Glycine Agonist Research Articles

Allosteric Cholesterol Site in Glycine Receptors Characterized through Molecular Simulations.

Glycine receptors are pentameric ligand-gated ion channels that conduct chloride ions across postsynaptic membranes to facilitate fast inhibitory neurotransmission. In addition to gating by the glycine agonist, interactions with lipids and other compounds in the surrounding membrane environment modulate their function, but molecular details of these interactions remain unclear, in particular, for cholesterol. Here, we report coarse-grained simulations in a model neuronal membrane for three zebrafish glycine receptor structures representing apparent resting, open, and desensitized states. We then converted the systems to all-atom models to examine detailed lipid interactions. Cholesterol bound to the receptor at an outer-leaflet intersubunit site, with a preference for the open and desensitized versus resting states, indicating that it can bias receptor function. Finally, we used short atomistic simulations and iterative amino acid perturbations to identify residues that may mediate allosteric gating transitions. Frequent cholesterol contacts in atomistic simulations clustered with residues identified by perturbation analysis and overlapped with mutations influencing channel function and pathology. Cholesterol binding at this site was also observed in a recently reported pig heteromeric glycine receptor. These results indicate state-dependent lipid interactions relevant to allosteric transitions of glycine receptors, including specific amino acid contacts applicable to biophysical modeling and pharmaceutical design.

Regulation of ethanol-mediated dopamine elevation by glycine receptors located on cholinergic interneurons in the nucleus accumbens.

Alcohol use disorder is one of the major psychiatric disorders worldwide, and there are many factors and effects contributing to the disorder, for example, the experience of ethanol reward. The rewarding and reinforcing properties of ethanol have been linked to activation of the mesolimbic dopamine system, an effect that appears to involve glycine receptors (GlyRs) in the nucleus accumbens. On which neuronal subtypes these receptors are located is, however, not known. The aim of this study was to explore the role of GlyRs on cholinergic interneurons (CIN) in sustaining extracellular dopamine levels and in ethanol-induced dopamine release. To this end, CIN were ablated by anti-choline acetyltransferase-saporin administered locally in the nucleus accumbens of male Wistar rats. Changes in dopamine levels induced by ablation, ethanol and/or a GlyR antagonist were monitored using in vivo microdialysis. The GlyRs antagonist strychnine depressed extracellular dopamine in a similar manner independent on local ablation, suggesting that GlyRs on CIN are not important for sustaining the extracellular dopamine tone. However, a low concentration of strychnine hampered ethanol-induced dopamine release in sham-treated animals, whilst no reduction was seen in ablated animals, suggesting that GlyRs located on CIN are involved in ethanol-induced dopamine release. Further, in ablated rats, ethanol-induced increases of the extracellular levels of the GlyR agonists glycine and taurine were attenuated. In conclusion, this study suggests that CIN are not important for GlyR-mediated regulation of basal dopamine output, but that CIN ablation blunts the ethanol-induced dopamine release, putatively by reducing the release of GlyR agonists.

The GlyT1-inhibitor Org 24598 facilitates the alcohol deprivation abolishing and dopamine elevating effects of bupropion + varenicline in rats

