective GluN1/GluN2A NMDAR antagonist. ► TCN 201 antagonism is dependent on the GluN1-agonist concentration. ► TCN 201 antagonism is independent on the GluN2-agonist concentration. ► TCN 201 blocks GluN2A-containing NMDARs in a non-competitive manner. ► TCN 201 allows pharmacological identification of native GluN2 A-containing NMDAR populations.
PMCID: PMC3384000  PMID: 22579927
TCN 201; TCN 213; NMDA receptor; GluN2A-selective; Glycine; d-serine; Schild analysis
3.  Mg2+ block properties of triheteromeric GluN1–GluN2B–GluN2D NMDA receptors on neonatal rat substantia nigra pars compacta dopaminergic neurones 
The Journal of Physiology  2014;592(10):2059-2078.
Native NMDA receptors (NMDARs) are tetrameric channels formed by two GluN1 and two GluN2 subunits. So far, seven NMDARs subunits have been identified and they can form diheteromeric or triheteromeric NMDARs (more than one type of GluN2 subunit). Extracellular Mg2+ is an important regulator of NMDARs, and particularly the voltage dependence of Mg2+ block is crucial to the roles of NMDARs in synaptic plasticity and the integration of synaptic activity with neuronal activity. Although the Mg2+ block properties of diheteromeric NMDARs are fully investigated, properties of triheteromeric NMDARs are still not clear. Our previous data suggested that dopaminergic neurones expressed triheteromeric GluN1–GluN2B–GluN2D NMDARs. Here, using NMDARs in dopaminergic neurones from postnatal day 7 (P7) rats as a model system, we characterize the voltage-dependent Mg2+ block properties of triheteromeric NMDARs. In control conditions, external Mg2+ significantly inhibits the whole cell NMDA-evoked current in a voltage-dependent manner with IC50 values of 20.9 μm, 53.3 μm and 173 μm at −90 mV, −70 mV and −50 mV, respectively. When the GluN2B-selective antagonist ifenprodil was applied, the Mg2+ sensitivity of the residual NMDA-mediated currents (which is mainly carried by GluN1–GluN2B–GluN2D NMDARs) is reduced to IC50 values of 45.9 μm (−90 mV), 104 μm (−70 mV) and 276 μm (−50 mV), suggesting that triheteromeric GluN1–GluN2B–GluN2D NMDARs have less affinity for external Mg2+ than GluN1–GluN2B receptors. In addition, fitting INMDA–V curves with a trapping Mg2+ block model shows the triheteromeric GluN1–GluN2B–GluN2D NMDARs have weaker voltage-dependent Mg2+ block (δ = 0.56) than GluN1–GluN2B NMDARs. Finally, our concentration jump and single channel recordings suggest that GluN1–GluN2B–GluN2D rather than GluN1–GluN2D NMDARs are present. These data provide information relevant to Mg2+ block characteristics of triheteromeric NMDARs and may help to better understand synaptic plasticity, which is dependent on these triheteromeric NMDARs.
PMCID: PMC4027860  PMID: 24614743
4.  Subunit-selective allosteric inhibition of glycine binding to NMDA receptors 
The Journal of Neuroscience  2012;32(18):6197-6208.
NMDA receptors are ligand-gated ion channels that mediate excitatory neurotransmission in the brain, and are involved in numerous neuropathological conditions. NMDA receptors are activated upon simultaneous binding of co-agonists glycine and glutamate to the GluN1 and GluN2 subunits, respectively. Subunit-selective modulation of NMDA receptor function by ligand binding to modulatory sites distinct from the agonist binding sites could allow pharmacological intervention with therapeutically beneficial mechanisms. Here, we show the mechanism of action for TCN-201, a new GluN1/GluN2A-selective NMDA receptor antagonist whose inhibition can be surmounted by glycine. Electrophysiological recordings from chimeric and mutant rat NMDA receptors suggest that TCN-201 binds to a novel allosteric site located at the dimer interface between the GluN1 and GluN2 agonist binding domains. Furthermore, we demonstrate that occupancy of this site by TCN-201 inhibits NMDA receptor function by reducing glycine potency. TCN-201 is therefore a negative allosteric modulator of glycine binding.
PMCID: PMC3355950  PMID: 22553026
5.  Structure-based discovery of antagonists for GluN3-containing N-methyl-D-aspartate receptors 
Neuropharmacology  2013;0:10.1016/j.neuropharm.2013.08.003.
NMDA receptors are ligand-gated ion channels that assemble into tetrameric receptor complexes composed of glycine-binding GluN1 and GluN3 subunits (GluN3A-B) and glutamate-binding GluN2 subunits (GluN2A-D). NMDA receptors can assemble as GluN1/N2 receptors and as GluN3-containing NMDA receptors, which are either glutamate/glycine-activated triheteromeric GluN1/N2/N3 receptors or glycine-activated diheteromeric GluN1/N3 receptors. The glycine-binding GluN1 and GluN3 subunits display strikingly different pharmacological selectivity profiles. However, the pharmacological characterization of GluN3-containing receptors has been hampered by the lack of methods and pharmacological tools to study GluN3 subunit pharmacology in isolation. Here, we have developed a method to study the pharmacology of GluN3 subunits in recombinant diheteromeric GluN1/N3 receptors by mutating the orthosteric ligand-binding pocket in GluN1. This method is suitable for performing compound screening and characterization of structure-activity relationship studies on GluN3 ligands. We have performed a virtual screen of the orthosteric binding site of GluN3A in the search for antagonists with selectivity for GluN3 subunits. In the subsequent pharmacological evaluation of 99 selected compounds, we identified 6-hydroxy-[1,2,5]oxadiazolo[3,4-b]pyrazin-5(4H)-one (TK80) a novel competitive antagonist with preference for the GluN3B subunit. Serendipitously, we also identified [2-hydroxy-5-((4-(pyridin-3-yl)thiazol-2-yl)amino]benzoic acid (TK13) and 4-(2,4-dichlorobenzoyl)-1H-pyrrole-2-carboxylic acid (TK30), two novel non-competitive GluN3 antagonists. These findings demonstrate that structural differences between the orthosteric binding site of GluN3 and GluN1 can be exploited to generate selective ligands.
