Rigorous Characterization of Allosteric Modulation of the Human Metabotropic Glutamate Receptor 1 Reveals Probe- and Assay-Dependent Pharmacology.

Allosteric modulation of metabotropic glutamate receptor subtype 1 (mGlu1) represents a viable therapeutic target for treating numerous central nervous system disorders. Although multiple chemically distinct mGlu1 positive (PAMs) and negative (NAMs) allosteric modulators have been identified, drug discovery paradigms have not included rigorous pharmacological analysis. In the present study, we hypothesized that existing mGlu1 allosteric modulators possess unappreciated probe-dependent or biased pharmacology. Using human embryonic kidney 293 (HEK293A) cells stably expressing human mGlu1, we screened mGlu1 PAMs and NAMs from divergent chemical scaffolds for modulation of different mGlu1 orthosteric agonists in intracellular calcium (iCa2+) mobilization and inositol monophosphate (IP1) accumulation assays. Operational models of agonism and allosterism were used to derive estimates for important pharmacological parameters such as affinity, efficacy, and cooperativity. Modulation of glutamate and quisqualate-mediated iCa2+ mobilization revealed probe dependence at the level of affinity and cooperativity for both mGlu1 PAMs and NAMs. We also identified the previously described mGlu5 selective NAM PF-06462894 as an mGlu1 NAM with a different pharmacological profile from other NAMs. Differential profiles were also observed when comparing ligand pharmacology between iCa2+ mobilization and IP1 accumulation. The PAMs Ro67-4853 and CPPHA displayed apparent negative cooperativity for modulation of quisqualate affinity, and the NAMs CPCCOEt and PF-06462894 had a marked reduction in cooperativity with quisqualate in IP1 accumulation and upon extended incubation in iCa2+ mobilization assays. These data highlight the importance of rigorous assessment of mGlu1 modulator pharmacology to inform future drug discovery programs for mGlu1 allosteric modulators. SIGNIFICANCE STATEMENT: Metabotropic glutamate receptor subtype 1 (mGlu1) positive and negative allosteric modulators have therapeutic potential in multiple central nervous system disorders. We show that chemically distinct modulators display differential pharmacology with different orthosteric ligands and across divergent signaling pathways at human mGlu1. Such complexities in allosteric ligand pharmacology should be considered in future mGlu1 allosteric drug discovery programs.

Increased [³H]quisqualic acid binding density in the dorsal striatum and anterior insula of alcoholics: A post-mortem whole-hemisphere autoradiography study.

The function of group I metabotropic glutamate receptors mGluR1 and mGluR5 is involved in the hyperglutamatergic state caused by chronic alcohol. Preclinical studies suggest that group I mGluR modulation could serve as a novel treatment of alcoholism. Considering the wide role of glutamatergic neurochemistry in addiction, group I mGluR binding was studied in brain areas involved in decision-making, learning and memory. Post-mortem whole hemisphere autoradiography was used to study the binding density of [³H]quisqualic acid, a potent group I mGluR agonist, in 9 Cloninger type 1 alcoholics, 8 Cloninger type 2 alcoholics and 10 controls. Binding was studied in the dorsal striatum, hippocampus and cortex. Alcoholics displayed a trend towards increased [³H]quisqualic acid binding in all brain areas. The most robust findings were in the putamen (p = 0.006) and anterior insula (p = 0.005), where both alcoholic subtypes displayed increased binding compared to the controls. These findings suggest altered group I mGluR function in alcoholic subjects in the dorsal striatum, which is involved in habitual learning, and in the anterior insula, which has a pivotal role in the perception of bodily sensations. Increased [³H]quisqualic acid binding might suggest a beneficial impact of mGluR1/5 modulators in the treatment of alcoholism.

Geranium intoxication induces detoxification enzymes in the Japanese beetle, Popillia japonica Newman.