Alcohol Use Disorder (AUD) is a relapsing brain disorder that involves perturbations of brain dopamine (DA) systems, and combined treatment with varenicline + bupropion produces additive effects on accumbal DA output and abolishes the alcohol deprivation effect (ADE) in rats. Also, direct and indirect glycine receptor (GlyR) agonists raise basal DA, attenuate alcohol-induced DA release in the nucleus Accumbens (nAc) and reduce alcohol consumption in rats. This study in rats examines whether the GlyT1-inhibitor Org 24598, an indirect GlyR agonist, enhances the ADE-reducing and DA elevating action of the combined administration of varenicline + bupropion in lower doses than previously applied. Effects on voluntary alcohol consumption, the ADE and extracellular levels of glycine and DA in nAc were examined following treatment with Org 24598 6 and 9 mg/kg i.p., bupropion 3.75 mg/kg i.p. and varenicline 1.5 mg/kg s.c., in monotherapy or combined, using a two-bottle, free-choice alcohol consumption paradigm with an ADE paradigm, and in vivo microdialysis in male Wistar rats. Notably, all treatment regimens appeared to abolish the ADE but only the effect produced by the triple combination (Org24598 + varenicline + bupropion) was significant compared to vehicle. Hence, addition of Org 24598 may enhance the ADE-reducing action of varenicline + bupropion and appears to allow for a dose reduction of bupropion. Treatment with Org 24598 raised accumbal glycine levels but did not significantly alter DA output in monotherapy. Varenicline + bupropion produced a substantial elevation in accumbal DA output that was slightly enhanced following addition of Org 24598. Conceivably, the blockade of the ADE is achieved by the triple combination enhancing accumbal DA transmission in complementary ways, thereby alleviating a hypothesized hypodopaminergia and negative reinforcement to drink. Ultimately, combining an indirect or direct GlyR agonist with varenicline + bupropion may constitute a new pharmacological treatment principle for AUD, although further refinement in dosing and evaluation of other glycinergic compounds are warranted.

Pharmacological Evaluation of Alcoholic Extract of Clitoria ternatea Root for Anxiolytic and Anticonvulsant Activity

Recent scientifi c studies have indicated that some of the same medicinal herbs used in traditional medicine to treat epilepsy may also have anticonvulsant qualities, suggesting that they may be a possible source of novel anticonvulsants. Anticonvulsant properties have been studied in animal models. Clitoria ternatea Linn’s ethanolic root extract was examined for antiepileptic, anxiolytic, and phytochemical properties. Phenytoin 25 mg/kg, as a reference medicine, the extract was tested against strychnine-induced convulsion in rats at oral doses of 200 and 400 mg/kg. The elevated plus maze and staircase test were used to examine mice’s anxiolytic activity with lorazepam (the gold standard) at 0.05 mg/kg. When using a strychnine-induced convulsion model, the ethanolic extract markedly accelerated the start of convulsions. In the elevated plus maze test, Clitoria ternatea Linn ethanolic root extract at 200, 400, and standard doses signifi cantly increased the time and number of the entry in open arms compared to the control group. In a staircase test, the ethanolic root extract of Clitoria ternatea increased the number of steps climbed and lowered rearing compared to the control group. Docking scores in mcule software indicate that interactions with the glycine agonist (PDB: 5I57) and the GABA agonist (PDB: 4MS3) receptors for epilepsy and anxiety, respectively, are highly eff ective. Evidence for the biochemical evaluation of GABA, anticonvulsant, and anxiolytic action, as well as in-silico activities, of the ethanolic extract of Clitoria ternatea was reported in this work.

N-methyl-d-aspartate receptors and glycinergic transmission, respectively, mediate muscle relaxation and immobility of pentobarbital in mice

Pentobarbital-induced anesthesia is believed to be mediated by enhancement of the inhibitory action of γ-aminobutyric acid (GABA)ergic neurons in the central nervous system. However, it is unclear whether all components of anesthesia induced by pentobarbital, such as muscle relaxation, unconsciousness, and immobility in response to noxious stimuli, are mediated only through GABAergic neurons. Thus, we examined whether the indirect GABA and glycine receptor agonists gabaculine and sarcosine, respectively, the neuronal nicotinic acetylcholine receptor antagonist mecamylamine, or the N-methyl-d-aspartate receptor channel blocker MK-801 could enhance pentobarbital-induced components of anesthesia. Muscle relaxation, unconsciousness, and immobility were evaluated by grip strength, the righting reflex, and loss of movement in response to nociceptive tail clamping, respectively, in mice. Pentobarbital reduced grip strength, impaired the righting reflex, and induced immobility in a dose-dependent manner. The change in each behavior induced by pentobarbital was roughly consistent with that in electroencephalographic power. A low dose of gabaculine, which significantly increased endogenous GABA levels in the central nervous system but had no effect on behaviors alone, potentiated muscle relaxation, unconsciousness, and immobility induced by low pentobarbital doses. A low dose of MK-801 augmented only the masked muscle-relaxing effects of pentobarbital among these components. Sarcosine enhanced only pentobarbital-induced immobility. Conversely, mecamylamine had no effect on any behavior. These findings suggest that each component of anesthesia induced by pentobarbital is mediated through GABAergic neurons and that pentobarbital-induced muscle relaxation and immobility may partially be associated with N-methyl-d-aspartate receptor antagonism and glycinergic neuron activation, respectively.

