NMDA-type Ionotropic Glutamate Receptors Research Articles

Selective Cell-Surface Expression of Triheteromeric NMDA Receptors.

NMDA-type ionotropic glutamate receptors are critically involved in many brain functions and are implicated in a variety of brain disorders. Seven NMDA receptor subunits exist (GluN1, GluN2A-D, and GluN3A-B) that assemble into tetrameric receptor subtypes with distinct functional properties and physiological roles. The majority NMDA receptors are composed of two GluN1 and two GluN2 subunits, which can assemble into four diheteromeric receptors subtypes composed of GluN1 and one type of GluN2 subunit (e.g., GluN1/2A), and presumably also six triheteromeric receptor subtypes composed of GluN1 and two different GluN2 subunits (e.g., GluN1/2A/2B). Furthermore, the GluN1 subunit exists as eight splice variants (e.g., GluN1-1a and GluN1-1b isoforms), and two different GluN1 isoforms can co-assemble to also form triheteromeric NMDA receptors (e.g., GluN1-1a/1b/2A). Here, we describe a method to faithfully express triheteromeric NMDA receptors in heterologous expression systems by controlling the identity of two of the four subunits. This method overcomes the problem that co-expression of three different NMDA receptor subunits generates two distinct diheteromeric receptor subtypes as well as one triheteromeric receptor subtype, thereby confounding studies that require a homogenous population of triheteromeric NMDA receptors. The method has been applied to selectively express recombinant triheteromeric GluN1/2A/2B, GluN1/2A/2C, GluN1/2B/2D, GluN1-1a/GluN1-1b/2A, GluN1-1a/GluN1-1b/2B receptors with negligible co-expression of the respective diheteromeric receptor subtypes. This method therefore enables quantitative evaluation of functional and pharmacological properties of triheteromeric NMDA receptors, some of which are abundant NMDA receptor subtypes in the adult brain.

Allosteric modulation of GluN1/GluN3 NMDA receptors by GluN1-selective competitive antagonists.

NMDA-type ionotropic glutamate receptors are critical for normal brain function and are implicated in central nervous system disorders. Structure and function of NMDA receptors composed of GluN1 and GluN3 subunits are less understood compared to those composed of GluN1 and GluN2 subunits. GluN1/3 receptors display unusual activation properties in which binding of glycine to GluN1 elicits strong desensitization, while glycine binding to GluN3 alone is sufficient for activation. Here, we explore mechanisms by which GluN1-selective competitive antagonists, CGP-78608 and L-689,560, potentiate GluN1/3A and GluN1/3B receptors by preventing glycine binding to GluN1. We show that both CGP-78608 and L-689,560 prevent desensitization of GluN1/3 receptors, but CGP-78608-bound receptors display higher glycine potency and efficacy at GluN3 subunits compared to L-689,560-bound receptors. Furthermore, we demonstrate that L-689,560 is a potent antagonist of GluN1FA+TL/3A receptors, which are mutated to abolish glycine binding to GluN1, and that this inhibition is mediated by a non-competitive mechanism involving binding to the mutated GluN1 agonist binding domain (ABD) to negatively modulate glycine potency at GluN3A. Molecular dynamics simulations reveal that CGP-78608 and L-689,560 binding or mutations in the GluN1 glycine binding site promote distinct conformations of the GluN1 ABD, suggesting that the GluN1 ABD conformation influences agonist potency and efficacy at GluN3 subunits. These results uncover the mechanism that enables activation of native GluN1/3A receptors by application of glycine in the presence of CGP-78608, but not L-689,560, and demonstrate strong intra-subunit allosteric interactions in GluN1/3 receptors that may be relevant to neuronal signaling in brain function and disease.