PMCID: PMC3865070  PMID: 23973313
NMDA receptor; GluN3 subunit; antagonist; selectivity; virtual screening; Xenopus oocyte electrophysiology
6.  Synaptic GluN2A and GluN2B Containing NMDA Receptors within the Superficial Dorsal Horn Activated following Primary Afferent Stimulation 
The Journal of Neuroscience  2014;34(33):10808-10820.
NMDA receptors are important elements in pain signaling in the spinal cord dorsal horn. They are heterotetramers, typically composed of two GluN1 and two of four GluN2 subunits: GluN2A-2D. Mice lacking some of the GluN2 subunits show deficits in pain transmission yet functional synaptic localization of these receptor subtypes in the dorsal horn has not been fully resolved. In this study, we have investigated the composition of synaptic NMDA receptors expressed in monosynaptic and polysynaptic pathways from peripheral sensory fibers to lamina I neurons in rats. We focused on substance P receptor-expressing (NK1R+) projection neurons, critical for expression of hyperalgesia and allodynia. EAB-318 and (R)-CPP, GluN2A/B antagonists, blocked both monosynaptic and polysynaptic NMDA EPSCs initiated by primary afferent activation by ∼90%. Physiological measurements exploiting the voltage dependence of monosynaptic EPSCs similarly indicated dominant expression of GluN2A/B types of synaptic NMDA receptors. In addition, at synapses between C fibers and NK1R+ neurons, NMDA receptor activation initiated a secondary, depolarizing current. Ifenprodil, a GluN2B antagonist, caused modest suppression of monosynaptic NMDA EPSC amplitudes, but had a widely variable, sometimes powerful, effect on polysynaptic responses following primary afferent stimulation when inhibitory inputs were blocked to mimic neuropathic pain. We conclude that GluN2B subunits are moderately expressed at primary afferent synapses on lamina I NK1R+ neurons, but play more important roles for polysynaptic NMDA EPSCs driven by primary afferents following disinhibition, supporting the view that the analgesic effect of the GluN2B antagonist on neuropathic pain is at least in part, within the spinal cord.
PMCID: PMC4131007  PMID: 25122884
disinhibition; ifenprodil; NK1 receptor; polysynaptic; spinal cord
7.  Mechanistic and structural determinants of NMDA receptor voltage-dependent gating and slow Mg2+ unblock 
NMDA receptor (NMDAR)-mediated currents depend on membrane depolarization to relieve powerful voltage-dependent NMDAR channel block by external magnesium (Mg2+o). Mg2+o unblock from native NMDARs exhibits a fast component consistent with rapid Mg2+o unbinding kinetics, and also a slower, ms time scale component (slow Mg2+o unblock). In recombinant NMDARs, slow Mg2+o unblock is prominent in GluN1/2A (NMDAR subtype composed of GluN1 and GluN2A subunits) and GluN1/2B receptors, with slower kinetics observed for GluN1/2B receptors, but absent from GluN1/2C and GluN1/2D receptors. Slow Mg2+o unblock from GluN1/2B receptors results from inherent voltage-dependent gating, which increases channel open probability with depolarization.
Here we examine the mechanisms responsible for NMDAR subtype dependence of slow Mg2+o unblock. We demonstrate that slow Mg2+o unblock from GluN1/2A receptors, like GluN1/2B receptors, results from inherent voltage-dependent gating. Surprisingly, GluN1/2A and GluN1/2B receptors exhibit equal inherent voltage dependence; faster Mg2+o unblock from GluN1/2A receptors can be explained by voltage-independent differences in gating kinetics. To investigate the absence of slow Mg2+o unblock in GluN1/2C and GluN1/2D receptors we examined the GluN2 S/L site, a site responsible for several NMDAR subtype-dependent channel properties. Mutating the GluN2 S/L site of GluN2A subunits from serine (found in GluN2A and GluN2B subunits) to leucine (found in GluN2C and GluN2D) greatly diminished both voltage-dependent gating and slow Mg2+o unblock. Thus, the residue at the GluN2 S/L site governs expression of both slow Mg2+o unblock and inherent voltage dependence.
PMCID: PMC3629906  PMID: 23447622
8.  Deletion of the N-terminal Domain (NTD) Alters the Ethanol Inhibition of NMDA Receptors in a Subunit-Dependent Manner 
Alcoholism, clinical and experimental research  2013;37(11):10.1111/acer.12168.
Ethanol inhibition of NMDA receptors is poorly understood due in part to the organizational complexity of the receptor that provides ample locations for sites of action. Among these the N-terminal domain of NMDA receptor subunits contains binding sites for a variety of modulatory agents including zinc, protons and GluN2B selective antagonists such as ifenprodil or Ro-25–6981. Ethanol inhibition of neuronal NMDA receptors expressed in some brain areas has been reported to be occluded by the presence of ifenprodil or similar compounds suggesting that the N-terminal domain may be important in regulating the ethanol sensitivity of NMDA receptors.
Wild-type GluN1 and GluN2 subunits and those in which the coding sequence for the N-terminal domain was deleted were expressed in HEK293 cells. Whole-cell voltage-clamp recording was used to assess ethanol inhibition of wild-type and mutant receptors lacking the N-terminal domain.
