Crystal structure of the GluR0 ligand-binding core from Nostoc punctiforme in complex with L-glutamate: structural dissection of the ligand interaction and subunit interface

GluR0 from Nostoc punctiforme (NpGluR0) is a bacterial homologue of the ionotropic glutamate receptor (iGluR). We have solved the crystal structure of the ligand-binding core of NpGluR0 in complex with l-glutamate at a resolution of 2.1 Å. The structure exhibits a noncanonical ligand interaction and two distinct subunit interfaces. The side-chain guanidium group of Arg80 forms a salt bridge with the γ-carboxyl group of bound l-glutamate: in GluR0 from Synechocystis (SGluR0) and other iGluRs, the equivalent residues are Asn or Thr, which cannot form a similar interaction. We suggest that the local positively charged environment and the steric constraint created by Arg80 mediate the selectivity of l-glutamate binding by preventing the binding of positively charged and hydrophobic amino acids. In addition, the NpGluR0 ligand-binding core forms a new subunit interface in which the two protomers are arranged differently than the known iGluR and SGluR0 dimer interfaces. The significance of there being two different dimer interfaces was investigated using analytical ultracentrifugation analysis.     

Molecular mechanism of agonist recognition by the ligand-binding core of the ionotropic glutamate receptor 4

The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) class of ionotropic glutamate receptors comprises four different subunits: iGluR1/iGluR2 and iGluR3/iGluR4 forming two subgroups. Three-dimensional structures have been reported only of the ligand-binding core of iGluR2. Here, we present two X-ray structures of a soluble construct of the R/G unedited flip splice variant of the ligand-binding core of iGluR4 (iGluR4_i(R)-S1S2) in complex with glutamate or AMPA. Subtle, but important differences are found in the ligand-binding cavity between the two AMPA receptor subgroups at position 724 (Tyr in iGluR1/iGluR2 and Phe in iGluR3/iGluR4), which in iGluR4 may lead to displacement of a water molecule and hence points to the possibility to make subgroup specific ligands.     

Crystal Structure of the GluR2 Amino-Terminal Domain Provides Insights into the Architecture and Assembly of Ionotropic Glutamate Receptors

Ionotropic glutamate receptors are functionally diverse but have a common architecture, including the 400-residue amino-terminal domain (ATD). We report a 1.8-Å resolution crystal structure of human GluR2-ATD. This dimeric structure provides a mechanism for how the ATDs can drive receptor assembly and subtype-restricted composition. Lattice contacts in a 4.1-Å resolution crystal form reveal a tetrameric (dimer-dimer) arrangement consistent with previous cellular and cryo-electron microscopic data for full-length AMPA receptors.     

Intrinsic motions in the N-terminal domain of an ionotropic glutamate receptor detected by fluorescence correlation spectroscopy

Ionotropic glutamate receptors (iGluRs) mediate excitatory neurotransmission in the central nervous system and play key roles in brain development and disease. iGluRs have two distinct extracellular domains, but the functional role of the distal N-terminal domain (NTD) is poorly understood. Crystal structures of the NTD from some non-N-methyl-d-aspartate (NMDA) iGluRs are consistent with a rigid body that facilitates receptor assembly but suggest an additional dynamic role that could modulate signaling. Here, we moved beyond spatial and temporal limitations of conventional protein single-molecule spectroscopy by employing correlation analysis of extrinsic oxazine fluorescence fluctuations. We observed nanosecond (ns)-to-microsecond (μs) motions of loop segments and helices within a region of an AMPA-type iGluR NTD, which has been identified previously to be structurally variable. Our data reveal that the AMPA receptor NTD undergoes rapid conformational fluctuations, suggesting an inherent allosteric capacity for this domain in addition to its established assembly function.     

Arabidopsis thaliana glutamate receptor ion channel function demonstrated by ion pore transplantation

Ionotropic glutamate receptors (iGluRs) are ligand-gated cation channels that mediate fast excitatory neurotransmission in the mammalian central nervous system. In the model plant Arabidopsis thaliana, a large family of 20 genes encoding proteins that share similarities with animal iGluRs in sequence and predicted secondary structure has been discovered. Members of this family, termed AtGLRs (A. thaliana glutamate receptors), have been implicated in root development, ion transport, and several metabolic and signalling pathways. However, there is still no direct proof of ligand-gated ion channel function of any AtGLR subunit. We used a domain transplantation technique to directly test whether the putative ion pore domains of AtGLRs can conduct ions. To this end, we transplanted the ion pore domains of 17 AtGLR subunits into rat α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (GluR1) and kainate (GluR6) receptor subunits and tested the resulting chimaeras for ion channel function in the Xenopus oocyte expression system. We show that AtGLR1.1 and AtGLR1.4 have functional Na⁺-, K⁺-, and Ca²⁺-permeable ion pore domains. The properties of currents through the AtGLR1.1 ion pore match those of glutamate-activated currents, depolarisations, and glutamate-triggered Ca²⁺ influxes observed in plant cells. We conclude that some AtGLRs have functional non-selective cation pores.     