Determination of binding affinity and efficacy for NMDA receptor agonists and GluN2A selective allosteric modulators

NMDA-type ionotropic glutamate receptors are composed of two glycine-binding GluN1 and two glutamate-binding GluN2 subunits. Simultaneous occupation of all 4 agonist binding domains allows the receptor to undergo all the conformational changes that lead to opening of the ion channel. Agonist-induced conformational changes in turn alter agonist affinity. Thus, the efficacy by which agonists induce conformational changes and channel gating influences the apparent affinity. Allosteric modulators influence the agonist-induced conformational changes and can therefore alter agonist binding affinity and efficacy.

Three-dimensional missense tolerance ratio analysis

A wealth of genetic information is available describing single-nucleotide variants in the human population that appear to be well-tolerated and in and of themselves do not confer disease. These variant data sets contain signatures about the protein structure-function relationships and provide an unbiased view of various protein functions in the context of human health. This information can be used to determine regional intolerance to variation, defined as the missense tolerance ratio (MTR), which is an indicator of stretches of the polypeptide chain that can tolerate changes without compromising protein function in a manner that impacts human health. This approach circumvents the lack of comprehensive data by averaging the data from adjacent residues on the polypeptide chain. We reasoned that many motifs in proteins consist of nonadjacent residues, but together function as a unit. We therefore developed an approach to analyze nearest neighbors in three-dimensional space as determined by crystallography rather than on the polypeptide chain. We used members of the GRIN gene family that encode subunits of NMDA-type ionotropic glutamate receptors (iGluRs) to exemplify the differences between these methods. Our method, 3DMTR, provides new information about regions of intolerance within iGluRs, allows consideration of protein–protein interfaces in multimeric proteins, and moves this important research tool from one-dimensional analysis to a structurally relevant tool. We validate the improved 3DMTR score by showing that it more accurately classifies the functional consequences of a set of newly measured and published point mutations of Grin family genes than existing methods.

Nitric Oxide Signaling Strengthens Inhibitory Synapses of Cerebellar Molecular Layer Interneurons through a GABARAP-Dependent Mechanism.

Nitric oxide (NO) is an important signaling molecule that fulfills diverse functional roles as a neurotransmitter or diffusible second messenger in the developing and adult CNS. Although the impact of NO on different behaviors such as movement, sleep, learning, and memory has been well documented, the identity of its molecular and cellular targets is still an area of ongoing investigation. Here, we identify a novel role for NO in strengthening inhibitory GABAA receptor-mediated transmission in molecular layer interneurons of the mouse cerebellum. NO levels are elevated by the activity of neuronal NO synthase (nNOS) following Ca2+ entry through extrasynaptic NMDA-type ionotropic glutamate receptors (NMDARs). NO activates protein kinase G with the subsequent production of cGMP, which prompts the stimulation of NADPH oxidase and protein kinase C (PKC). The activation of PKC promotes the selective strengthening of α3-containing GABAARs synapses through a GΑΒΑ receptor-associated protein-dependent mechanism. Given the widespread but cell type-specific expression of the NMDAR/nNOS complex in the mammalian brain, our data suggest that NMDARs may uniquely strengthen inhibitory GABAergic transmission in these cells through a novel NO-mediated pathway.SIGNIFICANCE STATEMENT Long-term changes in the efficacy of GABAergic transmission is mediated by multiple presynaptic and postsynaptic mechanisms. A prominent pathway involves crosstalk between excitatory and inhibitory synapses whereby Ca2+-entering through postsynaptic NMDARs promotes the recruitment and strengthening of GABAA receptor synapses via Ca2+/calmodulin-dependent protein kinase II. Although Ca2+ transport by NMDARs is also tightly coupled to nNOS activity and NO production, it has yet to be determined whether this pathway affects inhibitory synapses. Here, we show that activation of NMDARs trigger a NO-dependent pathway that strengthens inhibitory GABAergic synapses of cerebellar molecular layer interneurons. Given the widespread expression of NMDARs and nNOS in the mammalian brain, we speculate that NO control of GABAergic synapse efficacy may be more widespread than has been appreciated.

