Glutamate Binding Sites Research Articles

Glutamate acts on acid-sensing ion channels to worsen ischaemic brain injury.

Glutamate is traditionally viewed as the first messenger to activate NMDAR (N-methyl-D-aspartate receptor)-dependent cell death pathways in stroke1,2, but unsuccessful clinical trials with NMDAR antagonists implicate the engagement of other mechanisms3-7. Here we show that glutamate and its structural analogues, including NMDAR antagonist L-AP5 (also known as APV), robustly potentiate currents mediated by acid-sensing ion channels (ASICs) associated with acidosis-induced neurotoxicity in stroke4. Glutamate increases the affinity of ASICs for protons and their open probability, aggravating ischaemic neurotoxicity in both in vitro and in vivo models. Site-directed mutagenesis, structure-based modelling and functional assays reveal a bona fide glutamate-binding cavity in the extracellular domain of ASIC1a. Computational drug screening identified a small molecule, LK-2, that binds to this cavity and abolishes glutamate-dependent potentiation of ASIC currents but spares NMDARs. LK-2 reduces the infarct volume and improves sensorimotor recovery in a mouse model of ischaemic stroke, reminiscent of that seen in mice with Asic1a knockout or knockout of other cation channels4-7. We conclude that glutamate functions as a positive allosteric modulator for ASICs to exacerbate neurotoxicity, and preferential targeting of the glutamate-binding site on ASICs over that on NMDARs may be strategized for developing stroke therapeutics lacking the psychotic side effects of NMDAR antagonists.

Computational Investigation of BMAA and Its Carbamate Adducts as Potential GluR2 Modulators.

Beta-N-methylamino-l-alanine (BMAA) is a potential neurotoxic nonprotein amino acid, which can reach the human body through the food chain. When BMAA interacts with bicarbonate in the human body, carbamate adducts are produced, which share a high structural similarity with the neurotransmitter glutamate. It is believed that BMAA and its l-carbamate adducts bind in the glutamate binding site of ionotropic glutamate receptor 2 (GluR2). Chronic exposure to BMAA and its adducts could cause neurological illness such as neurodegenerative diseases. However, the mechanism of BMAA action and its carbamate adducts bound to GluR2 has not yet been elucidated. Here, we investigate the binding modes and the affinity of BMAA and its carbamate adducts to GluR2 in comparison to the natural agonist, glutamate, to understand whether these can act as GluR2 modulators. Initially, we perform molecular dynamics simulations of BMAA and its carbamate adducts bound to GluR2 to examine the stability of the ligands in the S1/S2 ligand-binding core of the receptor. In addition, we utilize alchemical free energy calculations to compute the difference in the free energy of binding of the beta-carbamate adduct of BMAA to GluR2 compared to that of glutamate. Our findings indicate that carbamate adducts of BMAA and glutamate remain stable in the binding site of the GluR2 compared to BMAA. Additionally, alchemical free energy results reveal that glutamate and the beta-carbamate adduct of BMAA have comparable binding affinity to the GluR2. These results provide a rationale that BMAA carbamate adducts may be, in fact, the modulators of GluR2 and not BMAA itself.

Serine protease inhibitors LmSPN2 and LmSPN3 co-regulate embryonic diapause in Locusta migratoria manilensis (Meyen) via the Toll pathway

Female adults of the migratory locust, Locusta migratoria manilensis (Meyen), can sense seasonal photoperiod changes, which induces embryonic diapause as a key strategy to overwinter. Serine protease inhibitor genes (SPNs) were thought to play key roles during diapause, while few SPNs were functionally characterized. LmSPN2 was one of those genes differentially expressed between diapause and non-diapause eggs; however, its biological function remained to be explored. So, we conducted RNAi knockdown of LmSPN2, resulting in a significant decrease of the egg diapause rate by 29.7%. Using yeast two-hybrid assays, co-immunoprecipitation, and pull-down methods, we found an interaction between LmSPN2 and LmSPN3, which was proved to be mediated by a glutamate (E331) binding site of LmSPN2. RNAi knockdown of LmSPN3 resulted in a significant increase in diapause rate by 14.6%, indicating an inverse function of LmSPN2 and LmSPN3 on diapause regulation. Double knockdown of two SPN genes resulted in a 26.4% reduction in diapause rate, indicating that LmSPN2 was the dominant regulatory signal. Moreover, we found four Toll pathway genes (easter, spätzle, pelle, and dorsal) upregulated significantly after the knockdown of LmSPN2 while downregulated after the knockdown of LmSPN3. Therefore, we speculate that two SPNs regulate diapause through the Toll pathway. Our results indicated that LmSPN2 positively regulates locust egg entry into diapause, while LmSPN3 is a negative regulator of embryonic commitment to diapause. Their interaction is mediated by the binding site of E331 and influences egg diapause through the Toll pathway. This mechanistic understanding of diapause regulation expands our understanding of insect developmental regulation and provides functional targets for developing locust management strategies.