Differential effects of chronic hyperammonemia on modulation of the glutamate-nitric oxide-cGMP pathway by metabotropic glutamate receptor 5 and low and high affinity AMPA receptors in cerebellum in vivo

Previous studies show that chronic hyperammonemia impairs learning ability of rats by impairing the glutamate-nitric oxide (NO)-cyclic guanosine mono-phosphate (cGMP) pathway in cerebellum. Three types of glutamate receptors cooperate in modulating the NO-cGMP pathway: metabotropic glutamate receptor 5 (mGluR5), (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-d-aspartic acid (NMDA) receptors. The aim of this work was to assess whether hyperammonemia alters the modulation of this pathway by mGluR5 and AMPA receptors in cerebellum in vivo. The results support that in control rats: (1) low AMPA concentrations (0.1mM) activate nearly completely Ca(2+)-permeable (glutamate receptor subunit 2 (GluR2)-lacking) AMPA receptors and the NO-cGMP pathway; (2) higher AMPA concentrations (0.3 mM) also activate Ca(2+)-impermeable (GluR2-containing) AMPA receptors, leading to activation of NMDA receptors and of NO-cGMP pathway. Moreover, the data support that chronic hyperammonemia: (1) reduces glutamate release and activation of the glutamate-NO-cGMP pathway by activation of mGluR5; (2) strongly reduces the direct activation by AMPA receptors of the NO-cGMP pathway, likely due to reduced entry of Ca(2+) through GluR2-lacking, high affinity AMPA receptors; (3) strongly increases the indirect activation of the NO-cGMP pathway by high affinity AMPA receptors, likely due to increased entry of Na(+) through GluR2-lacking AMPA receptors and NMDA receptors activation; (4) reduces the indirect activation of the NO-cGMP pathway by low affinity AMPA receptors, likely due to reduced activation of NMDA receptors.     

Membrane depolarization regulates AMPA receptor subunit expression in cerebellar granule cells in culture

The physiological responses of AMPA receptors can be modulated through the differential expression of their subunits and by modifying their number at the cell surface. Here we have studied the expression of AMPA receptor subunits (GluR1-4) mRNAs in cerebellar granule cells grown in depolarizing (25 mM K⁺) medium, and we have evaluated the effect of decreasing the [K⁺] in the culture medium for 24 h on both GluR1-4 expression (both mRNA and protein) and their presence at the plasma membrane. The expression of the four AMPAR subunits increases as the [K⁺] decreases, although the increase in GluR2 and GluR3 was only observed in the cell soma but not in the dendrites. Calcium entry through L-type calcium channel and CaMKIV activation are responsible for the reduction in the expression of AMPA receptor subunits in cells cultured in depolarizing conditions. Indeed, prolonged reduction of extracellular [K⁺] or blockage of L-type calcium channels enhanced both the surface insertion of the four AMPAR subunits and the AMPA response measured through intracellular calcium increase. These findings reveal a balanced increase in functional AMPA receptors at the surface of cells that can trigger strong increases in calcium in response to the persistent reduction of calcium entry.     

Nox4-dependent H2O2 production contributes to chronic glutamate toxicity in primary cortical neurons

Reactive oxygen species (ROS) can trigger neuronal cell death and has been implicated in a variety of neurodegenerative diseases as well as brain ischemia. Here, we demonstrate that chronic (but not acute) glutamate toxicity in primary cortical neuronal cultures is associated with hydrogen peroxide (H₂O₂) accumulation in the culture medium and that neurotoxicity can be eliminated by external catalase treatment. Neuronal cultures in Ca²⁺-free medium or treated with BAPTA showed reduced glutamate-induced H₂O₂ generation, indicating that H₂O₂ generation is Ca²⁺-dependent. Pharmacological and genetic approaches revealed that NADPH oxidase plays a role in glutamate-induced H₂O₂ generation and that activation of NMDA and AMPA receptors is involved in this H₂O₂ generation. The Nox4 siRNA reduced NMDA-induced H₂O₂ production by 54% and cytotoxicity in parallel, suggesting that Nox4-containing NADPH oxidase functions NMDA receptor-mediated H₂O₂ production resulting in neurotoxicity. These findings suggest that the modulation of NADPH oxidase can be used as a new therapeutic strategy for glutamate-induced neuronal diseases.     