Acidic pH reduces agonist efficacy and responses to synaptic-like glycine applications in zebrafish α1 and rat α1β recombinant glycine receptors.

Many pentameric ligand‐gated ion channels are modulated by extracellular pH. Glycine receptors (GlyRs) share this property, but it is not well understood how they are affected by pH changes. Whole cell experiments on HEK293 cells expressing zebrafish homomeric α1 GlyR confirmed previous reports that acidic pH (6.4) reduces GlyR sensitivity to glycine, whereas alkaline pH (8.4) has small or negligible effects. In addition to that, at pH 6.4 we observed a reduction in the maximum responses to the partial agonists β‐alanine and taurine relative to the full agonist glycine. In cell‐attached single‐channel recording, low pH reduced agonist efficacy, as the maximum open probability decreased from 0.97, 0.91 and 0.66 to 0.93, 0.57 and 0.34 for glycine, β‐alanine and taurine, respectively, reflecting a threefold decrease in efficacy equilibrium constants for all three agonists. We also tested the effect of pH 6.4 in conditions that replicate those at the native synapse, recording outside‐out currents elicited by fast application of millisecond pulses of agonists on α1 and α1β GlyR, at a range of intracellular chloride concentrations. Acidic pH reduced the area under the curve of the currents, by reducing peak amplitude, slowing activation and speeding deactivation. Our results show that acidification of the extracellular pH by one unit, as may occur in pathological conditions such as ischaemia, impairs GlyR gating and is likely to reduce the effectiveness of glycinergic synaptic inhibition. Key points Extracellular pH in the central nervous system (CNS) is known to shift towards acidic values during pathophysiological conditions such as ischaemia and seizures.Acidic extracellular pH is known to affect GABAergic inhibitory synapses, but its effect on signals mediated by glycine receptors (GlyR) is not well characterised.Moderate acidic conditions (pH 6.4) reduce the maximum single channel open probability of recombinant homomeric GlyRs produced by the neurotransmitter glycine or other agonists, such as β‐alanine and taurine.When glycine was applied with a piezoelectric stepper to outside out patches, to simulate its fast rise and short duration at the synapse, responses became shorter and smaller at pH 6.4. The effect was also observed with physiologically low intracellular chloride and in mammalian heteromeric GlyRs.This suggests that acidic pH is likely to reduce the strength of inhibitory signalling at glycinergic synapses.

Different Behaviors of a Glycine Receptor Channel Pore Residue between Wild-Type-Mimicking and Disease-Type-Mimicking Formats.

The glycine receptor (GlyR) is a neurotransmitter-gated chloride channel that mediates fast inhibitory neurotransmission, predominantly in the spinal cord and brain stem. Mutations of the GlyR are the major cause of hereditary hyperekplexia. Site-specific cysteine substitution followed by labeling with a fluorophore has previously been used to explore the behaviors of the hyperekplexia-related 271 (19') residue of the GlyR. However, this manipulation dramatically compromises sensitivity toward the agonist glycine and alters the pharmacological effects of various agents in manners similar to those of the hyperekplexia-causing R19'Q/L mutations, raising the question whether what is reported by the substituted and modified residue faithfully reflects what actually happens to the wild-type (WT) residue. In this study, a mechanism-rescuing second-site mutation was introduced to create a WT-mimicking GlyR (with the 19' residue cysteine substitution and modification still in place), in which the sensitivity toward glycine and pharmacological effects of various agents were restored. Further experiments revealed stark differences in the behaviors upon the various pharmacological treatments and consequently the underlying mechanisms of the 19' residue between this WT-mimicking GlyR and the GlyR without the mechanism rescue, which is correspondingly defined as the disease-type (DT)-mimicking GlyR. The data presented in this study warn generally that caution is required when attempting to deduce the behaviors of a WT residue from data based on substituted or modified residues that alter protein structure and function. Extra measures, such as rescuing mechanisms via alternative means as presented in this study, are needed to mitigate this challenge.