Chemerin-9 in paraventricular nucleus increases sympathetic outflow and blood pressure via glutamate receptor-mediated ROS generation

Chemerin is an adipokine involved in regulating energy homeostasis and reproductive function. Excessive sympathetic activity contributes to hypertension, chronic heart failure and chronic renal disease. Hypothalamic paraventricular nucleus (PVN) is crucial in regulating sympathetic activity and blood pressure. The present study is designed to investigate the roles of chemerin in the PVN in regulating sympathetic activity and blood pressure and underlying mechanisms. Microinjections were performed in the bilateral PVN in male adult rats under anesthesia. Renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were continuously recorded. The PVN microinjection of chemerin-9, an active fragment of chemerin, increased RSNA and MAP, which were abolished by chemokine-like receptor 1 (CMKLR1) antagonist α-NETA, a superoxide scavenger tempol, antioxidant N-acetylcysteine (NAC), NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) and apocynin. Immunofluorescence analyses showed that N-methyl-D-aspartate (NMDA) receptors existed in most of cells of the PVN, and some of them co-existed with chemerin. The effects of chemerin-9 on RSNA and MAP were prevented by glutamate-binding site antagonist L-phenylalanine, NMDA receptor antagonist MK-801, and calcium channel blocker verapamil or nifedipine, but only attenuated by non-NMDA receptor antagonist CNQX. Moreover, chemerin-9 increased NADPH oxidase activity and superoxide production, which were prevented by α-NETA, MK-801, or verapamil. These results indicate that chemerin-9 in the PVN increases sympathetic activity and blood pressure via CMKLR1-dependent calcium influx, and glutamate receptor-mediated NADPH oxidase activation and subsequent superoxide production.

Structural insights into inhibitory mechanism of human excitatory amino acid transporter EAAT2

Glutamate is a pivotal excitatory neurotransmitter in mammalian brains, but excessive glutamate causes numerous neural disorders. Almost all extracellular glutamate is retrieved by the glial transporter, Excitatory Amino Acid Transporter 2 (EAAT2), belonging to the SLC1A family. However, in some cancers, EAAT2 expression is enhanced and causes resistance to therapies by metabolic disturbance. Despite its crucial roles, the detailed structural information about EAAT2 has not been available. Here, we report cryo-EM structures of human EAAT2 in substrate-free and selective inhibitor WAY213613-bound states at 3.2 Å and 2.8 Å, respectively. EAAT2 forms a trimer, with each protomer consisting of transport and scaffold domains. Along with a glutamate-binding site, the transport domain possesses a cavity that could be disrupted during the transport cycle. WAY213613 occupies both the glutamate-binding site and cavity of EAAT2 to interfere with its alternating access, where the sensitivity is defined by the inner environment of the cavity. We provide the characterization of the molecular features of EAAT2 and its selective inhibition mechanism that may facilitate structure-based drug design for EAAT2.

6-Gingerol, a Major Constituent of Zingiber officinale Rhizoma, Exerts Anticonvulsant Activity in the Pentylenetetrazole-Induced Seizure Model in Larval Zebrafish.