Popillia japonica is a generalist herbivore that feeds on >300 host plant species in at least 72 plant families. It is unknown why P. japonica, despite possessing active detoxification enzymes in its gut, is paralyzed when feeding on the petals of one of its preferred host plant, Pelargonium×hortorum, or on artificial diet containing quisqualic acid (QA), the active compound in zonal geranium. We hypothesized that Pelargonium×hortorum or QA do not induce activity of the cytochrome P450, glutathione S transferase (GST), and carboxylesterase (CoE) detoxification enzymes in P. japonica. In this study, P. japonica were fed petals of zonal geranium or agar plugs containing QA, or rose petals, another preferred but non-toxic host. Midgut enzyme activities of P450, GST, and CoE were then assayed after 6, 12, or 24h of feeding. In most cases, P450, GST, and CoE activities were significantly induced in P. japonica midguts by geranium petals and QA, though the induction was slower than with rose petals. Induced enzyme activity reached a peak at 24h after consumption, which coincides with the period of highest recovery from geranium and QA paralysis. This study shows that toxic geranium and QA induce detoxification enzyme activity, but the induced enzymes do not effectively protect P. japonica from paralysis by QA. Further investigation is required through in vitro studies to know if the enzymes induced by geranium are capable of metabolizing QA. This study highlights a rare physiological mismatch between the detoxification tool kit of a generalist and its preferred host.

Activation and Desensitization Mechanism of AMPA Receptor-TARP Complex by Cryo-EM.

AMPA receptors mediate fast excitatory neurotransmission in the mammalian brain and transduce the binding of presynaptically released glutamate to the opening of a transmembrane cation channel. Within the postsynaptic density, however, AMPA receptors coassemble with transmembrane AMPA receptor regulatory proteins (TARPs), yielding a receptor complex with altered gating kinetics, pharmacology, and pore properties. Here, we elucidate structures of the GluA2-TARP γ2 complex in the presence of the partial agonist kainate or the full agonist quisqualate together with a positive allosteric modulator or with quisqualate alone. We show how TARPs sculpt the ligand-binding domain gating ring, enhancing kainate potency and diminishing the ensemble of desensitized states. TARPs encircle the receptor ion channel, stabilizing M2 helices and pore loops, illustrating how TARPs alter receptor pore properties. Structural and computational analysis suggests the full agonist and modulator complex harbors an ion-permeable channel gate, providing the first view of an activated AMPA receptor.

Glutamatergic modulation of noradrenaline release in the rat median preoptic area.

The present study was carried out to investigate whether glutamatergic receptor mechanisms modulate the release of noradrenaline (NA) in the region of the median preoptic nucleus (MnPO) using intracerebral microdialysis techniques in freely moving rats. Perfusion of N-methyl-d-asparatate (NMDA, 10 and 50µM) through the microdialysis probe significantly enhanced dialysate NA concentration in the region of the MnPO. Local perfusion of the NMDA antagonist dizocilpine (MK801, 10 and 50µM) did not change the basal release of NA in the MnPO area. MK801 (10µM) administered together with NMDA antagonized the stimulant effect of NMDA (50µM). Perfusion of the non-NMDA agonist quisqualic acid (QA, 10 and 50µM) or kainic acid (KA, 10 and 50µM) significantly increased the NA release in the MnPO area. Perfusion of the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 and 50µM) had no effect on the NA release. CNQX (10µM) administered together with either QA (50µM) or KA (50µM) in the MnPO area prevented the stimulant effect of the agonists on the NA release. Nonhypotensive hypovolemia following subcutaneous injections of polyethylene glycol (PEG, 30%, 5ml) significantly elevated the NA level in the MnPO area. The PEG-induced elevation in the NA release was attenuated by perfusion of either MK801 (10µM) or CNQX (10µM). The present results suggest that glutamatergic synaptic inputs may act to enhance the release of NA in the MnPO area through both NMDA and non-NMDA receptors, and imply that these glutamatergic receptor mechanisms may be involved in the noradrenergic reguratory system for the body fluid balance.

Novel Functional Properties of Drosophila CNS Glutamate Receptors.