Genetic deletion of NMDA receptors suppresses GABAergic synaptic transmission in two distinct types of central neurons

NMDA-type ionotropic glutamate receptors (NMDARs) play an important role in the regulation of synapse development and function in the brain. Recently we have shown that NMDARs are critical for GABAergic synapse development in developing hippocampal neurons. However, it remains unclear whether NMDARs are important for establishment of GABAergic synaptic transmission in other types of neurons in the brain. Here we report that in both cortical pyramidal neurons and midbrain dopamine neurons in ventral tegmental area (VTA), genetic deletion of the GluN1 subunit, which is required for assembly of functional NMDARs, leads to a strong reduction of GABAergic synaptic transmission. These data demonstrate that NMDARs play an important role in the development of GABAergic synaptic transmission in two types of neurons with distinct developmental origins, and suggest that NMDARs are commonly involved in development of GABAergic synaptic transmission in different types of neurons in the brain.

Super-resolution microscopy reveals functional organization of dopamine transporters into cholesterol and neuronal activity-dependent nanodomains

Dopamine regulates reward, cognition, and locomotor functions. By mediating rapid reuptake of extracellular dopamine, the dopamine transporter is critical for spatiotemporal control of dopaminergic neurotransmission. Here, we use super-resolution imaging to show that the dopamine transporter is dynamically sequestrated into cholesterol-dependent nanodomains in the plasma membrane of presynaptic varicosities and neuronal projections of dopaminergic neurons. Stochastic optical reconstruction microscopy reveals irregular dopamine transporter nanodomains (∼70 nm mean diameter) that were highly sensitive to cholesterol depletion. Live photoactivated localization microscopy shows a similar dopamine transporter membrane organization in live heterologous cells. In neurons, dual-color dSTORM shows that tyrosine hydroxylase and vesicular monoamine transporter-2 are distinctively localized adjacent to, but not overlapping with, the dopamine transporter nanodomains. The molecular organization of the dopamine transporter in nanodomains is reversibly reduced by short-term activation of NMDA-type ionotropic glutamate receptors, implicating dopamine transporter nanodomain distribution as a potential mechanism to modulate dopaminergic neurotransmission in response to excitatory input.

Inhibition of the NMDA and AMPA receptor channels by antidepressants and antipsychotics

It is known that some antidepressants and antipsychotics directly inhibit NMDA-type ionotropic glutamate receptors. In this study we systematically studied action of seven drugs (Fluoxetine, Citalopram, Desipramine, Amitriptyline, Atomoxetine, Chlorpromazine, and Clozapine) on NMDA receptors and Ca2+-permeable and -impermeable AMPA receptors in rat brain neurons by whole-cell patch-clamp technique. Except for weak effect of fluoxetine, all drugs were virtually inactive against Ca2+-impermeable AMPA receptors. Fluoxetine and desipramine significantly inhibited Ca2+-permeable AMPA receptors (IC50=43±7 and 105±12µM, respectively). Desipramine, atomoxetine and chlorpromazine inhibited NMDA receptors in clinically relevant low micromolar concentrations, while citalopram had only weak effect. All tested medicines have been clustered into two groups by their action on NMDA receptors: desipramine, amitriptyline, chlorpromazine, and atomoxetine display voltage- and magnesium-dependent open channel blocking mechanism. Action of fluoxetine and clozapine was found to be voltage- and magnesium-independent. All voltage-dependent compounds could be trapped in closed NMDA receptor channels. Possible contribution of NMDA receptor inhibition by certain antidepressants and antipsychotics to their analgesic effects in neuropathic pain is discussed.

Selective Cell-Surface Expression of Triheteromeric NMDA Receptors.

