Structure-based functional design of chemical ligands for AMPA-subtype glutamate receptors

Ionotropic glutamate receptors (GluRs) function as an excitatory transmitter system in the human brain, particularly in learning and memory. Development of small molecules that are capable of selectively potentiating the ion channel activity of AMPA-subtype GluRs holds promise for potential new treatment of neurodegenerative diseases such as Alzheimer’s. In working towards this goal, we obtained main-chain nuclear magnetic resonance (NMR) assignments of the extracellular ligand-binding domain of GluR2 that enables us to investigate receptor-ligand interactions in physiological conditions at atomic detail. With NMR structure-based methods, chemical compounds that can selectively modulate the ion chancel activity of GluR2 alone or synergistically with glutamate or kainate were identified. Our NMR structural analysis of GluR2 S1S2 further reveals that the regions of the receptor dimer interface exhibit distinct conformational dynamics, which we hypothesize to be linked to receptor functions in interactions with an agonist or antagonist. This coupling of ligand binding to receptor dimerization, gating, and desensitization may serve as an in vitro biophysical parameter to evaluate potential biological effects of the chemical ligands being developed here.     

Structure-based rational design of chemical ligands for AMPA-subtype glutamate receptors

Ionotropic glutamate receptors (GluRs) function as an excitatory transmitter system in human brain, particularly in learning and memory. Development of small-molecule chemical ligands that selectively potentiate the ion channel activity of AMPA-subtype GluRs would hold promise for treating an exceptionally wide range of disorders including neurodegenerative diseases such as Alzheimer’s. Toward this goal, we have obtained nearly complete main-chain NMR resonance assignments of the extracellular ligand-binding domain of GluR2, which enables us to investigate receptor-ligand interactions in physiological conditions at atomic detail. With our NMR structure-based methods, we have discovered several chemical compounds that bind specifically to the GluR2 protein. Notably, our initial lead compounds interact with GluR2 at sites near the interface of receptor dimerization, which plays a pivotal role in controlling receptor gating and desensitization. Our NMR structural analysis further reveals that the regions of GluR2 at the dimer interface exhibit distinct conformational dynamics as compared to the rest of the protein, which we hypothesize to be linked to the mechanisms by which the protein interacts with its ligand, either an agonist or antagonist. This newly discovered relationship of possibly coupling of ligand binding to receptor dimerization, gating and desensitization, which is being further validated, could serve as an excellent in vitro biophysical parameter to evaluate the potential biological effects of the chemical ligands being developed and optimized in our study.     

Antibodies to glutamate and aspartate recognize non-endogenous ligands for excitatory amino acid receptors

Antisera raised against glutaraldehyde conjugates of glutamate (Glu) and aspartate (Asp) with hemocyanin proved highly specific for their respective unconjugated amino acid haptens when tested in immunocytochemical blocking experiments on sections of the rat spinal cord. In addition, immunocytochemical staining by the Glu antiserum was effectively blocked by quisqualate but not by kainate or N-methyl-D-aspartate (NMDA); staining with the Asp antiserum was effectively blocked by kainate, to a lesser extent by quisqualate, and was not affected by NMDA. These results may be explained by assuming that the specific binding regions of the antibodies tested share certain recognition characteristics with endogenous binding sites or receptors for excitatory amino acids and their agonists.     

Retinal precursor cells express functional ionotropic glutamate and GABA receptors

R28 retinal progenitor cells offer the potential to replace damaged neurons; however, their ability to differentiate into the appropriate phenotype may depend on whether they express glutamatergic and GABAergic receptors. Whole-cell recordings and immunocytochemistry were used to identify glutamatergic and GABAergic receptors on proliferating R28 cells. R28 cells lacked voltage-gated channels; however, they produced inward currents when non-NMDA, NMDA, GABAa and GABAb receptor agonists were perfused onto the cells. R28 cells were immunoreactive to GluR1, 2 and 3, NMDA and GABAa receptors consistent with electrophysiological results. These results indicate that R28 progenitor cells express glutamatergic and GABAergic receptors capable of influencing their fate and function when grafted into retina or elsewhere in the nervous system.     

Early functional glutamate receptors in acutely dissociated embryonic raphe cells.