Phylogenetic analysis reveals AMPA, kainate, and NMDA receptor families in insect genomes, suggesting conserved functional properties corresponding to their vertebrate counterparts. However, heterologous expression of the Drosophila kainate receptor DKaiR1D and the AMPA receptor DGluR1A revealed novel ligand selectivity at odds with the classification used for vertebrate glutamate receptor ion channels (iGluRs). DKaiR1D forms a rapidly activating and desensitizing receptor that is inhibited by both NMDA and the NMDA receptor antagonist AP5; crystallization of the KaiR1D ligand-binding domain reveals that these ligands stabilize open cleft conformations, explaining their action as antagonists. Surprisingly, the AMPA receptor DGluR1A shows weak activation by its namesake agonist AMPA and also by quisqualate. Crystallization of the DGluR1A ligand-binding domain reveals amino acid exchanges that interfere with binding of these ligands. The unexpected ligand-binding profiles of insect iGluRs allows classical tools to be used in novel approaches for the study of synaptic regulation. VIDEO ABSTRACT.

Atypical signaling of metabotropic glutamate receptor 1 in human melanoma cells.

The metabotropic glutamate 1 (mGlu1) receptor has emerged as a novel target for the treatment of metastatic melanoma and various other cancers. Our laboratory has demonstrated that a selective, non-competitive mGlu1 receptor antagonist slows human melanoma growth in vitro and in vivo. In this study, we sought to determine if the activation of a canonical G protein-dependent signal transduction cascade, which is often used as an output of mGlu1 receptor activity in neuronal cells, correlated with mGlu1 receptor-mediated melanoma cell viability. Glutamate, the endogenous ligand of mGlu1 receptors, significantly increased melanoma cell viability, but did not stimulate phosphoinositide (PI) hydrolysis in several human melanoma cell lines. In contrast, melanoma cell viability was not increased by quisqualate, a highly potent mGlu1 receptor agonist, or DHPG, a selective group I mGlu receptor agonist. Similarly to glutamate, quisqualate also failed to stimulate PI hydrolysis in mGlu1 receptor-expressing melanoma cells. These results suggest that the canonical G protein-dependent signal transduction cascade is not coupled to mGlu1 receptors in all human melanoma cells. On the other hand, dynamin inhibition selectively decreased viability of mGlu1 receptor-expressing melanoma cells, suggesting that a mechanism requiring internalization may control melanoma cell viability. Taken together, these data demonstrate that the approaches commonly used to study mGlu1 receptor function and signaling in other systems may be inappropriate for studying mGlu1 receptor-mediated melanoma cell viability.

PI3K mediated activation of GSK-3ß reduces at-level primary afferent growth responses associated with excitotoxic spinal cord injury dysesthesias.

BACKGROUND: Neuropathic pain and sensory abnormalities are a debilitating secondary consequence of spinal cord injury (SCI). Maladaptive structural plasticity is gaining recognition for its role in contributing to the development of post SCI pain syndromes. We previously demonstrated that excitotoxic induced SCI dysesthesias are associated with enhanced dorsal root ganglia (DRG) neuronal outgrowth. Although glycogen synthase kinase-3ß (GSK-3ß) is a known intracellular regulator neuronal growth, the potential contribution to primary afferent growth responses following SCI are undefined. We hypothesized that SCI triggers inhibition of GSK-3ß signaling resulting in enhanced DRG growth responses, and that PI3K mediated activation of GSK-3ß can prevent this growth and the development of at-level pain syndromes. RESULTS: Excitotoxic SCI using intraspinal quisqualic acid (QUIS) resulted in inhibition of GSK-3ß in the superficial spinal cord dorsal horn and adjacent DRG. Double immunofluorescent staining showed that GSK-3ß(P) was expressed in DRG neurons, especially small nociceptive, CGRP and IB4-positive neurons. Intrathecal administration of a potent PI3-kinase inhibitor (LY294002), a known GSK-3ß activator, significantly decreased GSK-3ß(P) expression levels in the dorsal horn. QUIS injection resulted in early (3 days) and sustained (14 days) DRG neurite outgrowth of small and subsequently large fibers that was reduced with short term (3 days) administration of LY294002. Furthermore, LY294002 treatment initiated on the date of injury, prevented the development of overgrooming, a spontaneous at-level pain related dysesthesia. CONCLUSIONS: QUIS induced SCI resulted in inhibition of GSK-3ß in primary afferents and enhanced at-level DRG intrinsic growth (neurite elongation and initiation). Early PI3K mediated activation of GSK-3ß attenuated QUIS-induced DRG neurite outgrowth and prevented the development of at-level dysesthesias.