The NMDA-type ionotropic glutamate receptors play pivotal roles in many brain functions, but are also involved in numerous brain disorders. Seven NMDA receptor subunits exist (GluN1, GluN2A-D, and GluN3A-B) that assemble into a diverse array of tetrameric receptor subtypes with distinct functional properties and physiological roles. Most NMDA receptors are composed of two GluN1 and two GluN2 subunits, which can assemble into four diheteromeric receptor subtypes composed of GluN1 and one type of GluN2 subunit (e.g., GluN1/2A), and presumably also six triheteromeric receptor subtypes composed of GluN1 and two different GluN2 subunits (e.g., GluN1/2A/2B). Despite accumulating evidence that a large proportion of native NMDA receptors are triheteromers, little is known about their function and pharmacology due to the lack of methods to faithfully express triheteromeric NMDA receptors in heterologous expression systems. The problem is that co-expression of GluN1 with two different GluN2 subunits generates two distinct diheteromeric receptor subtypes as well as one triheteromeric receptor subtype, thereby confounding studies on a homogenous population of triheteromeric NMDA receptors. Here, we will describe a method to selectively express recombinant triheteromeric GluN1/2A/2B receptors without interfering co-expression of diheteromeric GluN1/2A and GluN1/2B receptors. This method enables quantitative evaluation of functional and pharmacological properties of triheteromeric GluN1/2A/2B receptors, which are presumably the most abundant NMDA receptors in the adult cortex and hippocampus.

An NMDA Receptor-Dependent Mechanism Underlies Inhibitory Synapse Development.

In the mammalian brain, GABAergic synaptic transmission provides inhibitory balance to glutamatergicexcitatory drive and controls neuronal output. The molecular mechanisms underlying the development of GABAergic synapses remain largely unclear. Here, we report that NMDA-type ionotropic glutamate receptors (NMDARs) in individual immature neurons are the upstream signaling moleculesessential for GABAergic synapse development, which requires signaling via Calmodulin binding motif in the C0 domain of the NMDAR GluN1 subunit. Interestingly, in neurons lacking NMDARs, whereas GABAergic synaptic transmission is strongly reduced, the tonic inhibition mediated by extrasynaptic GABAA receptors is increased, suggesting a compensatory mechanism for the lack of synaptic inhibition. These results demonstrate a crucial role for NMDARs in specifying the development of inhibitory synapses, and suggest an important mechanism for controlling the establishment of the balance between synaptic excitation and inhibition in the developing brain.

Homocysteine-induced membrane currents, calcium responses and changes in mitochondrial potential in rat cortical neurons

Homocysteine, a sulfur-containing amino acid, exhibits neurotoxic effects and is involved in the pathogenesis of several major neurodegenerative disorders. In contrast to well studied excitoxicity of glutamate, the mechanism of homocysteine neurotoxicity is not clearly understood. By using whole-cell patch-clamp, calcium imaging (fluo-3) and measurements of mitochondrial membrane potential (rhodamine 123) we studied transmembrane currents, calcium signals and changes in mitochondrial membrane potential induced by homocysteine versus responses induced by NMDA and glutamate in cultured rat cortical neurons. L-homocysteine (50 µM) induced inward currents that could be completely blocked by the selective antagonist of NMDA receptors - AP-5. In contrast to NMDA-induced currents, homocysteine-induced currents had a smaller steady-state amplitude. Comparison of calcium responses to homocysteine, NMDA or glutamate demonstrated that in all cortical neurons homocysteine elicited short, oscillatory-type calcium responses, whereas NMDA or glutamate induced sustained increase of intracellular calcium. Analysis of mitochondrial changes demonstrated that in contrast to NMDA homocysteine did not cause a drop of mitochondrial membrane potential at the early stages of action. However, after its long-term action, as in the case of NMDA and glutamate, the changes in mitochondrial membrane potential were comparable with the full drop of respiratory chain induced by protonophore FCCP. Our data suggest that in cultured rat cortical neuron homocysteine at the first stages of action induces neurotoxic effects through activation of NMDA-type ionotropic glutamate receptors with strong calcium influx through the channels of these receptors. The long-term action of homocysteine may lead to mitochondrial disfuction and appears as a drop of mitochondrial membrane potential.