We report for the first time, modulation of cytosolic calcium in response to glutamate and specific glutamate receptor agonists in early embryonic rat brain cells (raphe cells taken at gestation days 13 or 14). Metabotropic as well as ionotropic agonists were effective. Cells responding to kainic acid were particularly prominent in caudal raphe. We used very short post-plating delays (2 to 6 h); it may therefore be assumed that functional receptors already exist in the intact embryonic brain by gestation day 13. Since many developmental processes are influenced by cytosolic calcium modulation, glutamate receptors may play a key role in brain development, well before the extensively studied postnatal peak in receptor density.     

Mature hippocampal astrocytes exhibit functional metabotropic and ionotropic glutamate receptors in situ

Astrocytes closely contact neurons where they respond to neuronally released glutamate in immature brain slices. In previous studies, neither metabotropic nor ionotropic glutamate receptor-mediated responses were detected by imaging Ca2+ in astrocytes from mature (P21-P42) animals, suggesting astrocyte glutamate receptors only contribute to hippocampus physiology during development. In contrast to Ca2+ imaging, published electrophysiological experiments suggest P30-P35 astrocytes have alpha-amino-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. For this study, we imaged astrocytes in P31-P38 hippocampal slices to determine if metabotropic and ionotropic glutamate receptor activation elevates intracellular calcium in mature astrocytes. Drugs were perfused while [Ca2+]i was monitored (confocal imaging) in cells loaded with Calcium Green 1-AM. Imaged cells were subsequently identified as astrocytes by GFAP/S-100 immunostaining. Astrocytic Ca2+ increased after glutamate application in the presence of a glutamate uptake inhibitor. An agonist at group I/II metabotropic glutamate receptors, (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD), elicited Ca2+ increases as did group I agonist 3,5-dihydroxyphenylglycine (DHPG), suggesting that mature astrocytes respond to glutamate via metabotropic glutamate receptors. AMPA also elicited Ca2+ elevations that were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and occurred after treatment with omega-conotoxin MVIIC to block neurotransmitter release. These results demonstrate that astrocytes in mature hippocampus have functional ionotropic and metabotropic glutamate receptors that regulate astrocytic calcium levels. Glutamatergic regulation of astrocytic [Ca2+]i may be involved in synapse modeling, long-term potentiation, excitotoxicity and other events dependent on glutamatergic transmission in adult hippocampus.     

Cajal-Retzius cells in early postnatal mouse cortex selectively express functional metabotropic glutamate receptors

Glutamate receptors have been linked to the regulation of several developmental events in the CNS. By using cortical slices of early postnatal mice, we show that in layer I cells, glutamate produces intracellular calcium ([Ca(2+)](i)) elevations mediated by ionotropic and metabotropic glutamate receptors (mGluRs). The contribution of mGluRs to these responses was demonstrated by application of tACPD, an agonist to groups I and II mGluRs, which evoked [Ca(2+)](i) increases that could be reversibly blocked by MCPG, an antagonist to groups I and II mGluRs. In the absence of extracellular Ca(2+), repetitive applications of tACPD or quisqualate, an agonist to group I mGluRs, elicited decreasing [Ca(2+)](i) responses that were restored by refilling a thapsigargin-sensitive Ca(2+) store. The use of specific group I mGluR agonists CHPG and DHPG indicated that the functional mGluR in layer I was of the mGluR1 subtype. Subtype specific antibodies confirmed the presence of mGlur1 alpha, but not mGluR5, in Cajal-Retzius (Reelin-immunoreactive) neurons.     