Anti-cancer effect of metabotropic glutamate receptor 1 inhibition in human glioma U87 cells: involvement of PI3K/Akt/mTOR pathway.

BACKGROUND: Metabotropic glutamate receptors (mGluRs) are G-protein-coupled receptors that mediate neuronal excitability and synaptic plasticity in the central nervous system, and emerging evidence suggests a role of mGluRs in the biology of cancer. Previous studies showed that mGluR1 was a potential therapeutic target for the treatment of breast cancer and melanoma, but its role in human glioma has not been determined. METHODS: In the present study, we investigated the effects of mGluR1 inhibition in human glioma U87 cells using specific targeted small interfering RNA (siRNA) or selective antagonists Riluzole and BAY36-7620. The anti-cancer effects of mGluR1 inhibition were measured by cell viability, lactate dehydrogenase (LDH) release, TUNEL staining, cell cycle assay, cell invasion and migration assays in vitro, and also examined in a U87 xenograft model in vivo. RESULTS: Inhibition of mGluR1 significantly decreased the cell viability but increased the LDH release in a dose-dependent fashion in U87 cells. These effects were accompanied with the induction of caspase-dependent apoptosis and G0/G1 cell cycle arrest. In addition, the results of Matrigel invasion and cell tracking assays showed that inhibition of mGluR1 apparently attenuated cell invasion and migration in U87 cells. All these anti-cancer effects were ablated by the mGluR1 agonist L-quisqualic acid. The results of western blot analysis showed that mGluR1 inhibition overtly decreased the phosphorylation of PI3K, Akt, mTOR and P70S6K, indicating the mitigated activation of PI3K/Akt/mTOR pathway. Moreover, the anti-tumor activity of mGluR1 inhibition in vivo was also demonstrated in a U87 xenograft glioma model in athymic nude mice. CONCLUSION: The remarkable efficiency of mGluR1 inhibition to induce cell death in U87 cells may find therapeutic application for the treatment of glioma patients.

Biphasic modulation by mGlu5 receptors of TRPV1-mediated intracellular calcium elevation in sensory neurons contributes to heat sensitivity.

BACKGROUND AND PURPOSE: Elevation of glutamate, an excitatory amino acid, during inflammation and injury plays a crucial role in the reception and transmission of sensory information via ionotropic and metabotropic receptors. This study aimed to investigate the mechanisms underlying the biphasic effects of metabotropic glutamate mGlu5 receptor activation on responses to noxious heat. EXPERIMENTAL APPROACH: We assessed the effects of intraplantar quisqualate, a non-selective glutamate receptor agonist, on heat and mechanical pain behaviours in mice. In addition, the effects of quisqualate on the intracellular calcium response and on membrane currents mediated by TRPV1 channels, were examined in cultured dorsal root ganglion neurons from mice. KEY RESULTS: Activation of mGlu5 receptors in hind paw transiently increased, then decreased, the response to noxious heat. In sensory neurons, activation of mGlu5 receptors potentiated TRPV1-mediated intracellular calcium elevation, while terminating activation of mGlu5 receptors depressed it. TRPV1-induced currents were potentiated by activation of mGlu5 receptors under voltage clamp conditions and these disappeared after washout. However, voltage-gated calcium currents were inhibited by the mGlu5 receptor agonist, even after washout. CONCLUSIONS AND IMPLICATIONS: These results suggest that, in sensory neurons, mGlu5 receptors biphasically modulate TRPV1-mediated intracellular calcium response via transient potentiation of TRPV1 channel-induced currents and persistent inhibition of voltage-gated calcium currents, contributing to heat hyper- and hypoalgesia.

Enteral supplements of a carbon monoxide donor CORM-A1 protect against cerebrovascular dysfunction caused by neonatal seizures.