Functional expression of glycine receptors in DRG neurons of mice

Glycine receptor is one of the chloride-permeable ion channels composed of combinations of four α subunits and one β subunit. In adult spinal cord, the glycine receptor α1 subunit is crucial for the generation of inhibitory neurotransmission. The reduced glycinergic inhibition is regarded as one of the key spinal mechanisms underlying pathological pain symptoms. However, the expression and function of glycine receptors in the peripheral system are largely unknown as yet. Here we found that glycine receptor α1 subunit was prevalent in the dorsal root ganglia (DRG) neurons as well as in the sciatic nerves of adult mice. Intraganglionar or intraplantar injection of glycine receptor antagonist strychnine caused the hypersensitivity to mechanical, thermal and cold stimuli, suggesting the functional importance of peripheral glycine receptors in the control of nociceptive signal transmission. Our data showed that peripheral inflammation induced by formalin decreased the expression of glycine receptor α1 subunit on the plasma membrane of DRG neurons, which was attributed to the activation of protein kinase C signaling. Intraplantar application of glycine receptor agonist glycine or positive modulator divalent zinc ion alleviated the first-phase painful behaviors induced by formalin. These data suggested that peripheral glycine receptor might serve as an effective target for pain therapy.

Mechanism of gating and partial agonist action in the glycine receptor

Ligand-gated ion channels mediate signal transduction at chemical synapses and transition between resting, open, and desensitized states in response to neurotransmitter binding. Neurotransmitters that produce maximum open channel probabilities (Po) are full agonists, whereas those that yield lower than maximum Po are partial agonists. Cys-loop receptors are an important class of neurotransmitter receptors, yet a structure-based understanding of the mechanism of partial agonist action has proven elusive. Here, we study the glycine receptor with the full agonist glycine and the partial agonists taurine and γ-amino butyric acid (GABA). We use electrophysiology to show how partial agonists populate agonist-bound, closed channel states and cryo-EM reconstructions to illuminate the structures of intermediate, pre-open states, providing insights into previously unseen conformational states along the receptor reaction pathway. We further correlate agonist-induced conformational changes to Po across members of the receptor family, providing a hypothetical mechanism for partial and full agonist action at Cys-loop receptors.

The intracellular domain of homomeric glycine receptors modulates agonist efficacy

Like other pentameric ligand-gated channels, glycine receptors (GlyRs) contain long intracellular domains (ICDs) between transmembrane helices 3 and 4. Structurally characterized GlyRs are generally engineered to have a very short ICD. We show here that for one such construct, zebrafish GlyREM, the agonists glycine, β-alanine, taurine, and GABA have high efficacy and produce maximum single-channel open probabilities greater than 0.9. In contrast, for full-length human α1 GlyR, taurine and GABA were clearly partial agonists, with maximum open probabilities of 0.46 and 0.09, respectively. We found that the elevated open probabilities in GlyREM are not due to the limited sequence differences between the human and zebrafish orthologs, but rather to replacement of the native ICD with a short tripeptide ICD. Consistent with this interpretation, shortening the ICD in the human GlyR increased the maximum open probability produced by taurine and GABA to 0.90 and 0.70, respectively, but further engineering it to resemble GlyREM (by introducing the zebrafish transmembrane helix 4 and C terminus) had no effect. Furthermore, reinstating the native ICD to GlyREM converted taurine and GABA to partial agonists, with maximum open probabilities of 0.66 and 0.40, respectively. Structural comparison of transmembrane helices 3 and 4 in short- and long-ICD GlyR subunits revealed that ICD shortening does not distort the orientation of these helices within each subunit. This suggests that the effects of shortening the ICD stem from removing a modulatory effect of the native ICD on GlyR gating, revealing a new role for the ICD in pentameric ligand-gated channels.