Exposure to novel environment is characterized by an interaction of D1/NMDA receptors underlined by phosphorylation of the NMDA and AMPA receptor subunits and activation of ERK1/2 signaling, leading to epigenetic changes and gene expression in rat hippocampus

Interactions between dopamine and glutamate receptors are essential for prefrontal cortical (PFC) and hippocampal cognitive functions. The hippocampus has been identified as a detector of a novel stimulus, where an association between incoming information and stored memories takes place. Further to our previous results which showed a strong synergistic interaction of dopamine D1 and glutamate NMDA receptors, the present study is going to investigate the functional status of that interaction in rats, following their exposure to a novel environment. Our results showed that the “spatial” novelty induced in rat hippocampus and PFC (a) a significant increase in phosphorylation of NMDA and AMPA receptor subunits, as well as a robust phosphorylation/activation of ERK1/2 signaling, which are both dependent on the concomitant stimulation of D1/NMDA receptors and are both abolished by habituation procedure, (b) chromatin remodeling events (phosphorylation-acetylation of histone H3) and (c) an increase in the immediate early genes (IEGs) c-Fos and zif-268 expression in the CA1 region of hippocampus, which is dependent on the co-activation of D1/NMDA and acetylcholine muscarinic receptors. In conclusion, our results clearly show that a strong synergistic interaction of D1/NMDA receptor is required for the novelty-induced phosphorylation of NMDA and AMPA receptor subunits and for the robust activation of ERK1/2 signaling, leading to chromatin remodeling events and the expression of the IEGs c-Fos and zif-268, which are involved in the regulation of synaptic plasticity and memory consolidation.     

The combination of glutamate receptor antagonist MK-801 with tamoxifen and its active metabolites potentiates their antiproliferative activity in mouse melanoma K1735-M2 cells

Recent reports suggest that N-methyl-d-aspartate receptor (NMDAR) blockade by MK-801 decreases tumor growth. Thus, we investigated whether other ionotropic glutamate receptor (iGluR) antagonists were also able to modulate the proliferation of melanoma cells. On the other hand, the antiestrogen tamoxifen (TAM) decreases the proliferation of melanoma cells, and is included in combined therapies for melanoma. As the efficacy of TAM is limited by its metabolism, we investigated the effects of the NMDAR antagonist MK-801 in combination with TAM and its active metabolites, 4-hydroxytamoxifen (OHTAM) and endoxifen (EDX). The NMDAR blockers MK-801 and memantine decreased mouse melanoma K1735-M2 cell proliferation. In contrast, the NMDAR competitive antagonist APV and the AMPA and kainate receptor antagonist NBQX did not affect cell proliferation, suggesting that among the iGluR antagonists only the NMDAR channel blockers inhibit melanoma cell proliferation. The combination of antiestrogens with MK-801 potentiated their individual effects on cell biomass due to diminished cell proliferation, since it decreased the cell number and DNA synthesis without increasing cell death. Importantly, TAM metabolites combined with MK-801 promoted cell cycle arrest in G1. Therefore, the data obtained suggest that the activity of MK-801 and antiestrogens in K1735-M2 cells is greatly enhanced when used in combination.     

Modulation of synaptic plasticity by brain estrogen in the hippocampus

The hippocampus is a center for learning and memory as well as a target of Alzheimer's disease in aged humans. Synaptic modulation by estrogen is essential to understand the molecular mechanisms of estrogen replacement therapy. Because the local synthesis of estrogen occurs in the hippocampus of both sexes, in addition to the estrogen supply from the gonads, its functions are attracting much attention.     

Crystal structure of the kainate receptor GluR5 ligand-binding core in complex with (S)-glutamate

The X-ray structure of the ligand-binding core of the kainate receptor GluR5 (GluR5-S1S2) in complex with (S)-glutamate was determined to 1.95 A resolution. The overall GluR5-S1S2 structure comprises two domains and is similar to the related AMPA receptor GluR2-S1S2J. (S)-glutamate binds as in GluR2-S1S2J. Distinct features are observed for Ser741, which stabilizes a highly coordinated network of water molecules and forms an interdomain bridge. The GluR5 complex exhibits a high degree of domain closure (26 degrees) relative to apo GluR2-S1S2J. In addition, GluR5-S1S2 forms a novel dimer interface with a different arrangement of the two protomers compared to GluR2-S1S2J.     