Cerebral blood flow dysregulation caused by oxidative stress contributes to adverse neurologic outcome of seizures. A carbon monoxide (CO) donor CORM-A1 has antioxidant and cytoprotective properties. We investigated whether enteral supplements of CORM-A1 can improve cerebrovascular outcome of bicuculline-induced seizures in newborn piglets. CORM-A1 (2 mg/kg) was given to piglets via an oral gastric tube 10 minutes before or 20 minutes after seizure onset. Enteral CORM-A1 elevated CO in periarachnoid cerebrospinal fluid and produced a dilation of pial arterioles. Postictal cerebral vascular responses to endothelium-, astrocyte-, and vascular smooth muscle-dependent vasodilators were tested 48 hours after seizures by intravital microscopy. The postictal responses of pial arterioles to bradykinin, glutamate, the AMPA receptor agonist quisqualic acid, ADP, and heme were greatly reduced, suggesting that seizures cause injury to endothelial and astrocyte components of the neurovascular unit. In contrast, in the two groups of piglets receiving enteral CORM-A1, the postictal cerebral vascular responsiveness to these dilators was improved. Overall, enteral supplements of CORM-A1 before or during seizures offer a novel effective therapeutic option to deliver cytoprotective mediator CO to the brain, reduce injury to endothelial and astrocyte components of cerebral blood flow regulation and to improve the cerebrovascular outcome of neonatal seizures.

Agrin requires specific proteins to selectively activate γ-aminobutyric acid neurons for pain suppression.

Agrin, a heparan sulfate proteoglycan functioning as a neuro-muscular junction inducer, has been shown to inhibit neuropathic pain in sciatic nerve injury rat models, via phosphorylation of N-Methyl-d-aspartate receptor NR1 subunits in gamma-aminobutyric acid neurons. However, its effects on spinal cord injury-induced neuropathic pain, a debilitating syndrome frequently encountered after various spine traumas, are unknown. In the present investigation, we studied the 50kDa agrin isoform effects in a quisqualic acid dorsal horn injection rat model mimicking spinal cord injury-induced neuropathic pain. Our results indicate that 50kDa agrin decreased only in the dorsal horn of neuropathic animals and increased 50kDa agrin expression in the dorsal horn, via intra-spinal injection of adeno-associated virus serum type two, suppressed spinal cord injury-induced neuropathic pain. Also, the reason why 50kDa agrin only activates the N-Methyl-d-aspartate receptor NR1 subunits in the GABA neurons, but not in sensory neurons, is unknown. Using immunoprecipitation and Western-blot analysis, two dimensional gel separation, and mass spectrometry, we identified several specific proteins in the reaction protein complex, such as neurofilament 200 and mitofusin 2, that are required for the activation of the NR1 subunits of gamma-aminobutyric acid inhibitory neurons by 50kDa agrin. These findings indicate that 50kDa agrin is a promising agent for neuropathic pain treatment.

Extracellular calcium modulates actions of orthosteric and allosteric ligands on metabotropic glutamate receptor 1α.

Metabotropic glutamate receptor 1α (mGluR1α), a member of the family C G protein-coupled receptors, is emerging as a potential drug target for various disorders, including chronic neuronal degenerative diseases. In addition to being activated by glutamate, mGluR1α is also modulated by extracellular Ca(2+). However, the underlying mechanism is unknown. Moreover, it has long been challenging to develop receptor-specific agonists due to homologies within the mGluR family, and the Ca(2+)-binding site(s) on mGluR1α may provide an opportunity for receptor-selective targeting by therapeutics. In the present study, we show that our previously predicted Ca(2+)-binding site in the hinge region of mGluR1α is adjacent to the site where orthosteric agonists and antagonists bind on the extracellular domain of the receptor. Moreover, we found that extracellular Ca(2+) enhanced mGluR1α-mediated intracellular Ca(2+) responses evoked by the orthosteric agonist l-quisqualate. Conversely, extracellular Ca(2+) diminished the inhibitory effect of the mGluR1α orthosteric antagonist (S)-α-methyl-4-carboxyphenylglycine. In addition, selective positive (Ro 67-4853) and negative (7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester) allosteric modulators of mGluR1α potentiated and inhibited responses to extracellular Ca(2+), respectively, in a manner similar to their effects on the response of mGluR1α to glutamate. Mutations at residues predicted to be involved in Ca(2+) binding, including E325I, had significant effects on the modulation of responses to the orthosteric agonist l-quisqualate and the allosteric modulator Ro 67-4853 by extracellular Ca(2+). These studies reveal that binding of extracellular Ca(2+) to the predicted Ca(2+)-binding site in the extracellular domain of mGluR1α modulates not only glutamate-evoked signaling but also the actions of both orthosteric ligands and allosteric modulators on mGluR1α.