As compared to wild-type GluN1/GluN2A receptors, ethanol inhibition was slightly greater in cells expressing GluN2A subunits lacking the N-terminal domain. In contrast, GluN2B N-terminal deletion mutants showed normal ethanol inhibition while those lacking the N-terminal domain in both GluN1 and GluN2B subunits had decreased ethanol inhibition as compared to wild-type receptors. N-terminal domain lacking GluN2B receptors were insensitive to ifenprodil but retained normal sensitivity to ethanol.
These findings indicate that the N-terminal domain modestly influences the ethanol sensitivity of NMDA receptors in a subunit-dependent manner. They also show that ifenprodil’s actions on GluN2B containing receptors can be dissociated from those of ethanol. These results suggest that while the N-terminal domain is not a primary site of action for ethanol on NMDA receptors, it likely affects sensitivity via actions on intrinsic channel properties.
PMCID: PMC3812356  PMID: 23905549
Ethanol; NMDA receptors; N-terminal domain; glutamate
9.  The Effects of NMDA Subunit Composition on Calcium Influx and Spike Timing-Dependent Plasticity in Striatal Medium Spiny Neurons 
PLoS Computational Biology  2012;8(4):e1002493.
Calcium through NMDA receptors (NMDARs) is necessary for the long-term potentiation (LTP) of synaptic strength; however, NMDARs differ in several properties that can influence the amount of calcium influx into the spine. These properties, such as sensitivity to magnesium block and conductance decay kinetics, change the receptor's response to spike timing dependent plasticity (STDP) protocols, and thereby shape synaptic integration and information processing. This study investigates the role of GluN2 subunit differences on spine calcium concentration during several STDP protocols in a model of a striatal medium spiny projection neuron (MSPN). The multi-compartment, multi-channel model exhibits firing frequency, spike width, and latency to first spike similar to current clamp data from mouse dorsal striatum MSPN. We find that NMDAR-mediated calcium is dependent on GluN2 subunit type, action potential timing, duration of somatic depolarization, and number of action potentials. Furthermore, the model demonstrates that in MSPNs, GluN2A and GluN2B control which STDP intervals allow for substantial calcium elevation in spines. The model predicts that blocking GluN2B subunits would modulate the range of intervals that cause long term potentiation. We confirmed this prediction experimentally, demonstrating that blocking GluN2B in the striatum, narrows the range of STDP intervals that cause long term potentiation. This ability of the GluN2 subunit to modulate the shape of the STDP curve could underlie the role that GluN2 subunits play in learning and development.
Author Summary
The striatum of the basal ganglia plays a key role in fluent motor control; pathology in this structure causes the motor symptoms of Parkinson's Disease and Huntington's Chorea. A putative cellular mechanism underlying learning of motor control is synaptic plasticity, which is an activity dependent change in synaptic strength. A known mediator of synaptic potentiation is calcium influx through the NMDA-type glutamate receptor. The NMDA receptor is sensitive to the timing of neuronal activity, allowing calcium influx only when glutamate release and a post-synaptic depolarization coincide temporally. The NMDA receptor is comprised of specific subunits that modify its sensitivity to neuronal activity and these subunits are altered in animal models of Parkinson's disease. Here we use a multi-compartmental model of a striatal neuron to investigate the effect of different NMDA subunits on calcium influx through the NMDA receptor. Simulations show that the subunit composition changes the temporal intervals that allow coincidence detection and strong calcium influx. Our experiments manipulating the dominate subunit in brain slices show that the subunit effect on calcium influx predicted by our computational model is mirrored by a change in the amount of potentiation that occurs in our experimental preparation.
PMCID: PMC3334887  PMID: 22536151
10.  GluN2B-containing NMDA receptors regulate depression-like behavior and are critical for the rapid antidepressant actions of ketamine 
eLife  null;3:e03581.
A single, low dose of the NMDA receptor antagonist ketamine produces rapid antidepressant actions in treatment-resistant depressed patients. Understanding the cellular mechanisms underlying this will lead to new therapies for treating major depression. NMDARs are heteromultimeric complexes formed through association of two GluN1 and two GluN2 subunits. We show that in vivo deletion of GluN2B, only from principal cortical neurons, mimics and occludes ketamine's actions on depression-like behavior and excitatory synaptic transmission. Furthermore, ketamine-induced increases in mTOR activation and synaptic protein synthesis were mimicked and occluded in 2BΔCtx mice. We show here that cortical GluN2B-containing NMDARs are uniquely activated by ambient glutamate to regulate levels of excitatory synaptic transmission. Together these data predict a novel cellular mechanism that explains ketamine's rapid antidepressant actions. In this model, basal glutamatergic neurotransmission sensed by cortical GluN2B-containing NMDARs regulates excitatory synaptic strength in PFC determining basal levels of depression-like behavior.
eLife digest
Depression is the leading cause of disability worldwide, with hundreds of millions of people living with the condition. The ‘gold standard’ for depression treatment involves a combination of psychotherapy and medication. Unfortunately, current antidepressant medications do not help everyone, waiting lists for psychotherapy are often long, and both normally take a number of weeks of regular treatment before they begin to have an effect. As patients are often at a high risk of suicide, it is crucial that treatments that act more quickly, and that are safe and effective, are developed.
One substance that may fulfill these requirements is a drug called ketamine. Studies have shown that depression symptoms can be reduced within hours by a single low dose of ketamine, and this effect on mood can last for more than a week. However, progress has been hindered by a lack of knowledge about what ketamine actually does inside the brain.
Neurons communicate with one another by releasing chemicals known as neurotransmitters, which transfer information by binding to receptor proteins on the surface of other neurons. Drugs such as ketamine also bind to these receptors. Ketamine works by blocking a specific receptor called the n-methyl d-aspartate (NMDA) receptor, but how this produces antidepressant effects is not fully understood.