Glutamate-induced deregulation of calcium homeostasis and mitochondrial dysfunction in mammalian central neurones

Delayed neuronal death following prolonged (10-15 min) stimulation of Glu receptors is known to depend on sustained elevation of cytosolic Ca(2+) concentration ([Ca(2+)](i)) which may persist far beyond the termination of Glu exposure. Mitochondrial depolarization (MD) plays a central role in this Ca(2+) deregulation: it inhibits the uniporter-mediated Ca(2+) uptake and reverses ATP synthetase which enhances greatly ATP consumption during Glu exposure. MD-induced inhibition of Ca(2+) uptake in the face of continued Ca(2+) influx through Glu-activated channels leads to a secondary increase of [Ca(2+)](i) which, in its turn, enhances MD and thus [Ca(2+)](i). Antioxidants fail to suppress this pathological regenerative process which indicates that reactive oxygen species are not involved in its development. In mature nerve cells (>11 DIV), the post-glutamate [Ca(2+)](i) plateau associated with profound MD usually appears after 10-15 min Glu (100 microM) exposure. In contrast, in young cells (<9 DIV) delayed Ca(2+) deregulation (DCD) occurs only after 30-60 min Glu exposure. This difference is apparently determined by a dramatic increase in the susceptibility of mitochondia to Ca(2+) overload during nerve cells maturation. The exact mechanisms of Glu-induced profound MD and its coupling with the impairment of Ca(2+) extrusion following toxic Glu challenge is not clarified yet. Their elucidation demands a study of dynamic changes in local concentrations of ATP, Ca(2+), H(+), Na(+) and protein kinase C using novel methodological approaches.     

Oxidative stress induced by P2X7 receptor stimulation in murine macrophages is mediated by c-Src/Pyk2 and ERK1/2

Background

Activation of ATP-gated P2X7 receptors (P2X7R) in macrophages leads to production of reactive oxygen species (ROS) by a mechanism that is partially characterized. Here we used J774 cells to identify the signaling cascade that couples ROS production to receptor stimulation.

Methods

J774 cells and mP2X7-transfected HEK293 cells were stimulated with Bz-ATP in the presence and absence of extracellular calcium. Protein inhibitors were used to evaluate the physiological role of various kinases in ROS production. In addition, phospho-antibodies against ERK1/2 and Pyk2 were used to determine activation of these two kinases.

Results

ROS generation in either J774 or HEK293 cells (expressing P2X7, NOX2, Rac1, p47phox and p67phox) was strictly dependent on calcium entry via P2X7R. Stimulation of P2X7R activated Pyk2 but not calmodulin. Inhibitors of MEK1/2 and c-Src abolished ERK1/2 activation and ROS production but inhibitors of PI3K and p38 MAPK had no effect on ROS generation. PKC inhibitors abolished ERK1/2 activation but barely reduced the amount of ROS produced by Bz-ATP. In agreement, the amount of ROS produced by PMA was about half of that produced by Bz-ATP.

Conclusions

Purinergic stimulation resulted in calcium entry via P2X7R and subsequent activation of the PKC/c-Src/Pyk2/ERK1/2 pathway to produce ROS. This signaling mechanism did not require PI3K, p38 MAPK or calmodulin.

General significance

ROS is generated in order to kill invading pathogens, thus elucidating the mechanism of ROS production in macrophages and other immune cells allow us to understand how our body copes with microbial infections.     

The astrocyte excitability brief: from receptors to gliotransmission

Astrocytes do not merely serve as the supporting cast and scenery against which starring roles would be played by neurons. Rather, these glial cells are intimately involved in many of the brain's functions by responding to neuronal activity and modulating it. Such interplay between two principle neural cells, neurons and astrocytes, is evidenced in bi-directional glutamatergic astrocyte-neuron signaling. A key feature in this signaling pathway is astrocytic excitability based on variations of cytosolic Ca(2+). It enables astrocytes, through the activation of their glutamatergic receptors, to respond to the same signal used by nearby neurons in synaptic transmission. Furthermore, increases in cytosolic Ca(2+) in astrocytes can subsequently lead to Ca(2+)-dependent exocytotic secretion of gliotransmitter glutamate that in turn can signal to adjacent neurons. Astrocytic secretory machinery includes an assortment of exocytotic proteins which governs a merger of secretory vesicles to the plasma membrane. A cumulative knowledge on astrocytic excitability will aid better understanding of operating procedures in the brain in health and disease.     

Pharmacological modulation of NMDA receptor activity and the advent of negative and positive allosteric modulators

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 identified 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 and 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.     

The role of protein phosphatase-1 in the modulation of synaptic and structural plasticity

Synaptic plasticity is a phenomenon contributing to changes in the efficacy of neuronal transmission. These changes are widely believed to be a major cellular basis for learning and memory. Protein phosphorylation is a key biochemical process involved in synaptic plasticity that operates through a tight balance between the action of protein kinases and protein phosphatases (PPs). Although the majority of research in this field has concentrated primarily on protein kinases, the significant role of PPs is becoming increasingly apparent. This review examines one such phosphatase, PP1, and highlights recent advances in the understanding of its intervention in synaptic and structural plasticity and the mechanisms of learning and memory.