Thermophoretic analysis of ligand-specific conformational states of the inhibitory glycine receptor embedded in copolymer nanodiscs

The glycine receptor (GlyR), a member of the pentameric ligand-gated ion channel family (pLGIC), displays remarkable variations in the affinity and efficacy of the full agonist glycine and the partial agonist taurine depending on the cell system used. Despite detailed insights in the GlyR three-dimensional structure and activation mechanism, little is known about conformational rearrangements induced by these agonists. Here, we characterized the conformational states of the α1 GlyR upon binding of glycine and taurine by microscale thermophoresis expressed in HEK293 cells and Xenopus oocytes after solubilization in amphipathic styrene-maleic acid copolymer nanodiscs. Our results show that glycine and taurine induce different conformational transitions of the GlyR upon ligand binding. In contrast, the variability of agonist affinity is not mediated by an altered conformational change. Thus, our data shed light on specific agonist induced conformational features and mechanisms of pLGIC upon ligand binding determining receptor activation in native environments.

Trans-Synaptic Signaling through the Glutamate Receptor Delta-1 Mediates Inhibitory Synapse Formation in Cortical Pyramidal Neurons

SummaryFine orchestration of excitatory and inhibitory synaptic development is required for normal brain function, and alterations may cause neurodevelopmental disorders. Using sparse molecular manipulations in intact brain circuits, we show that the glutamate receptor delta-1 (GluD1), a member of ionotropic glutamate receptors (iGluRs), is a postsynaptic organizer of inhibitory synapses in cortical pyramidal neurons. GluD1 is selectively required for the formation of inhibitory synapses and regulates GABAergic synaptic transmission accordingly. At inhibitory synapses, GluD1 interacts with cerebellin-4, an extracellular scaffolding protein secreted by somatostatin-expressing interneurons, which bridges postsynaptic GluD1 and presynaptic neurexins. When binding to its agonist glycine or D-serine, GluD1 elicits non-ionotropic postsynaptic signaling involving the guanine nucleotide exchange factor ARHGEF12 and the regulatory subunit of protein phosphatase 1 PPP1R12A. Thus, GluD1 defines a trans-synaptic interaction regulating postsynaptic signaling pathways for the proper establishment of cortical inhibitory connectivity and challenges the dichotomy between iGluRs and inhibitory synaptic molecules.

β-Amyloid Peptide Antagonizes the Effect of Protons on Taurine-Induced Chloride Current in Rat Hippocampal Pyramidal Neurons.

Taurine is an important endogenous agonist of glycine receptors (GlyR). Using the patchclamp technique, we measured chloride current induced by a short (600 msec) application of taurine (ITau) on isolated rat pyramidal neurons. pH of taurine solution in the applicator pipette was neutral (7.4) or acidic (7.0-5.0). Application of protons to a neuron causes a dosedependent decrease in the peak amplitude and acceleration of ITau desensitization. Addition of 100 nM β-amyloid peptide (Aβ) to the perfusate caused acceleration of ITau desensitization. The effects of Aβ and H+ on the rate of ITau desensitization were not additive. In addition, Aβ attenuated the effect of H+ on the peak amplitude of ITau. We also studied the effect of protons on the chloride current caused by activation of GABA receptors. In contrast to H+ effects on GlyR, Aβ did not modulate the effects of H+ on GABA receptors.

Modulation of extrasynaptic GABAergic receptor activity influences glutamate release and neuronal survival following excitotoxic damage to mouse spinal cord neurons

Excitotoxic levels of released glutamate trigger a cascade of deleterious cellular events leading to delayed neuronal death. This phenomenon implies extensive dysregulation in the balance between network excitation and inhibition. Our hypothesis was that enhancing network inhibition should prevent excitotoxicity and provide neuroprotection. To test this notion, we used mouse organotypic spinal slice cultures and explored if excitotoxicity caused by the potent glutamate analogue kainate was blocked by pharmacological increase in GABAA receptor activity. To this end we monitored (with a biosensor) real-time glutamate release following 1 h kainate application and quantified neuronal survival 24 h later. Glutamate release evoked by kainate was strongly decreased by the allosteric GABAA modulator midazolam (10 nM) or the GABA agonist THIP (10 μM), leading to neuroprotection. On the contrary, much higher glutamate release was induced by the GABA antagonist bicuculline (20 μM) that inhibits synaptic and extrasynaptic GABAA receptors. Gabazine (20 μM), an antagonist of synaptic GABAA receptors, had no effect on glutamate release or neuroprotection. No effect was observed with the glycine antagonist strychnine or the glycine agonist L-alanine. These findings indicate that enhancement of GABA receptor activity was an effective tool to counteract excitotoxic death in spinal networks. In view of the potent activity by THIP, preferentially acting on extrasynaptic GABAA receptors, the present data imply a significant role for extrasynaptic GABAA receptors in sparing spinal cord neurons from injury.