Genetic analysis of neuronal ionotropic glutamate receptor subunits

In the brain, fast, excitatory synaptic transmission occurs primarily through AMPA- and NMDA-type ionotropic glutamate receptors. These receptors are composed of subunit proteins that determine their biophysical properties and trafficking behaviour. Therefore, determining the function of these subunits and receptor subunit composition is essential for understanding the physiological properties of synaptic transmission. Here, we discuss and evaluate various genetic approaches that have been used to study AMPA and NMDA receptor subunits. These approaches have demonstrated that the GluA1 AMPA receptor subunit is required for activity-dependent trafficking and contributes to basal synaptic transmission, while the GluA2 subunit regulates Ca(2+) permeability, homeostasis and trafficking to the synapse under basal conditions. In contrast, the GluN2A and GluN2B NMDA receptor subunits regulate synaptic AMPA receptor content, both during synaptic development and plasticity. Ongoing research in this field is focusing on the molecular interactions and mechanisms that control these functions. To accomplish this, molecular replacement techniques are being used, where native subunits are replaced with receptors containing targeted mutations. In this review, we discuss a single-cell molecular replacement approach which should arguably advance our physiological understanding of ionotropic glutamate receptor subunits, but is generally applicable to study of any neuronal protein.

Memory in Caenorhabditis elegans Is Mediated by NMDA-Type Ionotropic Glutamate Receptors

Learning and memory are essential processes of both vertebrate and invertebrate nervous systems that allow animals to survive and reproduce. The neurotransmitter glutamate signals via ionotropic glutamate receptors (iGluRs) that have been linked to learning and memory formation; however, the signaling pathways that contribute to these behaviors are still not well understood. We therefore undertook a genetic and electrophysiological analysis of learning and memory in the nematode Caenorhabditis elegans. Here, we show that two genes, nmr-1 and nmr-2, are predicted to encode the subunits of an NMDA-type (NMDAR) iGluR that is necessary for memory retention in C. elegans. We cloned nmr-2, generated a deletion mutation in the gene, and showed that like nmr-1, nmr-2 is required for in vivo NMDA-gated currents. Using an associative-learning paradigm that pairs starvation with the attractant NaCl, we also showed that the memory of a learned avoidance response is dependent on NMR-1 and NMR-2 and that expression of NMDARs in a single pair of interneurons is sufficient for normal memory. Our results provide new insights into the molecular and cellular mechanisms underlying the memory of a learned event.

Protective effect of rhynchophylline and isorhynchophylline on in vitro ischemia-induced neuronal damage in the hippocampus: putative neurotransmitter receptors involved in their action

Rhynchophylline and isorhynchophylline are major tetracyclic oxindole alkaloid components of Uncaira species, which have been long used as medicinal plants. In this study we examined the protective effects of rhynchophylline and isorhynchophylline on in vitro ischemia-induced neuronal damage in the hippocampus and interaction of these alkaloids with neurotransmitter receptors in a receptor expression model of Xenopus oocytes. In vitro ischemia was induced by exposing the hippocampal slices to oxygen- and D-glucose-deprived medium over 8 min. The resultant neuronal damage was elucidated as deterioration of population spike (PS) amplitudes evoked trans-synaptically by electrical stimulation of Schäffer collaterals and recorded in the CA1 area. Rhynchophylline and isorhynchophylline, as well as the N-methyl-D-aspartate (NMDA) antagonist (±)-2-amino-5-phosphono-valeric acid (APV), the muscarinic M 1 receptor antagonist pirenzepine, and the 5-HT 2 receptor antagonist ketanserin, attenuated the in vitro ischemia-induced neuronal damage in a concentration-dependent manner. There was no difference in the extent of protection against the neuronal damage between rhynchophylline and isorhynchophylline treatment. In Xenopus oocytes expressing the rat brain receptors encoded by total RNA, both rhynchophylline and isorhynchophylline reduced muscarinic receptor- and 5-HT 2 receptor-mediated current responses in a competitive manner. Together with our previous findings that rhynchophylline and isorhynchophylline have a non-competitive antagonistic effect on the NMDA-type ionotropic glutamate receptors, the present results suggest that these alkaloids exert their protective action against ischemia-induced neuronal damage by preventing NMDA, muscarinic M 1, and 5-HT 2 receptors-mediated neurotoxicity during ischemia.