The NMDA receptor is actually formed from a combination of individual protein subunits, including one called GluN2B. Now Miller, Yang et al. have created mice that lack receptors containing these GluN2B subunits in neurons in their neocortex, including the prefrontal cortex, a brain region involved in complex mental processes such as decision-making. This allowed Miller, Yang et al. to discover that when the neurotransmitter glutamate binds to GluN2B-containing NMDA receptors, it limits the production of certain proteins that make it easier for signals to be transmitted between neurons. Suppressing the synthesis of these proteins too much may cause depressive effects by reducing communication between the neurons in the prefrontal cortex.
Both mice lacking GluN2B-containing receptors in their cortical neurons and normal mice treated with ketamine showed a reduced amount of depressive-like behavior. This evidence supports Miller, Yang et al.'s theory that by blocking these NMDA receptors, ketamine restricts their activation. This restores normal levels of protein synthesis, improves communication between neurons in the cortex, and reduces depression.
Understanding how ketamine works to alleviate depression is an important step towards developing it into a safe and effective treatment. Further research is also required to determine the conditions that cause overactivation of the GluN2B-containing NMDA receptors.
PMCID: PMC4270067  PMID: 25340958
depression; cortex; synapse; ketamine; electrophysiology; protein synthesis; mouse; rat
11.  Tyrosine phosphorylation regulates the endocytosis and surface expression of GluN3A-containing NMDA receptors 
Selective control of receptor trafficking provides a mechanism for remodeling the receptor composition of excitatory synapses, and thus supports synaptic transmission, plasticity and development. GluN3A (formerly NR3A) is a non-conventional member of the NMDA receptor (NMDAR) subunit family which endows NMDAR channels with low calcium permeability and reduced magnesium sensitivity compared to NMDARs comprising only GluN1 and GluN2 subunits. Because of these special properties, GluN3A subunits act as a molecular brake to limit the plasticity and maturation of excitatory synapses, pointing towards GluN3A removal as a critical step in the development of neuronal circuitry. However, the molecular signals mediating GluN3A endocytic removal remain unclear. Here we define a novel endocytic motif (YWL) that is located within the cytoplasmic carboxy-terminal tail of GluN3A and mediates its binding to the clathrin adaptor AP2. Alanine mutations within the GluN3A endocytic motif inhibited clathrin-dependent internalization and led to accumulation of GluN3A-containing NMDARs at the cell surface, whereas mimicking phosphorylation of the tyrosine residue promoted internalization and reduced cell-surface expression as shown by immunocytochemical and electrophysiological approaches in recombinant systems and rat neurons in primary culture. We further demonstrate that the tyrosine residue is phosphorylated by Src family kinases, and that Src-activation limits surface GluN3A expression in neurons. Together, our results identify a new molecular signal for GluN3A internalization that couples the functional surface expression of GluN3A-containing receptors to the phosphorylation state of GluN3A subunits, and provide a molecular framework for the regulation of NMDAR subunit composition with implications for synaptic plasticity and neurodevelopment.
PMCID: PMC3682218  PMID: 23447623
12.  Structure-activity relationships for allosteric NMDA receptor inhibitors based on 2-naphthoic acid 
Neuropharmacology  2011;62(4):1730-1736.
Over-activation of N-methyl-D-aspartate (NMDA) receptors is critically involved in many neurological conditions, thus there has been considerable interest in developing NMDA receptor antagonists. We have recently identified a series of naphthoic and phenanthroic acid compounds that allosterically modulate NMDA receptors through a novel mechanism of action. In the present study, we have determined the structure-activity relationships of 18 naphthoic acid derivatives for the ability to inhibit the four GluN1/GluN2(A-D) NMDA receptor subtypes. 2-Naphthoic acid has low activity at GluN2A-containing receptors and yet lower activity at other NMDA receptors. 3-Amino addition, and especially 3-hydroxy addition, to 2-naphthoic acid increased inhibitory activity at GluN1/GluN2C and GluN1/GluN2D receptors. Further halogen and phenyl substitutions to 2-hydroxy-3-naphthoic acid leads to several relatively potent inhibitors, the most potent of which is UBP618 (1-bromo-2-hydroxy-6-phenylnaphthalene-3-carboxylic acid) with an IC50 ~ 2 μM at each of the NMDA receptor subtypes. While UBP618 is non-selective, elimination of the hydroxyl group in UBP618, as in UBP628 and UBP608, leads to an increase in GluN1/GluN2A selectivity. Of the compounds evaluated, specifically those with a 6-phenyl substitution were less able to fully inhibit GluN1/GluN2A, GluN1/GluN2B and GluN1/GluN2C responses (maximal % inhibition of 60 – 90%). Such antagonists may potentially have reduced adverse effects by not excessively blocking NMDA receptor signaling. Together, these studies reveal discrete structure-activity relationships for the allosteric antagonism of NMDA receptors that may facilitate the development of NMDA receptor modulator agents for a variety of neuropsychiatric and neurological conditions.
PMCID: PMC3269548  PMID: 22155206
13.  Pharmacological Modulation of NMDA Receptor Activity and the Advent of Negative and Positive Allosteric Modulators 
Neurochemistry international  2012;61(4):581-592.