Metabolomics analysis explores the rescue to neurobehavioral disorder induced by maternal PM2.5 exposure in mice

Reproductive epidemiological studies have suggested associations between perinatal exposure to fine particulate matter (PM2.5) and adverse birth outcomes. To explore the effects of early prenatal exposure to PM2.5 on subsequent generations, pregnant mice were exposed to PM2.5 or filtered clean air in whole body dynamic exposure chambers for 14 consecutive days from gestation day (GD) 1.5 to GD15.5. Neurobehavioral tests showed that spontaneous locomotion and exploratory behaviors in the offspring were significantly enhanced in the open field test. Meanwhile, metabolomics analysis suggested activation of dopamine pathway while inhibition of glycine pathway in murine brains. Administration of the DRD4 antagonist, clozapine; or supplementation of glycine receptor agonist, taurine, to mice offspring attenuated the locomotor hyperactivities to levels indistinguishable from controls. These data provide strong evidence that maternal exposure to air pollution might increase the risk for neural disorders in the offspring during critical periods of brain development.

Trpv4 Mediates Hypotonic Inhibition of Central Osmosensory Neurons via Taurine Gliotransmission

The maintenance of hydromineral homeostasis requires bidirectional detection of changes in extracellular fluid osmolality by primary osmosensory neurons (ONs) in the organum vasculosum laminae terminalis (OVLT). Hypertonicity excites ONs in partthrough the mechanical activation of a variant transient receptor potential vanilloid-1 channel (dn-Trpv1). However, the mechanism by which local hypotonicity inhibits ONs in the OVLT remains unknown. Here, we show that hypotonicity can reduce the basal activity of dn-Trpv1 channels and hyperpolarize acutely isolated ONs. Surprisingly, we found that mice lacking dn-Trpv1 maintain normal inhibitory responses to hypotonicity when tested in situ. In the intact setting, hypotonicity inhibits ONs through a non-cell-autonomous mechanism that involves glial release of the glycine receptor agonist taurine through hypotonicity activated anion channels (HAAC) that are activated subsequent to Ca2+ influx through Trpv4 channels. Our study clarifies how Trpv4 channels contribute to the inhibition of OVLT ONs during hypotonicity in situ.

The LILI Motif of M3-S2 Linkers Is a Component of the NMDA Receptor Channel Gate.

N-methyl-D-aspartate receptors (NMDARs) mediate excitatory synaptic transmission in the central nervous system, underlie the induction of synaptic plasticity, and their malfunction is associated with human diseases. Native NMDARs are tetramers composed of two obligatory GluN1 subunits and various combinations of GluN2A-D or, more rarely, GluN3A-B subunits. Each subunit consists of an amino-terminal, ligand-binding, transmembrane and carboxyl-terminal domain. The ligand-binding and transmembrane domains are interconnected via polypeptide chains (linkers). Upon glutamate and glycine binding, these receptors undergo a series of conformational changes leading to the opening of the Ca2+-permeable ion channel. Here we report that different deletions and mutations of amino acids in the M3-S2 linkers of the GluN1 and GluN2B subunits lead to constitutively open channels. Irrespective of whether alterations were introduced in the GluN1 or the GluN2B subunit, application of glutamate or glycine promoted receptor channel activity; however, responses induced by the GluN1 agonist glycine were larger, on average, than those induced by glutamate. We observed the most prominent effect when residues GluN1(L657) and GluN2B(I655) were deleted or altered to glycine. In parallel, molecular modeling revealed that two interacting pairs of residues, the LILI motif (GluN1(L657) and GluN2B(I655)), form a functional unit with the TTTT ring (GluN1(T648) and GluN2B(T647)), described earlier to control NMDAR channel gating. These results provide new insight into the structural organization and functional interplay of the LILI and the TTTT ring during the course of NMDAR channel opening and closing.