Zingiber officinale is one of the most frequently used medicinal herbs in Asia. Using rodent seizure models, it was previously shown that Zingiber officinale hydroethanolic extract exerts antiseizure activity, but the active constituents responsible for this effect have not been determined. In this paper, we demonstrated that Zingiber officinale methanolic extract exerts anticonvulsant activity in the pentylenetetrazole (PTZ)-induced hyperlocomotion assay in larval zebrafish. Next, we isolated 6-gingerol (6-GIN)—a major constituent of Zingiber officinale rhizoma. We observed that 6-GIN exerted potent dose-dependent anticonvulsant activity in the PTZ-induced hyperlocomotion seizure assay in zebrafish, which was confirmed electroencephalographically. To obtain further insight into the molecular mechanisms of 6-GIN antiseizure activity, we assessed the concentration of two neurotransmitters in zebrafish, i.e., inhibitory γ-aminobutyric acid (GABA) and excitatory glutamic acid (GLU), and their ratio after exposure to acute PTZ dose. Here, 6-GIN decreased GLU level and reduced the GLU/GABA ratio in PTZ-treated fish compared with only PTZ-bathed fish. This activity was associated with the decrease in grin2b, but not gabra1a, grin1a, gria1a, gria2a, and gria3b expression in PTZ-treated fish. Molecular docking to the human NR2B-containing N-methyl-D-aspartate (NMDA) receptor suggests that 6-GIN might act as an inhibitor and interact with the amino terminal domain, the glutamate-binding site, as well as within the ion channel of the NR2B-containing NMDA receptor. In summary, our study reveals, for the first time, the anticonvulsant activity of 6-GIN. We suggest that this effect might at least be partially mediated by restoring the balance between GABA and GLU in the epileptic brain; however, more studies are needed to prove our hypothesis.

Study Of The Neuroprotective Properties Of Biologically Active Compounds

The works show that, using fluorescent probes, it was used to study the effect of PC-8 on changes in the dynamics of the intracellular Ca2+ content in synaptosomes of the rat brain, depending on the site of the binding of glutamate on calcium channels by a specific mediator with glutamate. To measure the amount of cytosolic Ca2+ synaptosomes, we calculated using the Grinkevich equation. It has been shown that polyphenol PC-8 binds to the glutamate-binding site of NMDA receptors, so that the conductance for Ca2+ ions is reduced through a channel blocking the effect of polyphenol PC-8 can be explained. due to its binding to the competing action of the site, the binding of glutamate to NMDA receptors.

Esmethadone (REL-1017) Antagonizes NMDA Receptors and Reduces Ca2+ Entry in Presence of Quinolinic Acid and Gentamicin

Esmethadone (dextromethadone; REL-1017) is a novel NMDA receptor (NMDAR) antagonist currently in clinical development for major depressive disorder (MDD). To better understand its role as an NMDAR channel blocker, we examined the functional interaction between REL-1017 and quinolinic acid, an endogenous partial agonist at the glutamate binding site of the NMDAR, and REL-1017 and gentamicin, an antibiotic that modulates NMDAR potentiation.

Stereoselective synthesis of novel 2'-(S)-CCG-IV analogues as potent NMDA receptor agonists.

We developed a versatile stereoselective route for the synthesis of new 2'-(S)-CCG-IV analogues. The route allows for late stage diversification and thereby provides access to a great variety of conformationally restricted cyclopropyl glutamate analogues. A selection of the 2'-(S)-CCG-IV analogues were evaluated using two-electrode voltage-clamp electrophysiology at recombinant GluN1/GluN2A-D receptors, demonstrating that agonists can be developed with GluN2 subunit-dependent potency and agonist efficacy. We also describe a crystal structure of the GluN2A agonist binding domain in complex with 2'-butyl-(S)-CCG-IV that determines the position of 2'-substituents in (S)-CCG-IV agonists in the glutamate binding site and provides further insight to the structural determinants of their agonist efficacy. The stereoselective synthesis described here enables versatile and straight-forward modifications to diverse analogues of interest for the development of potent subtype-specific NMDA receptor agonists and other applications.

Mechanism and potential sites of potassium interaction with glutamate transporters.

In the mammalian glutamate transporters, countertransported intracellular K+ is essential for relocating the glutamate binding site to the extracellular side of the membrane. This K+-dependent process is believed to be rate limiting for the transport cycle. In contrast, extracellular K+ induces glutamate release upon transporter reversal. Here, we analyzed potential K+ binding sites using molecular dynamics (MD) simulations and site-directed mutagenesis. Two candidate sites were identified by spontaneous K+ binding in MD simulations, one site (K1 site) overlapping with the Na1 Na+ binding site and the K2 site being localized under hairpin loop 2 (HP2). Mutations to conserved amino acid residues in these sites resulted in several transporters that were defective in K+-induced reverse transport and which bound K+ with reduced apparent affinity compared with the wild-type transporter. However, external K+ interaction was abolished in only one mutant transporter EAAC1D454A in the K1 site. Our results, for the first time, directly demonstrate effects of K1-site mutations on K+ binding, in contrast to previous reports on K+ binding sites based on indirect evidence. We propose that K+ binding to the K1 site is responsible for catalyzing the relocation step, whereas binding to the K2 site may have an as-of-yet unidentified regulatory function.