Differences in agonist and antagonist activities for two indices of metabotropic glutamate receptor‐stimulated phosphoinositide turnover

1. The abilities of the four diastereoisomers of 1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) to stimulate, and the metabotropic glutamate receptor (mGluR) antagonist (+/-)-alpha-methylcarboxyphenylglycine (MCPG) to inhibit, phosphoinositide turnover in neonatal rat cerebral cortex have been studied. Two indices of phosphoinositide cycle activity were assessed; inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) mass accumulation, and total inositol phosphate [3H]-InsPx accumulation (in the presence of Li+) in myo-[3H]-inositol prelabelled slices. 2. The diastereoisomers of ACPD stimulated each response with a rank order of potency of 1S, 3R > 1R, 3R > 1S, 3S >> 1R, 3S. The response to 1R, 3R-ACPD was largely prevented by pre-addition of the NMDA-receptor antagonist, MK-801, or omission of extracellular Ca2+, suggesting that this isomer acts indirectly on phosphoinositide responses through activation of NMDA-type ionotropic glutamate receptors. In contrast, the responses to 1S, 3R- and 1S, 3S-ACPD were unaffected by prior addition of MK-801, but were blocked by MCPG. 3. The concentration of 1S, 3R-ACPD required to half-maximally stimulate the Ins(1,4,5)P3 response (-log EC50 (M), -4.09 +/- 0.10) was significantly higher than that required to exert a similar effect on [3H]-InsPx accumulation (-log EC50 (M), -4.87 +/- 0.07; P < 0.01; n = 4). A similar marked 8-9 fold discrepancy between these two values was observed for the 1S, 3S isomer, which elicited similar maximal responses to those caused by 1S, 3R-ACPD. 4. Significant differences were also observed with respect to the ability of (+/-)-MCPG (1 mM) to cause a rightward shift in the concentration-response relationships for 1S, 3R-ACPD-stimulated Ins(1,4,5)P3 (5.59 +/- 0.24 fold shift) and [3H]-InsPx (3.04 +/- 0.34 fold shift; P < 0.01; n = 4) responses, giving rise to Kd values of 218 and 490 microM for (+/-)-MCPG antagonism of the respective responses. 5. The potency difference between the 1S, 3R-ACPD-stimulated Ins(1,4,5)P3 and [3H]-InsPx responses was reduced when experiments were performed in nominally calcium-free medium ([Ca2+]e = 2 - 5 microM) and EC50 values were almost identical when extracellular calcium was reduced further by EGTA addition ([Ca2+]e < or = 100 nM). Similarly, the Kd value for (+/-)-MCPG antagonism of the 1S, 3R-ACPD-stimulated [3H]-InsPx response decreased under [Ca2+]e-free conditions, approaching those obtained for the 1S, 3R-ACPD-stimulated Ins(1,4,5)P3 response in the presence of normal [Ca2+]e. 6. These data suggest that estimates of the activities of mGluR agonists and antagonists, derived by measuring phosphoinositide turnover, can differ significantly depending on whether Ins(1,4,5)P3 mass or [3H]-InsPx responses are measured. In particular, the possibility that the mGluR-mediated [3H]-InsPx response may not simply reflect direct receptor/G protein/phosphoinositidase C (PIC) activation, but may also be the consequence of stimulation of a facilitatory Ca2+-influx pathway is discussed.