Expression and functional properties of group I metabotropic glutamate receptors in bovine chromaffin cells

We demonstrate the presence and functional properties of Group I metabotropic glutamate receptors (mGluRs) expressed in chromaffin cells. Immunocytochemical techniques revealed that two mGluR subtypes (mGluR1alpha and mGluR5) are expressed in chromaffin cells, located in both the cytoplasmic membrane and the cytosol surrounding the nucleus. These mGluRs are functionally active on catecholamine (CA) secretion in chromaffin cells because both (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD) and the specific agonist of Group I mGluRs, (S)-3,5-dihydroxyphenylglycine (DHPG), were able to stimulate the release of CAs (adrenaline and noradrenaline) in a dose-response manner. These effects were specifically reversed by L-(+)-2-amino-3-phosphonopropionic acid (L-AP3), a selective antagonist of the Group I metabotropic glutamate receptors. t-ACPD induced an increase in CA secretion in both the presence and absence of extracellular calcium, the former effect being accompanied by cell membrane depolarization. Noradrenaline (NA) release was higher in the presence of extracellular calcium than in its absence, whereas adrenaline release was of the same order under both conditions. These results indicate that different subtypes of Group I mGluRs are present in noradrenergic and adrenergic cells. Fluorescence imaging techniques in single cells showed different t-ACPD-induced increases in intracellular calcium in different chromaffin cells: in chromaffin cells, 67% expressed functional metabotropic glutamate receptors and with nicotinic receptors, whereas the remaining 33% expressed only nicotinic receptors. In the absence of external calcium, only about 25% of cells responded to t-ACPD-increased intracellular calcium by increasing inositol 1,4,5-trisphosphate (IP(3)) concentration and subsequent calcium mobilization from intracellular stores, whereas the remaining 75% increased intracellular calcium by promoting Ca(2+) influx from the extracellular medium through L- and N- but not P/Q voltage-dependent calcium channels.     

Ammonium acetate inhibits ionotropic receptors and differentially affects metabotropic receptors for glutamate

Summary The effects of ammonium salts in concentration similar to those found in plasma in course of hepatic encephalopathy (2–4 mM) were studied in brain slices in order to clarify how glutamate synapses are affected by this pathological situation. Electrophysiological (mice cortical wedge preparations) and biochemical techniques (inositol phosphates and cyclic AMP measurements) were used so that the function of both the ionotropic and metabotropic glutamate receptors was evaluated. Ammonium acetate (2–4 mM), but not sodium acetate reduced the degree of depolarization of cortical wedges induced by different concentrations of N-methyl-D-aspartic acid (NMDA) or (S)-alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). This reduction was non-competitive in nature and did not reverse during the experimental period (90 min). In a similar manner, ammonium acetate reduced the formation of inositol phosphates induced by (1S,3R)-1-amynocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) (100μM), the prototype agonist of metabotropic glutamate receptors. When the metabotropic glutamate receptors negatively linked to the forskolin-stimulated cyclic AMP formation were evaluated, ammonium acetate significantly hampered forskolin effects and its actions were additive with those of the metabotropic glutamate receptor agonist 1S,3R-ACPD. In conclusion, our results suggest that toxic concentrations of ammonium impair the function of glutamate receptors of NMDA and AMPA type and of the metabotropic glutamate receptors linked to inositol phosphate formation while they functionally potentiate the action of glutamate agonists on the receptors negatively linked to adenylyl cyclase.     

Transmembrane topology of the glutamate receptors

The glutamate receptor subunits were first thought to cross the cell membrane four times in a manner analogous to the neuronal nicotinic acetylcholine, GABA_A, and glycine receptors. This model led the field for nearly five years, although it was frequently in conflict with the data. Recently, comparisons with bacterial proteins, epitope tagging experiments, and the construction of chimeras has produced a new model of glutamate receptor topology that is novel and quite unlike any of the other receptors.     

Reducing conditions differentially affect the functional and structural properties of group-I and -II metabotropic glutamate receptors