The GluN3 subunit regulates ion selectivity within native N-methyl-d-aspartate receptors

Glutamatergic N-methyl-d-aspartate receptors (NMDARs) are heterotetrameric proteins whose subunits are derived from three gene families, GRIN1 (codes for GluN1), GRIN2 (GluN2) and GRIN3 (GluN3). In addition to providing binding sites for glutamate and the co-agonist glycine, these subunits in their di (d-) and tri (t-) heteromeric configurations regulate various aspects of receptor function in the brain. For example, the decay kinetics of NMDAR-mediated synaptic currents depend on the type of GluN2 subunit (GluN2A-GluN2D) in the receptor subunit composition. While much is known about the contributions of GluN1 and GluN2 to d-NMDAR function, we know comparatively little about how GluN3 influences the function of t-NMDARs composed of one or more subunits from each of the three gene families. We report here that in addition to altering kinetics and voltage-dependent properties, the GluN3 subunit endows these receptors with ion selectivity wherein influx of Ca2+ is preferred over Na+. This became apparent in the process of assessing Ca2+ permeability through these receptors and is of significance given that NMDARs are generally believed to be nonselective to cations and increased selectivity can lead to enhanced permeability. This was true of two independent brain regions where t-NMDARs are expressed, the somatosensory cortex, where both receptor subtypes are expressed at separate inputs onto single neurons, and the entorhinal cortex, where they are co-expressed at individual synaptic inputs. Based on this data and the sequence of amino acids lining selectivity filters within these subunits, we propose GluN3 to be a regulatory subunit for ion selectivity in t-NMDARs.

The Human SLC1A5 Neutral Amino Acid Transporter Catalyzes a pH-Dependent Glutamate/Glutamine Antiport, as Well.

ASCT2 is a neutral amino acid transporter, which catalyzes a sodium-dependent obligatory antiport among glutamine and other neutral amino acids. The human ASCT2 over-expressed in Pichia pastoris and reconstituted in proteoliposomes has been employed for identifying alternative substrates of the transporter. The experimental data highlighted that hASCT2 also catalyzes a sodium-dependent antiport of glutamate with glutamine. This unconventional antiport shows a preferred sidedness: glutamate is inwardly transported in exchange for glutamine transported in the counter direction. The orientation of the transport protein in proteoliposomes is the same as in the cell membrane; then, the observed sidedness corresponds to the transport of glutamate from the extracellular to the intracellular compartment. The competitive inhibition exerted by glutamate on the glutamine transport together with the docking analysis indicates that the glutamate binding site is the same as that of glutamine. The affinity for glutamate is lower than that for neutral amino acids, while the transport rate is comparable to that measured for the asparagine/glutamine antiport. Differently from the neutral amino acid antiport that is insensitive to pH, the glutamate/glutamine antiport is pH-dependent with optimal activity at acidic pH on the external (extracellular) side. The stimulation of glutamate transport by a pH gradient suggests the occurrence of a proton flux coupled to the glutamate transport. The proton transport has been detected by a spectrofluorometric method. The rate of proton transport correlates well with the rate of glutamate transport indicating a 1:1 stoichiometry H+: glutamate. The glutamate/glutamine antiport is also active in intact HeLa cells. On a physiological point of view, the described antiport could have relevance in some districts in which a glutamate/glutamine cycling is necessary, such as in placenta.

Binding of a negative allosteric modulator and competitive antagonist can occur simultaneously at the ionotropic glutamate receptor GluA2

Ionotropic glutamate receptors are ligand-gated ion channels governing neurotransmission in the central nervous system. Three major types of antagonists are known for the AMPA-type receptor GluA2: competitive, noncompetitive (i.e., negative allosteric modulators; NAMs) used for treatment of epilepsy, and uncompetitive antagonists. We here report a 4.65Å resolution X-ray structure of GluA2, revealing that four molecules of the competitive antagonist ZK200775 and four molecules of the NAM GYKI53655 are capable of binding at the same time. Using negative stain electron microscopy, we show that GYKI53655 alone or ZK200775/GYKI53655 in combination predominantly results in compact receptor forms. The agonist AMPA provides a mixed population of compact and bulgy shapes of GluA2 not impacted by addition of GYKI53655. Taken together, this suggests that the two different mechanisms of antagonism that lead to channel closure are independent and that the distribution between bulgy and compact receptors primarily depends on the ligand bound in the glutamate binding site. DATABASE: The atomic coordinates and structure factors from the crystal structure determination have been deposited in the Protein Data Bank under accession code https://doi.org/10.2210/pdb6RUQ/pdb. The electron microscopy 3D reconstruction volumes have been deposited in EMDB (EMD-4875: Apo; EMD-4920: ZK200775/GYKI53655; EMD-4921: AMPA compact; EMD-4922: AMPA/GYKI53655 bulgy; EMD-4923: GYKI53655; EMD-4924: AMPA bulgy; EMD-4925: AMPA/GYKI53655 compact).