Excitotoxic injury to thoracolumbar gray matter alters sympathetic activation and thermal pain sensitivity.

Studies of humans, monkeys and rodents have implicated combined gray and white matter damage as important for development of chronic pain following spinal cord injury (SCI). Below-level chronic pain and hyperalgesia following injury to the spinal white matter, including the spinothalamic tract (STT), can be enhanced by excitotoxic influences within the gray matter at the site of SCI. Also, excitotoxic injury of thoracic gray matter without interruption of the STT results in below-level heat hyperalgesia. The present study evaluates the possibility that thoracolumbar gray matter injury increases sensitivity to nociceptive heat stimulation by altering spinal sympathetic outflow. Thermal preferences of rats for heat (45 °C) versus cold (15 °C) were evaluated before and after thoracolumbar injections of quisqualic acid (QUIS). A pre-injury preference for heat changed to a post-injury preference for cold. Systemic activation of the sympathetic nervous system by restraint stress decreased the heat preference pre-injury and increased the cold preference post-injury. The heat aversive effect of stress was magnified and prolonged post-injury, compared to pre-injury. Also, peripheral sympathetic activation by nociceptive stimulation was evaluated pre- and post-injury by measuring thermal transfer through a hindpaw during stimulation with 44.5 °C. Skin temperature recordings revealed enhanced sympathetic activation by nociceptive heat stimulation following spinal QUIS injury. However, increased sympathetic activation with peripheral vasoconstriction should enhance cold aversion, in contrast to the observed increase in heat aversion. Thus, peripheral sympathetic vasoconstriction can be ruled out as a mechanism for heat hyperalgesia following excitotoxic gray matter injury.

Molecular dynamic simulation of mGluR5 amino terminal domain: essential dynamics analysis captures the agonist or antagonist behaviour of ligands.

We describe the application of molecular dynamics followed by principal component analysis to study the inter-domain movements of the ligand binding domain (LBD) of mGluR5 in response to the binding of selected agonists or antagonists. Our results suggest that the method is an attractive alternative to current approaches to predict the agonist-induced or antagonist-blocked LBD responses. The ratio between the eigenvalues of the first and second eigenvectors (R1,2) is also proposed as a numerical descriptor for discriminating the ligand behavior as a mGluR5 agonist or antagonist.

Excitotoxic spinal cord injury induced dysesthesias are associated with enhanced intrinsic growth of sensory neurons.

Sensory dysesthesias and pain are common sequelae following spinal cord injury (SCI). While efforts to understand the mechanisms involved in SCI pain syndromes have focused on spinal and supraspinal regions, recent evidence suggests that SCI induces pathological responses in primary afferent neurons that may contribute to the development of sensory abnormalities. The purpose of this study was to investigate if excitotoxic spinal lesions lead to abnormal growth responses of cultured dorsal root ganglia (DRG) neurons, and to examine if the degree of neurite growth correlated with the presence of dysesthesias. Long-Evans rats underwent excitotoxic spinal cord lesions by injection of quisqualic acid at spinal level T12. Animals were examined daily for overgrooming behavior. Fourteen days after injury, DRG neurons were removed from at and below the level of injury, cultured and analyzed for soma size and neurite length. Grooming animals showed robust neurite growth in small, medium, and large neurons compared to nongrooming and control animals. Enhanced neuronal growth responses also occurred several segments caudal to the level of injury. This study provides the first evidence that excitotoxic spinal lesions result in DRG neurite outgrowth that correlated with the presence of sensory dysesthesias, providing support for the role of maladaptive peripheral afferent responses contributing to SCI pain syndromes.

Anxiolytic properties of Valeriana officinalis in the zebrafish: a possible role for metabotropic glutamate receptors.