We have examined the influence of reducing conditions on the activity of group-I or -II metabotropic glutamate receptors. In cultured cerebellar granule cells or in hippocampal slices, the reducing agent dithiothreitol (DTT) inhibited the stimulation of polyphosphoinositide (PPI) hydrolysis elicited by group-I mGlu receptor agonists without affecting responses to norepinephrine or carbamylcholine. Similarly, DTT reduced the increase in intracellular free Ca(2+) induced by glutamate in HEK-293 cells expressing mGlu5 receptors. In adult hippocampal slices, the selective group-II mGlu receptor agonist, (2S,1'R,2'R,3'R)-2-(2, 3-dicarboxycyclopropyl)glycine (DCG-IV) had no effect per se on PPI hydrolysis, but potentiated the response to quisqualate. Although DTT substantially attenuated the action of quisqualate, it did not affect the potentiation by DCG-IV, suggesting that group-II mGlu receptors are resistant to extracellular reduction. Accordingly, DTT did not affect the inhibition of forskolin-stimulated cAMP formation induced by maximally effective concentrations of group-II mGlu receptor agonists in hippocampal slices or in CHO cells expressing mGlu2 receptors. At structural level, DTT differentially affected the aggregation state of mGlu1a, -2/3 or -5 receptors. In immunoblots performed under non-reducing conditions, mGlu1a, -2/3 or -5 antibodies labeled exclusively a high-molecular weight band, corresponding to receptor dimers. Under reducing conditions, mGlu1a or -5 receptors were detected as monomers, whereas a large proportion of mGlu2/3 receptors was still present in a dimeric form. We conclude that reducing conditions differentially influence the aggregation state of group-I and -II mGlu receptors and suggest that dimerization affects the functional activity of native mGlu receptors.     

Bilirubin does not modulate ionotropic glutamate receptors or glutamate transporters

Bilirubin, a product of haemoglobin metabolism, has been suggested to damage neurons by increasing activation of N-methyl-D-aspartate (NMDA) receptors when it reaches high levels in the blood [15,19], as occurs in neonatal jaundice [7]. Bilirubin is also generated in the brain following synthesis of the messenger carbon monoxide (CO) by haem oxygenase, and haem oxygenase is upregulated in Alzheimer's disease [23]. We examined the effect of bilirubin on currents generated by NMDA and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors in hippocampal pyramidal cells, and on glutamate transporter currents in retinal glial cells. Bilirubin did not modulate either receptor-gated currents or transporter currents. These data show the negative, but important result that bilirubin does not induce neuronal death by acting directly on NMDA or AMPA receptors, nor indirectly by blocking glutamate uptake and raising the extracellular concentration of glutamate.     

Ionotropic glutamate receptors & CNS disorders

Disorders of the central nervous system (CNS) are complex disease states that represent a major challenge for modern medicine. Although aetilogy is often unknown, it is established that multiple factors such as defects in genetics and/or epigenetics, the environment as well as imbalance in neurotransmitter receptor systems are all at play in determining an individual's susceptibility to disease. Gene therapy is currently not available and therefore, most conditions are treated with pharmacological agents that modify neurotransmitter receptor signaling. Here, I provide a review of ionotropic glutamate receptors (iGluRs) and the roles they fulfill in numerous CNS disorders. Specifically, I argue that our understanding of iGluRs has reached a critical turning point to permit, for the first time, a comprehensive re-evaluation of their role in the cause of disease. I illustrate this by highlighting how defects in AMPA receptor (AMPAR) trafficking are important to fragile X mental retardation and ectopic expression of kainate receptor (KAR) synapses contributes to the pathology of temporal lobe epilepsy. Finally, I discuss how parallel advances in studies of other neurotransmitter systems may allow pharmacologists to work towards a cure for many CNS disorders rather than developing drugs to treat their symptoms.     

GPCR drug discovery: novel ligands for CNS receptors

G protein-coupled receptors (GPCRs) are the largest class of cell surface receptors in humans. They convey extracellular signals into the cell interior by activating intracellular processes such as heterotrimeric G protein-dependent signaling pathways. They are widely distributed in the nervous system, and mediate key physiological processes including cognition, mood, appetite, pain and synaptic transmission. With at least 30% of marketed drugs being GPCR modulators, they are a major therapeutic target in the pharmaceutical industry's drug discovery programs. This review will survey recently patented ligands for GPCRs implicated in CNS disorders, in particular the metabotropic glutamate, adenosine and cannabinoid receptors. Metabotropic glutamate receptors regulate signaling by glutamate, the major excitatory brain neurotransmitter, while adenosine is a ubiquitous neuromodulater mediating diverse physiological effects. Recent patents for ligands of these receptors include mGluR5 antagonists and adenosine A(1) receptor agonists. Cannabinoid receptors remain one of the most important GPCR drug discovery target due to the intense interest in CB(1) receptor antagonists for treating obesity and metabolic syndrome. Such small molecule ligands are the outcome of the continuing focus of many pharmaceutical companies to identify novel GPCR agonist, antagonist or allosteric modulators useful for CNS disorders, for which more effective drugs are eagerly awaited.     