The NMDA receptor (NMDAR) family of L-glutamate receptors are well known to have diverse roles in CNS function as well as in various neuropathological and psychiatric conditions. Until recently, the types of agents available to pharmacologically regulate NMDAR function have been quite limited in terms of mechanism of action and subtype selectivity. This has changed significantly in the past two years. The purpose of this review is to summarize the many drug classes now available for modulating NMDAR activity. Previously, this included competitive antagonists at the L-glutamate and glycine binding sites, high and low affinity channel blockers, and GluN2B-selective N-terminal domain binding site antagonists. More recently, we and others have identifed new classes of NMDAR agents that are either positive or negative allosteric modulators (PAMs and NAMs, respectively). These compounds include the pan potentiator UBP646, the GluN2A-selective potentiator/GluN2C & GluN2D inhibitor UBP512, the GluN2D-selective potentiator UBP551, the GluN2C/GluN2D-selective potentiator CIQ as well as the new NMDAR-NAMs such as the pan-inhibitor UBP618, the GluN2C/GluN2D-selective inhibitor QZN46 and the GluN2A inhibitors UBP608 and TCN201. These new agents do not bind within the L-glutamate or glycine binding sites, the ion channel pore or the N-terminal regulatory domain. Collectively, these new allosteric modulators appear to be acting at multiple novel sites on the NMDAR complex. Importantly, these agents display improved subtype-selectivity and as NMDAR PAMs and NAMs, they represent a new generation of potential NMDAR therapeutics.
PMCID: PMC3360989  PMID: 22269804
NMDA receptors; allosteric modulators; glycine; potentiators; competitive inhibitors; channel blockers; antagonists
14.  Selective inhibition of GluN2D-containing N-methyl-D-aspartate receptors prevents tissue plasminogen activator-promoted neurotoxicity both in vitro and in vivo 
Tissue plasminogen activator (tPA) exerts multiple functions in the central nervous system, depending on the partner with which it interacts. In particular, tPA acts as a positive neuromodulator of N-methyl-D-aspartate glutamatergic receptors (NMDAR). At the molecular level, it has been proposed that the pro-neurotoxicity mediated by tPA might occur through extrasynaptic NMDAR containing the GluN2D subunit. Thus, selective antagonists targeting tPA/GluN2D-containing NMDAR signaling would be of interest to prevent noxious effects of tPA.
Here, we compared three putative antagonists of GluN2D-containing NMDAR and we showed that the new compound UBP145 ((2R*,3S*)-1-(9-bromophenan-threne-3-carbonyl)piperazine-2,3-dicarboxylic acid) is far more selective for GluN2D subunits than memantine and PPDA (phenanthrene derivative (2S*, 3R*)-1-(phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid). Indeed, in vitro, in contrast to the two other compounds, UBP145 prevented NMDA toxicity only in neurons expressing GluN2D (ie, in cortical but not hippocampal neurons). Furthermore, in cultured cortical neurons, UBP145 fully prevented the pro-excitotoxic effect of tPA. In vivo, we showed that UBP145 potently prevented the noxious action of exogenous tPA on excitotoxic damages. Moreover, in a thrombotic stroke model in mice, administration of UBP145 prevented the deleterious effect of late thrombolysis by tPA.
In conclusion, tPA exerts noxious effects on neurons by acting on GluN2D-containing NMDAR and pharmacological antagonists of GluN2D-containing NMDAR could be used to prevent the ability of tPA to promote neurotoxicity.
PMCID: PMC3204249  PMID: 21975018
Tissue plasminogen activator; GluN2D; UBP145; excitotoxicity; stroke; glutamatergic neurotransmission
15.  Subunit Arrangement and Phenylethanolamine Binding in GluN1/GluN2B NMDA Receptors 
Nature  2011;475(7355):249-253.
Since it was unexpectedly discovered that the anti-hypertensive agent, ifenprodil, has neuroprotective activity through effects to N-methyl-D-aspartate (NMDA) receptors1, enormous efforts have been made to understand the mechanism of action and to develop improved therapeutic compounds based on this knowledge2–4. Neurotransmission mediated by NMDA receptors is essential for basic brain development and function5. These receptors form heteromeric ion channels and become activated upon concurrent binding of glycine and glutamate to the GluN1 and GluN2 subunits, respectively. A functional hallmark of NMDA receptors is that their ion channel activity is allosterically regulated by binding of small compounds to the amino terminal domain (ATD) in a subtype specific manner. Ifenprodil and related phenylethanolamine compounds, which specifically inhibit GluN1/GluN2B NMDA receptors6,7, have been intensely studied for their potential use in treatment of various neurological disorders and diseases including depression, Alzheimer’s disease and Parkinson’s disease2,4. Despite great enthusiasm, mechanisms underlying recognition of phenylethanolamines and the ATD-mediated allosteric inhibition remain limited due to lack of structural information. Here we report that the GluN1 and GluN2B ATDs form heterodimer and that phenylethanolamine binds at the GluN1-GluN2B subunit interface rather than within the GluN2B cleft. The crystal structure of the GluN1b/GluN2B ATD heterodimer shows a highly distinct pattern of subunit arrangement that is different from those observed in homodimeric non-NMDA receptors and reveals the molecular determinants for phenylethanolamine binding. Restriction of domain movement in the bi-lobed structures of the GluN2B ATD by engineering an inter-subunit disulfide bond dramatically decreases ifenprodil-sensitivity indicating that conformational freedom in the GluN2B ATD is essential for ifenprodil-mediated allosteric inhibition in NMDA receptors.
PMCID: PMC3171209  PMID: 21677647
16.  The Subtype of GluN2 C-terminal Domain Determines the Response to Excitotoxic Insults 
Neuron  2012;74(3):543-556.
It is currently unclear whether the GluN2 subtype influences NMDA receptor (NMDAR) excitotoxicity. We report that the toxicity of NMDAR-mediated Ca2+ influx is differentially controlled by the cytoplasmic C-terminal domains of GluN2B (CTD2B) and GluN2A (CTD2A). Studying the effects of acute expression of GluN2A/2B-based chimeric subunits with reciprocal exchanges of their CTDs revealed that CTD2B enhances NMDAR toxicity, compared to CTD2A. Furthermore, the vulnerability of forebrain neurons in vitro and in vivo to NMDAR-dependent Ca2+ influx is lowered by replacing the CTD of GluN2B with that of GluN2A by targeted exon exchange in a mouse knockin model. Mechanistically, CTD2B exhibits stronger physical/functional coupling to the PSD-95-nNOS pathway, which suppresses protective CREB activation. Dependence of NMDAR excitotoxicity on the GluN2 CTD subtype can be overcome by inducing high levels of NMDAR activity. Thus, the identity (2A versus 2B) of the GluN2 CTD controls the toxicity dose-response to episodes of NMDAR activity.