Valerian extract is used in complementary and alternative medicine for its anxiolytic and sedative properties. Our previous research demonstrated valerian interactions with glutamate receptors. The purpose of this study was to determine if valerian anxiolytic properties are mediated by metabotropic glutamate receptors (mGluR) such as mGluR (1/5) (mGluR I) and mGluR (2/3) (mGluR II). Adult wild-type zebrafish (Danio rerio) prefer the black compartment and avoid the white compartment in the dark/light preference task. Zebrafish exposed to 1 mg/mL of valerian extract or 0.00117 mg/mL valerenic acid increased their residence time in the white side by 84.61 ± 6.55 % and 58.30 ± 8.97 %, respectively. LAP3 (mGluR I antagonist) and EGLU (mGluR II antagonist) significantly inhibited the effects of valerian and valerenic acid. These results demonstrated that valerian and valerenic acid have anxiolytic properties in the zebrafish. Moreover, the selective interaction of valerian with mGluR I and II represent an alternative explanation for the anxiolytic properties of this plant and support the role of mGluR in anxiety.

Orthosteric and allosteric drug binding sites in the Caenorhabditis elegans mgl-2 metabotropic glutamate receptor.

The metabotropic glutamate receptors (mGluRs) are evolutionarily conserved from nematodes to vertebrates. The Caenorhabditis elegans (C. elegans) genome contains three mGluR genes referred to as mgl-1, mgl-2, and mgl-3. The aim of this study was to characterize the pharmacological profiles of orthosteric and allosteric mGluR ligands on mgl-2. A phylogenetic analysis revealed that mgl-2 is closely associated with the mammalian Group 1 mGluRs (mGluR1 and mGluR5) and is distinct from Group 2 and 3 mGluRs. The ligand binding domain of mgl-2 displayed higher homology to the rat Group 1 mGluRs binding domains compared to the level of homology in the heptahelical transmembrane domain regions. We found that, when transiently expressed in human embryonic kidney 293 cells, mgl-2 can be activated by glutamate and couples to human G-proteins to induce the release of intracellular calcium. Dose-response analyses revealed that mgl-2 has approximately a 15-20-fold lower affinity for glutamate and quisqualate compared to rat mGluR5. In contrast to orthosteric agonists, Group 1 negative allosteric modulators that target the transmembrane domain were ineffective at mgl-2. Surprisingly, CDPPB, an mGluR5 positive allosteric modulator, potentiated glutamate mediated activation of mgl-2, although MPEP and fenobam, two mGluR5 antagonists that share similar binding residues with CDPPB were ineffective at mgl-2. These findings indicate that selective pressures on mGluR protein structures have resulted in conservation of the glutamate binding site, whereas the allosteric modulator sites have been subjected to greater divergent evolutionary changes.

The loss of an electrostatic contact unique to AMPA receptor ligand binding domain 2 slows channel activation.

Ligand-gated ion channels undergo conformational changes that transfer the energy of agonist binding to channel opening. Within ionotropic glutamate receptor (iGluR) subunits, this process is initiated in their bilobate ligand binding domain (LBD) where agonist binding to lobe 1 favors closure of lobe 2 around the agonist and allows formation of interlobe hydrogen bonds. AMPA receptors (GluAs) differ from other iGluRs because glutamate binding causes an aspartate-serine peptide bond in a flexible part of lobe 2 to rotate 180° (flipped conformation), allowing these residues to form cross-cleft H-bonds with tyrosine and glycine in lobe 1. This aspartate also contacts the side chain of a lysine residue in the hydrophobic core of lobe 2 by a salt bridge. We investigated how the peptide flip and electrostatic contact (D655-K660) in GluA3 contribute to receptor function by examining pharmacological and structural properties with an antagonist (CNQX), a partial agonist (kainate), and two full agonists (glutamate and quisqualate) in the wildtype and two mutant receptors. Alanine substitution decreased the agonist potency of GluA3(i)-D655A and GluA3(i)-K660A receptor channels expressed in HEK293 cells and differentially affected agonist binding affinity for isolated LBDs without changing CNQX affinity. Correlations observed in the crystal structures of the mutant LBDs included the loss of the D655-K660 electrostatic contact, agonist-dependent differences in lobe 1 and lobe 2 closure, and unflipped D(A)655-S656 bonds. Glutamate-stimulated activation was slower for both mutants, suggesting that efficient energy transfer of agonist binding within the LBD of AMPA receptors requires an intact tether between the flexible peptide flip domain and the rigid hydrophobic core of lobe 2.