Probing the Role of Chloride in Facilitating System xc− Function

System xc− is a heterodimeric transporter that functions as a Na+‐independent antiporter exchanging extracellular cystine for intracellular glutamate. The transporter functions as an obligate heterodimer and is comprised of two proteins, xCT, which functions specifically in amino acid exchange, and 4F2HC, which appears to play a role in transporter stability. System xc− belongs to the SLC7 family of transporters, which is further subdivided into the cationic amino acid transporters (CAT) and the L‐type amino acid transporters (LAT), the latter of which is the subfamily which includes System xc−. In recent years, there have been advances in our understanding of the structure of the SLC7 transporters as crystal structures of bacterial homologs of CAT and LAT transporters have been published. As a result, we can now begin to ask important questions about the mechanism by which System xc− exchanges cystine and glutamate across the membrane. Previous studies in the Chase lab have demonstrated that System xc− is Cl−‐dependent. More recently we have shown that transport of one molecule of cystine requires more than one Cl− ion, and that Cl− binding affects the affinity of the transporter for cystine. We have also shown that chloride dependence of cystine transport is pH dependent. The K0.5 of chloride increases as the pH increases, suggesting that the Cl− binding affinity decreases at higher pH. Using this collective information, we examined the structure of xCT to identify the most likely binding site for cystine and glutamate. Specifically, we used Chimera 1.13.1 and Pyrx to dock cystine and glutamate into its most energetically favorable positions on xCT and identified several ionizable side chains within the putative permeation pathway associated with transmembrane domains 3 and 6 that appear to interact with substrate. These residues include R135, T138, Y246 and Y251. We are currently assessing this model in the presence of Cl− to identify putative residues which may be important for the docking of Cl− near the substrate binding site and using site‐directed mutagenesis to determine whether these residues are responsible for Cl−‐dependent cystine transport.Support or Funding InformationThis research is supported by the Campbell Foundation and Schaap Endowed Funds for Undergraduate Research.

Structure-based discovery and development of metabotropic glutamate receptor 5 negative allosteric modulators.

The metabotropic glutamate (mGlu) receptors are a family of eight class C G protein-coupled receptors (GPCRs) which modulate cell signaling and synaptic transmission to the major excitatory neurotransmitter l-glutamate (l-glutamic acid). Due to their role in modulating glutamate response, their widespread distribution in the central nervous system (CNS) and some evidence of dysregulation in disease, the mGlu receptors have become attractive pharmacological targets. As the orthosteric (glutamate) binding site is highly conserved across the eight mGlu receptors, it is difficult not only to generate ligands with subtype selectivity but, due to the nature of the binding site, with suitable drug-like properties to allow oral bioavailability and CNS penetration. Selective pharmacological targeting of a single receptor subtype can be achieved by targeting alternative (allosteric) binding sites. The nature of the allosteric binding pockets allows ligands to be developed that have good physical chemical properties as evidenced by several allosteric modulators of mGlu receptors entering clinical trials. The first negative allosteric modulators of the metabotropic glutamate 5 (mGlu5) receptor were discovered from high throughput screening activities. An alternative approach to drug discovery is to use structural knowledge to enable structure-based drug design (SBDD), which allows the design of molecules in a more rational, rather than empirical, fashion. Here we will describe the process of SBDD in the discovery of the mGlu5 negative allosteric modulator HTL0014242 and describe how knowledge of receptor structure can also be used to gain insights into the receptor activation mechanisms.