► The CTD of GluN2B promotes excitotoxicity better than that of GluN2A ► GluN2 CTD subtype differences are seen in both WT and chimeric 2A/2B subunits ► The GluN2B CTD couples to a prodeath PSD-95/nNOS-dependent CREB shut-off pathway
Martel et al. find that the two subtypes (2A versus 2B) of the GluN2 C-terminal domain differentially couple to the CREB shut-off pathway, causing distinct effects on NMDA receptor-mediated neuronal death both in vitro and in vivo.
PMCID: PMC3398391  PMID: 22578505
17.  Structural and mechanistic determinants of a novel site for non-competitive inhibition of GluN2D-containing NMDA receptors 
N-methyl-D-aspartate (NMDA) receptors are ionotropic glutamate receptors that mediate excitatory synaptic transmission and have been implicated in several neurological diseases. We have evaluated the mechanism of action of a class of novel subunit-selective NMDA receptor antagonists, typified by (E)-4-(6-methoxy-2-(3-nitrostyryl)-4-oxoquinazolin-3(4H)-yl)-benzoic acid (QNZ46). We found that QNZ46 inhibits NMDA receptor function in a non-competitive and voltage-independent manner by an unconventional mechanism that requires binding of glutamate to the GluN2 subunit, but not glycine binding to the GluN1 subunit. This dependency of antagonist association on glutamate binding to GluN2 renders these compounds nominally use-dependent, since inhibition will rely on synaptic release of glutamate. Evaluation of the structural determinants responsible for the subunit-selectivity of QNZ46 revealed that these compounds act at a new site that has not previously been described. Residues residing in the part of the agonist binding domain immediately adjacent to the transmembrane helices appear to control selectivity of QNZ46 for GluN2C- and GluN2D-containing receptors. These residues are well-positioned to sense glutamate binding to GluN2 and thus mediate glutamate-dependent actions. This new class of non-competitive antagonists could provide an opportunity for the development of pharmacological tools and therapeutic agents that target NMDA receptors at a new site and modulate function by a novel mechanism.
PMCID: PMC3063124  PMID: 21389220
patch-clamp electrophysiology; Xenopus oocytes; pharmacology; allosteric modulation
18.  Double Dissociation of Spike Timing–Dependent Potentiation and Depression by Subunit-Preferring NMDA Receptor Antagonists in Mouse Barrel Cortex 
Cerebral Cortex (New York, NY)  2009;19(12):2959-2969.
Spike timing–dependent plasticity (STDP) is a strong candidate for an N-methyl-D-aspartate (NMDA) receptor-dependent form of synaptic plasticity that could underlie the development of receptive field properties in sensory neocortices. Whilst induction of timing-dependent long-term potentiation (t-LTP) requires postsynaptic NMDA receptors, timing-dependent long-term depression (t-LTD) requires the activation of presynaptic NMDA receptors at layer 4-to-layer 2/3 synapses in barrel cortex. Here we investigated the developmental profile of t-LTD at layer 4-to-layer 2/3 synapses of mouse barrel cortex and studied their NMDA receptor subunit dependence. Timing-dependent LTD emerged in the first postnatal week, was present during the second week and disappeared in the adult, whereas t-LTP persisted in adulthood. An antagonist at GluN2C/D subunit–containing NMDA receptors blocked t-LTD but not t-LTP. Conversely, a GluN2A subunit–preferring antagonist blocked t-LTP but not t-LTD. The GluN2C/D subunit requirement for t-LTD appears to be synapse specific, as GluN2C/D antagonists did not block t-LTD at horizontal cross-columnar layer 2/3-to-layer 2/3 synapses, which was blocked by a GluN2B antagonist instead. These data demonstrate an NMDA receptor subunit-dependent double dissociation of t-LTD and t-LTP mechanisms at layer 4-to-layer 2/3 synapses, and suggest that t-LTD is mediated by distinct molecular mechanisms at different synapses on the same postsynaptic neuron.
PMCID: PMC2774397  PMID: 19363149
development; LTD; LTP; rodent; synaptic plasticity
19.  GluN2B protein deficits in the left, but not the right, hippocampus in schizophrenia 
BMC Psychiatry  2014;14(1):274.
Increasing evidence indicates that alterations to the function and subunit composition of the glutamatergic NMDA receptor are associated with the pathophysiology of schizophrenia. The GluN2B protein is a structural and functional subunit of the NMDA receptor, with a growing body of evidence indicating it plays a critical role in cognitive functions mediated by the NMDA receptor. The hippocampus plays a key role in cognitive function, with studies suggesting lateralised glutamatergic dysfunction in this region may contribute to the cognitive deficits observed in schizophrenia patients. The present study, for the first time, investigated GluN2B protein and binding density in the left and right hippocampus of 20 schizophrenia subjects compared to 20 matched controls.
The dentate gyrus of 20 schizophrenia and 20 control subjects, matched for age, post-mortem interval, and pH, was obtained from the NSW Tissue Resource Centre, Australia. Each group consisted of dentate gyrus from the left hemisphere (n = 10) and right hemisphere (n = 10). GluN2B protein density was measured via immunoblotting. GluN2B binding density was measured using the GluN2B antagonist, [3H] Ifenprodil. Analyses of covariance, covarying for demographic variables that influenced the data, were used to test for statistical significance between schizophrenia and control groups. Pearson’s correlations were used to determine the association of GluN2B protein and binding density with demographic and clinical variables, including lifetime antipsychotic drug exposure.