Role of glutamate receptors in periventricular leukomalacia

Periventricular leukomalacia is a form of white-matter injury that occurs in the setting of either primary or secondary hypoxia-ischemia in the premature infant. Hypoxia-ischemia induces increases in cerebral extracellular glutamate levels, thereby activating glutamate receptors on a variety of cell types within the white matter. This review examines the evidence of a role for glutamate receptors in white-matter injury and periventricular leukomalacia. Multiple glutamate receptor subtypes exist, and these appear to play differential roles depending on cell type and time after injury. Glutamate receptors are developmentally regulated on neurons and glia, and certain subtypes are transiently overexpressed in developing rodent brain and are expressed on immature oligodendrocytes in human white matter in the premature period. Pharmacologic agents acting on glutamate receptors might represent age-specific therapeutic strategies for the treatment of periventricular leukomalacia.     

Neurosteroid modulation of recombinant ionotropic glutamate receptors

Pregnenolone sulfate (PS) is an abundant neurosteroid that can potentiate or inhibit ligand gated ion channel activity and thereby alter neuronal excitability. Whereas PS is known to inhibit kainate and AMPA responses while potentiating NMDA responses, the dependence of modulation on receptor subunit composition remains to be determined. Toward this end, the effect of PS on recombinant kainate (GluR6), AMPA (GluR1 or GluR3), and NMDA (NR1₁₀₀+NR2A) receptors was characterized electrophysiologically with respect to efficacy and potency of modulation. With Xenopus oocytes expressing GluR1, GluR3 or GluR6 receptors, PS reduces the efficacy of kainate without affecting its potency, indicative of a noncompetitive mechanism of action. Conversely, with oocytes expressing NR1₁₀₀+NR2A subunits, PS enhances the efficacy of NMDA without affecting its potency. Whereas the modulatory efficacy, but not the potency, of PS is increased two-fold by co-injection of NR1₁₀₀+NR2A cRNAs as compared with NR1₁₀₀ cRNA alone, there is little or no effect of the NR2A subunit on efficacy or potency of pregnanolone (or epipregnanolone) sulfate as an inhibitor of the NMDA response. This suggests that the NR2A subunit controls the efficacy of neurosteroid enhancement, but not inhibition, which is consistent with our previous finding that potentiating and inhibitory steroids act at distinct sites on the NMDA receptor. This represents a first step towards understanding the role of subunit composition in determining neurosteroid modulation of ionotropic glutamate receptor function.     

Expression of glutamate transporters and ionotropic glutamate receptors in GLAST knockout mice

In order to investigate the molecular mechanism underlying high seizure susceptibility of GLAST knockout mice, we carried out Western blotting for the expression of GLT-1, EAAC-1, and several kinds of glutamate receptors in the hippocampus and the cortex. Although no significant difference was observed between GLAST (+/+) and (-/-) mice in terms of expression of GLT-1 and EAAC-1 in the hippocampus, these proteins were over-expressed in the frontal cortex in GLAST (-/-) mice (GLT-1, about 210% increase; EAAC-1, about 180% increase). Expression of hippocampal Glu-R1 and Glu-R2 in GLAST (-/-) mice was remarkably increased (Glu-R1, about 140% increase; Glu-R2, about 160% increase), while Glu-R3 and NMDA receptors levels (NMDA-R1, 2A and 2B) were equal to those in control. Cortical levels of Glu-R1, -R2 and -R3 receptors in GLAST (-/-) mice were remarkably decreased (Glu-R1, about 60% decrease; Glu-R2, about 60% decrease; Glu-R3, about 70% decrease), while NMDA receptors were remarkably increased in comparison to those in GLAST (+/+) mice (N-R1, about 150% increase; N-R2A, about 150% increase; N-R2B, about 140% increase). These data suggest that the increased susceptibility to seizures in GLAST (-/-) mice might be derived from increased expression of Glu-R1 in the hippocampus coupled with decreased cortical expression of Glu-R2 and increased NMDA-R1 and -2A, -2B expression.