Förster Resonance Energy Transfer (FRET) Analysis of the C-Terminal Domain of Fp-Tagged Homomeric and Heteromeric AMPARs Upon Phosphorylation

AMPA receptors (AMPARs) are glutamate-gated cation channels that mediate the majority of fast excitatory neurotransmission in the central nervous system. AMPARs are tetrameric assemblies of GluA1 to GluA4 subunits. Recently Cryo-EM and X-ray crystal structures of homomeric and heteromeric AMPARs has been provided; showing a highly modular architecture with an extracellular domain containing four glutamate binding sites coupled to a transmembrane domain containing the central ion channel. AMPARs also contain an intracellular domain (ICD) formed of C-terminals from each subunit. The ICD is important for interactions with intracellular proteins and contains multiple regulatory phosphorylation sites, but its structural and mechanistic role is poorly understood as the ICD is not resolved in present structures. We have used a Förster Resonance Energy Transfer (FRET) approach to study intramolecular distances and dynamics of the ICD in homomeric and heteromeric AMPARs by insertion of fluorescent proteins (FPs) at various intracellular positions in the GluA1 and GluA2 subunits. Using fluorescence life-time imaging (FLIM) to determine FRET efficiencies between FPs or a membrane dye, we determine relative distances within the ICD and to the membrane. Furthermore, we investigate the effect of phosphorylation mimicking mutations in the GluA1 subunit on FRET and identify mutation-induced changes that indicate phosphorylation state of the ICD can change its structure. Our preliminary data provides new insight into the structural architecture and potential role of the ICD.

A Light-Controlled Allosteric Modulator Unveils a Role for mGlu4 Receptors During Early Stages of Ischemia in the Rodent Cerebellar Cortex.

Metabotropic glutamate receptors (mGlus) are G Protein coupled-receptors that modulate synaptic transmission and plasticity in the central nervous system. Some act as autoreceptors to control neurotransmitter release at excitatory synapses and have become attractive targets for drug therapy to treat certain neurological disorders. However, the high degree of sequence conservation around the glutamate binding site makes the development of subtype-specific orthosteric ligands difficult to achieve. This problem can be circumvented by designing molecules that target specific less well conserved allosteric sites. One such allosteric drug, the photo-switchable compound OptoGluNAM4.1, has been recently employed to reversibly inhibit the activity of metabotropic glutamate 4 (mGlu4) receptors in cell cultures and in vivo. We studied OptoGluNAM4.1 as a negative modulator of neurotransmission in rodent cerebellar slices at the parallel fiber – Purkinje cell synapse. Our data show that OptoGluNAM4.1 antagonizes pharmacological activation of mGlu4 receptors in a fully reversible and photo-controllable manner. In addition, for the first time, this new allosteric modulator allowed us to demonstrate that, in brain slices from the rodent cerebellar cortex, mGlu4 receptors are endogenously activated in excitotoxic conditions, such as the early phases of simulated cerebellar ischemia, which is associated with elevated levels of extracellular glutamate. These findings support OptoGluNAM4.1 as a promising new tool for unraveling the role of mGlu4 receptors in the central nervous system in physio-pathological conditions.

Evidences on the role of the lid loop of γ-glutamyltransferases (GGT) in substrate selection

γ-Glutamyltransferase (GGT) catalyzes the transfer of the γ-glutamyl moiety from a donor substrate such as glutathione to water (hydrolysis) or to an acceptor amino acid (transpeptidation) through the formation of a γ-glutamyl enzyme intermediate.The vast majority of the known GGTs has a short sequence covering the glutamate binding site, called lid-loop. Although being conserved enzymes, both B. subtilis GGT and the related enzyme CapD from B. anthracis lack the lid loop and, differently from other GGTs, both accept poly-γ-glutamic acid (γ-PGA) as a substrate. Starting from this observation, in this work the activity of an engineered mutant enzyme containing the amino acid sequence of the lid loop from E. coli GGT inserted into the backbone of B. subtilis GGT was compared to that of the lid loop-deficient B. subtilis GGT and the lid loop-carrier E. coli GGT. Results indicate that the absence of the lid loop seems not to be the sole structural feature responsible for the recognition of a polymeric substrate by GGTs. Nevertheless, time course of hydrolysis reactions carried out using oligo-γ-glutamyl glutamines as substrates showed that the lid loop acts as a gating structure, allowing the preferential selection of the small glutamine with respect to the oligomeric substrates. In this respect, the mutant B. subtilis GGT revealed to be more similar to E. coli GGT than to its wild-type counterpart. In addition, the transpeptidase activity of the newly produced mutant enzyme revealed to be higher with respect to that of both E. coli and wild-type B. subtilis GGT. These findings can be helpful in selecting GGTs intended as biocatalysts for preparative purposes as well as in designing mutant enzymes with improved transpeptidase activity.