GluN2B protein levels were decreased by 43% in the left hemisphere of schizophrenia subjects compared to controls (p = 0.012). There was no difference in GluN2B protein levels in the right hemisphere of schizophrenia subjects compared to controls. There were no differences in [3H] Ifenprodil binding according to diagnosis or hemisphere. There were no associations between GluN2B measures and lifetime antipsychotic drug exposure.
Our findings provide the first evidence of GluN2 protein abnormalities in the hippocampus in schizophrenia, highlighting the hippocampal lateralisation in this disorder. We suggest this deficit could contribute to the cognitive dysfunctions that arise in patients. These findings provide preliminary support for the development of therapeutics that target the GluN2B subunit, as a novel therapy for schizophrenia, especially the cognitive dysfunctions.
PMCID: PMC4195867  PMID: 25292222
GluN2B; NR2B; NMDA receptor; hippocampus; dentate gyrus; human brain
20.  Involvement of the N-methyl-d-aspartate receptor GluN2D subunit in phencyclidine-induced motor impairment, gene expression, and increased Fos immunoreactivity 
Molecular Brain  2013;6:56.
Noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonists evoke a behavioral and neurobiological syndrome in experimental animals. We previously reported that phencyclidine (PCP), an NMDA receptor antagonist, increased locomotor activity in wildtype (WT) mice but not GluN2D subunit knockout mice. Thus, the aim of the present study was to determine whether the GluN2D subunit is involved in PCP-induced motor impairment.
PCP or UBP141 (a GluN2D antagonist) induced potent motor impairment in WT mice but not GluN2D KO mice. By contrast, CIQ, a GluN2C/2D potentiator, induced severe motor impairment in GluN2D KO mice but not WT mice, suggesting that the GluN2D subunit plays an essential role in the effects of PCP and UBP141, and an appropriate balance between GluN2C and GluN2D subunits might be needed for appropriate motor performance. The level of the GluN2D subunit in the mature mouse brain is very low and restricted. GluN2D subunits exist in brainstem structures, the globus pallidus, thalamus, and subthalamic nucleus. We found that the expression of the c-fos gene increased the most among PCP-dependent differentially expressed genes between WT and GluN2D KO mice, and the number of Fos-positive cells increased after PCP administration in the basal ganglia motor circuit in WT mice but not GluN2D KO mice.
These results suggest that the GluN2D subunit within the motor circuitry is a key subunit for PCP-induced motor impairment, which requires an intricate balance between GluN2C- and GluN2D-mediated excitatory outputs.
PMCID: PMC3878647  PMID: 24330819
GluN2C; GluN2D; Motor impairment; Motor loop; PCP
21.  Long-Term Ethanol and Corticosterone Co-Exposure Sensitize the Hippocampal CA1 Region Pyramidal Cells to Insult During Ethanol Withdrawal in an NMDA GluN2B Subunit-Dependent Manner 
Chronic ethanol (EtOH) exposure produces neuroadaptations in NMDA receptor function and/or abundance and alterations in hypothalamic–pituitary–adrenal (HPA) axis functioning that contribute to neuronal excitation and neurotoxicity during ethanol withdrawal (EWD). Both EtOH and corticosterone (CORT) promote synthesis of polyamines, which allosterically potentiate NMDA receptor function at the GluN2B subunit. The current studies investigated the effect of 10-day EtOH and CORT co-exposure on toxicity during EWD in rat hippocampal explants and hypothesized that alterations in function and/or density of GluN2B subunits contribute to the toxicity.
Organotypic hippocampal slice cultures were exposed to CORT (0.01–1.0 μM) during 10-day EtOH exposure (50 mM) and 1 day of EWD. EtOH-naïve cultures were exposed to CORT for 11 days. Additional cultures were exposed to a membrane impermeable form of CORT (BSA-CORT) with and without 10-day EtOH exposure and EWD. Cytotoxicity (uptake of propidium iodide) was assessed in the pyramidal cell layer of the CA1 region. Western blot analysis was employed to assess the density of GluN2B subunits following EtOH and CORT exposure.
EWD did not produce overt neurotoxicity. However, co-exposure to EtOH/EWD and CORT produced significant neurotoxicity in the CA1 region pyramidal cell layer. Ifenprodil, a GluN2B polyamine site antagonist, significantly reduced toxicity from EtOH and CORT (0.1 μM) co-exposure during EWD. However, Western blots did not reveal differences in GluN2B subunit density among groups. Exposure to BSA-CORT did not produce toxicity, suggesting that membrane-bound CORT receptors did not significantly contribute to the observed toxicity.
These data suggest that CORT and EtOH co-exposure result in increased function of polyamine-sensitive GluN2B subunits, but this toxicity does not appear dependent on the abundance of hippocampal NMDA GluN2B subunits or membrane-bound CORT receptor function.
PMCID: PMC4166417  PMID: 23889203
Alcohol Dependence; HPA Axis; Ifenprodil; NMDA Receptor
22.  Antagonist Properties of Conus parius Peptides on N-Methyl-D-Aspartate Receptors and Their Effects on CREB Signaling 
PLoS ONE  2013;8(11):e81405.