Liposome-encapsulated clodronate specifically depletes spinal microglia and reduces initial neuropathic pain

Liposome-encapsulated clodronate (LEC) is a specific depletor of macrophages. Our study characterized the LEC depletory effects, given intrathecally, on spinal microglia and assessed its effects on initiation and maintenance of neuropathic pain. Measured by using the MTT assay, LEC treatment specifically inhibited cell viability of cultured primary microglia, but not astrocytes or neurons, from neonatal rats, with an IC50 of 43 μg/mL. In spinal nerve ligation-induced neuropathic rats, pretreatment (1 day but not 5 days earlier) with intrathecal LEC specifically depleted microglia (but not astrocytes or neurons) in both contralateral and ipsilateral dorsal horns by the same degree (63% vs. 71%). Intrathecal injection of LEC reversibly blocked the antinociceptive effects of the GLP-1 receptor agonist exenatide and dynorphin A stimulator bulleyaconitine, which have been claimed to be mediated by spinal microglia, whereas it failed to alter morphine- or the glycine receptor agonist gelsemine-induced mechanical antiallodynia which was mediated via the neuronal mechanisms. Furthermore, intrathecal LEC injection significantly attenuated initial (one day after nerve injury) but not existing (2 weeks after nerve injury) mechanical allodynia. Our study demonstrated that LEC, given intrathecally, is a specific spinal microglial inhibitor and significantly reduces initiation but not maintenance of neuropathic pain, highlighting an opposite role of spinal microglia in different stages of neuropathic pain.

Probing Dynamic Heterogeneity in Aggregated Ion Channels in Live Cells

N-Methyl-d-aspartate (NMDA) receptor is a crucial ion channel requiring the concurrent binding of both agonists glycine and glutamate along with the membrane depolarization for its opening and mediates calcium influx, which triggers the signal transduction important for synaptic transmission and plasticity. The efficiency and regulations of such cellular physiological processes are crucially relying on inherent molecular spatial organization and interactions of these channels with ligands as well as trafficking in the confined local environment. To decipher their organization, we have combined two imaging microscopic approaches, photobleaching step counting and single particle tracking analysis, that allow detection of unique organized patterns of NMDA receptor ion channel in the cell membrane. A broad range of photobleaching steps is observed ranging from 1 to 16. Single particle tracking analysis is performed to see the surface mobility of NMDA receptors and measured diffusion coefficient for all the tr...

Novel Mode of Antagonist Binding in NMDA Receptors Revealed by the Crystal Structure of the GluN1-GluN2A Ligand-Binding Domain Complexed to NVP-AAM077

Competitive antagonists against N-methyl-D-aspartate (NMDA) receptors have played critical roles throughout the history of neuropharmacology and basic neuroscience. There are currently numerous NMDA receptor antagonists containing a variety of chemical groups. Among those compounds, a GluN2-specific antagonist, (R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl-phosphonic acid (NVP-AAM077), contains a unique combination of a dioxoquinoxalinyl ring, a bromophenyl group, and a phosphono group. In this study, we present the crystal structure of the isolated ligand-binding domain of the GluN1-GluN2A NMDA receptor in complex with the GluN1 agonist glycine and the GluN2A antagonist NVP-AAM077. The structure shows placement of the dioxoquinoxalinyl ring and the phosphono group of NVP-AAM077 in the glutamate-binding pocket in GluN2A and the novel interaction between the bromophenyl group and GluN1-Glu781 at the GluN1-GluN2A subunit interface. Site-directed mutagenesis of GluN1-Glu781 reduced the potency of inhibition by NVP-AAM077, thus confirming the involvement of the GluN1 subunit for binding of NVP-AAM077. The unique antagonist-binding pattern shown in this study provides a novel dimension to design and create antagonists with potential therapeutic values.