F11. TRANSLATIONAL STUDY OF GRIN1, GRIN2A AND 2B GENE EXPRESSION IN PATIENTS WITH SCHIZOPHRENIA AND ANIMAL MODELS

BackgroundChanges in glutamatergic system, specifically the ionotropic receptor N-methyl-D-aspartate (NMDAR), are involved in psychosis. NMDARs could be composed of two NR1 and two NR2 subunits. NR1 is one obligatory subunit and is the glycine binding site; and NR2 subunit contain the binding site for the neurotransmitter glutamate and have four different subtypes including NR2A-D. NR1 and NR2A-B are essential subunits of NMDAR, which are encoded by genes Grin1, Grin2A and Grin2B, and have been identified as candidate genes for psychiatric disorders. NMDARs dysfunction disrupts neural excitation and to contribute to the altered brain function underlying, especially in schizophrenia and other psychosis. The aims of this work were 1) to evaluate the expression of Grin1, Grin2A and 2B genes by qPCR of patients with first episode of schizophrenia compared with the siblings and controls; 2) to quantify the NR1 and NR2 subunits plasma concentrations by ELISA; 3) to evaluate the Grin1, Grin2A and 2B gene expression by qPCR in peripheral blood and animals brain tissue.MethodsParticipants will be 30 patients diagnosed with schizophrenia or schizophreniform disorder, including the shorter illness without substance addiction; those participants with siblings who agreed to participate (n = 30) and 30 controls, matched to patients by sex, age and education. Male Wistar rats were kept isolated (n = 10) or grouped (n = 10) from weaning for 10 weeks. After this period the animals were exposed to the Open Field and soon afterwards they were sacrificed, hippocampus and prefrontal cortex (PFC) were dissected to RNA extraction. RT-PCR was performed using probes and TaqMan mastermix to evaluate the mRNA expression. One-way ANOVA with a Bonferroni correction was used for statistical analysis.ResultsHumans: Regarding the glutamatergic system, none of the chosen genes were expressed in the studied sample. Animals: Isolated reared animals presented hyperlocomotion at the two first time bins (0–5 and 5–10 min) in periphery of the arena when compared to the grouped [0–5 min: p = 0.025; 5–10 min: p = 0.002], respectively. Decreased expression of Grin1 (31%), Grin2A (45%) and Grin2B (52%) were found in PFC of isolated animals when compared to grouped (p < 0.05), while no changes were found in the hippocampus.DiscussionChanges in the expression of essential isoforms (NR1 and NR2) that make up NMDAR in PFC suggest abnormalities of glutamatergic neurotransmission in the pathophysiology of schizophrenia, corroborating recent studies. In addition, this study reinforces the validity of the social isolation rearing model from weaning with a better understanding of the mechanisms of NMDAR hypofunction and the influence of the environment on gene expression in this disorder.

Investigation of Agonist Recognition and Channel Properties in a Flatworm Glutamate-Gated Chloride Channel.

Glutamate-gated chloride channels (GluCls) are neurotransmitter receptors that mediate crucial inhibitory signaling in invertebrate neuromuscular systems. Their role in invertebrate physiology and their absence from vertebrates make GluCls a prime target for antiparasitic drugs. GluCls from flatworm parasites are substantially different from and are much less understood than those from roundworm and insect parasites, hindering the development of potential therapeutics targeting GluCls in flatworm-related diseases such as schistosomiasis. Here, we sought to dissect the molecular and chemical basis for ligand recognition in the extracellular glutamate binding site of SmGluCl-2 from Schistosoma mansoni, using site-directed mutagenesis, noncanonical amino acid incorporation, and electrophysiological recordings. Our results indicate that aromatic residues in ligand binding loops A, B, and C are important for SmGluCl-2 function. Loop C, which differs in length compared to other pentameric ligand-gated ion channels (pLGICs), contributes to ligand recognition through both an aromatic residue and two vicinal threonine residues. We also show that, in contrast to other pLGICs, the hydrophobic channel gate in SmGluCl-2 extends from the 9' position to the 6' position in the channel-forming M2 helix. The 6' and 9' positions also seem to control sensitivity to the pore blocker picrotoxin.