Three members of a family of small neurotoxic peptides from the venom of Conus parius, conantokins (Con) Pr1, Pr2, and Pr3, function as antagonists of N-methyl-D-aspartate receptors (NMDAR). We report structural characterizations of these synthetic peptides, and also demonstrate their antagonistic properties toward ion flow through NMDAR ion channels in primary neurons. ConPr1 and ConPr2 displayed moderate increases in α-helicity after addition of Mg2+. Native apo-ConPr3 possessed an α-helical conformation, and the helicity increased only slightly on addition of Mg2+. Additionally, these peptides diminished NMDA/Gly-mediated currents and intracellular Ca2+ (iCa2+) influx in mature rat primary hippocampal neurons. Electrophysiological data showed that these peptides displayed slower antagonistic properties toward the NMDAR than conantokins from other species of cone snails, e.g., ConT and ConG. Furthermore, to demonstrate selectivity of the C. parius-derived conantokins towards specific NMDAR subunits, cortical neurons from GluN2A-/- and GluN2B-/- mice were utilized. Robust inhibition of NMDAR-mediated stimulation in GluN2A-/--derived mouse neurons, as compared to those isolated from GluN2B-/--mouse brains, was observed, suggesting a greater selectivity of these antagonists towards the GluN2B subunit. These C. parius conantokins mildly inhibited NMDAR-induced phosphorylation of CREB at Ser133, suggesting that the peptides modulated iCa2+ entry and, thereby, activation of CREB, a transcription factor that is required for maintaining long-term synaptic activity. Our data mechanistically show that while these peptides effectively antagonize NMDAR-directed current and iCa2+ influx, receptor-coupled CREB signaling is maintained. The consequence of sustained CREB signaling is improved neuronal plasticity and survival during neuropathologies.
PMCID: PMC3832412  PMID: 24260577
23.  Distinct modes of AMPA receptor suppression at developing synapses by GluN2A and GluN2B: analysis of single-cell GluN2 subunit deletion in vivo 
Neuron  2011;71(6):1085-1101.
During development there is an activity-dependent switch in synaptic NMDA receptor subunit composition from predominantly GluN2B to GluN2A, though the precise role of this switch remains unknown. By deleting GluN2 subunits in single neurons during synaptogenesis, we find both GluN2B and GluN2A suppress AMPA receptor expression, albeit by distinct means. Similar to GluN1, GluN2B deletion increases the number of functional synapses, while GluN2A deletion increases the strength of unitary connections without affecting the number of functional synapses. We propose a model of excitatory synapse maturation in which baseline activation of GluN2B-containing receptors prevents premature synapse maturation until correlated activity allows induction of functional synapses. This activity also triggers the switch to GluN2A which dampens further potentiation. Furthermore, we analyze the subunit composition of synaptic NMDA receptors in CA1 pyramidal cells, provide electrophysiological evidence for a large population of synaptic triheteromeric receptors, and estimate the subunit-dependent open probability.
PMCID: PMC3183990  PMID: 21943605
24.  Amino Terminal Domains of the NMDA Receptor Are Organized as Local Heterodimers 
PLoS ONE  2011;6(4):e19180.
The N-methyl-D-aspartate (NMDA) receptor, an obligate heterotetrameric assembly organized as a dimer of dimers, is typically composed of two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits. Despite the crucial role that the NMDA receptor plays in the nervous system, the specific arrangement of subunits within the dimer-of-dimer assemblage is not conclusively known. Here we studied the organization of the amino terminal domain (ATD) of the rat GluN1/GluN2A and GluN1/GluN2B NMDA receptors by cysteine-directed, disulfide bond-mediated cross-linking. We found that GluN1 ATDs and GluN2 ATDs spontaneously formed disulfide bond-mediated dimers after introducing cysteines into the L1 interface of GluN2A or GluN2B ATD. The formation of dimer could be prevented by knocking out endogenous cysteines located near the L1 interface of GluN1. These results indicate that GluN1 and GluN2 ATDs form local heterodimers through the interactions in the L1-L1 interface and further demonstrate a dimer-of-heterodimer arrangement in GluN1/GluN2A and GluN1/GluN2B NMDA receptors.
PMCID: PMC3081335  PMID: 21544205
25.  Open-channel blockade is less effective on GluN3B than GluN3A subunit-containing NMDA receptors 
European Journal of Pharmacology  2012;686(1-3):22-31.
The GluN3 subunits of the N-methyl-d-aspartate (NMDA) receptor are known to reduce its Ca2+ permeability and Mg2+ sensitivity, however, little is known about their effects on other channel blockers. cRNAs for rat NMDA receptor subunits were injected into Xenopus oocytes and responses to NMDA and glycine were recorded using two electrode voltage clamp. Channel block of receptors containing GluN1-1a/2A, GluN1-1a/2A/3A or GluN1-1a/2A/3B subunits was characterised using Mg2+, memantine, MK-801, philanthotoxin-343 and methoctramine. IC50 values for Mg2+ and memantine increased when receptors contained GluN3A subunits and were further increased when they contained GluN3B, e.g. IC50s at − 75 mV for block of GluN1-1a/2A, GluN1-1a/2A/3A and GluN1-1a/2A/3B receptors respectively were 4.2, 22.4 and 40.1 μM for Mg2+, and 2.5, 7.5 and 17.5 μM for memantine. Blocking activity was found to be fully or partially restored when G or R (at the N and N + 1 sites respectively) were mutated to N in GluN3A. Thus, the changes cannot be attributed to the loss of the N or N + 1 sites alone, but rather involve both sites or residues elsewhere. Block by MK-801 and philanthotoxin-343 was also reduced by GluN3A, most strongly at − 100 mV but not at − 50 mV, and by GluN3B at all Vh. Methoctramine was the least sensitive to introduction of GluN3 subunits suggesting a minimal interaction with the N and N + 1 sites. We conclude that GluN3B-containing receptors provide increased resistance to channel block compared to GluN3A-containing receptors and this must be due to differences outside the deep pore region (N site and deeper).
Graphical abstract
PMCID: PMC3657159  PMID: 22564863
N-methyl-d-aspartate receptors; GluN3 subunit; Channel block; Magnesium; Memantine; MK-801; Philanthotoxin-343; Methoctramine

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