Amélie S Tora

Xavier Rovira

Anne-Marinette Cao

Alexandre Cabaye

Linnea Olofsson

Fanny Malhaire

Pauline Scholler

Hayeon Baik

Ann Van Eeckhaut

Ilse Smolders

Alexandre Cabayé

Linnéa Olofsson

Philippe Rondard

Emmanuel Margeat

Francine Acher
email: francine.acher@parisdescartes.fr

Jean-Philippe Pin
email: jean-philippe.pin@igf.cnrs.fr

Cyril Goudet
email: cyril.goudet@igf.cnrs.fr

Chloride ions stabilize the glutamate-induced active state of the metabotropic glutamate receptor 3

Keywords: Glutamate, Receptor, Chloride, Constitutive activity, Allostery, GPCR

INTRODUCTION

The amino acid L-glutamate is an essential element of life, and has been very early used as a messenger.

During evolution, L-glutamate became one of the major neurotransmitters, being the mediator of the neuromuscular junction in arthropods and being responsible for transmitting signals at most fast excitatory synapses in the brain [START_REF] Fonnum | Glutamate: a neurotransmitter in mammalian brain[END_REF]. Such an essential role of glutamate, associated with all the other functions of this amino acid, imposes a tight regulation of its extracellular concentrations. Indeed, an inaccurate control of extracellular glutamate levels can lead to epilepsy and brain damage through excitotoxicity [START_REF] Meldrum | Glutamate as a neurotransmitter in the brain: review of physiology and pathology[END_REF].

Essential mechanisms have then been selected during evolution to monitor extracellular glutamate concentrations, including active uptake mechanisms and a control of glutamate entry at the level of the blood brain barrier. Defects in these mechanisms have been proposed to be responsible for a number of brain diseases, including neurodegenerative disorders as well as psychiatric diseases or addiction [START_REF] Kalivas | The glutamate homeostasis hypothesis of addiction[END_REF][START_REF] Lewerenz | Chronic Glutamate Toxicity in Neurodegenerative Diseases-What is the Evidence?[END_REF][START_REF] Miladinovic | Overview of Glutamatergic Dysregulation in Central Pathologies[END_REF].

Glutamate activates cationic glutamate-gated channels (i.e. ionotropic glutamate receptors) that mediate the fast excitatory actions of glutamate. It also activate G protein-coupled receptors, named metabotropic glutamate receptors (mGluRs) [START_REF] Pin | Organization and functions of mGlu and GABAB receptor complexes[END_REF]. The eight subtypes of mGluRs are key modulators of transmission at most synapses in the brain. Some mGluRs are located post-synaptically (the group-I mGlu 1 and mGlu5), allowing a tight control of the post-synaptic response, while others (group-II, mGlu2 & 3, and group- III,mGlu4,7 & 8) are mainly found in pre-synaptic terminals where they inhibit the release of various neurotransmitters. Aside from the group-III mGlu6, which is responsible of the ON-bipolar neuronal transmission in the retina [START_REF] Vardi | Localization of mGluR6 to dendrites of ON bipolar cells in primate retina[END_REF], mGluRs can be considered as glutamate sensors allowing cells to adapt their response as a function of the extracellular glutamate concentration. Indeed, mGluRs are not only found at glutamatergic synapses, but also at other types of synapses, including dopaminergic (DA) and GABAergic synapses, as well as in other regulatory cells such as astrocytes and microglial cells [START_REF] Nicoletti | Metabotropic glutamate receptors: from the workbench to the bedside[END_REF].

All mGluRs share a common structural architecture [START_REF] Pin | Organization and functions of mGlu and GABAB receptor complexes[END_REF]. They are obligate dimers, each protomer being composed of a large bi-lobed extracellular domain (ECD) containing a Venus Flytrap (VFT) and a cysteine-rich domain (CRD) that connects to the seven helices transmembrane spanning domain (TMD).

The VFT domain binds orthosteric ligands and also endogenous allosteric modulators, notably extracellular ions, such as calcium and chloride, that potentiate receptor activity [START_REF] Diraddo | A real-time method for measuring cAMP production modulated by Galphai/o-coupled metabotropic glutamate receptors[END_REF][START_REF] Jiang | Extracellular calcium modulates actions of orthosteric and allosteric ligands on metabotropic glutamate receptor 1alpha[END_REF][START_REF] Kuang | Ion dependence of ligand binding to metabotropic glutamate receptors[END_REF][START_REF] Kubo | Structural basis for a Ca2+-sensing function of the metabotropic glutamate receptors[END_REF][START_REF] Tora | Allosteric modulation of metabotropic glutamate receptors by chloride ions[END_REF][START_REF] Vafabakhsh | Conformational dynamics of a class C G-proteincoupled receptor[END_REF].

The group-II mGlu3 receptor is found at the post-synapse and in pre-synaptic elements in neurons and in astrocytes, facing the micro-vessels, making this receptor a likely key component of the detection of the extracellular glutamate concentration [START_REF] Mudo | Cellular localization of mGluR3 and mGluR5 mRNAs in normal and injured rat brain[END_REF][START_REF] Ohishi | Distribution of the mRNA for a metabotropic glutamate receptor (mGluR3) in the rat brain: an in situ hybridization study[END_REF][START_REF] Tamaru | Distribution of metabotropic glutamate receptor mGluR3 in the mouse CNS: differential location relative to pre-and postsynaptic sites[END_REF]. Genetic studies revealed a possible involvement of this receptor in various diseases, including schizophrenia and other psychiatric disorders [START_REF] Consortium | Biological insights from 108 schizophrenia-associated genetic loci[END_REF][START_REF] De Filippis | The role of group II metabotropic glutamate receptors in cognition and anxiety: comparative studies in GRM2(-/-), GRM3(-/-) and GRM2/3(-/-) knockout mice[END_REF][START_REF] Egan | Variation in GRM3 affects cognition, prefrontal glutamate, and risk for schizophrenia[END_REF][START_REF] Harrison | The group II metabotropic glutamate receptor 3 (mGluR3, mGlu3, GRM3): expression, function and involvement in schizophrenia[END_REF][START_REF] O'brien | The functional GRM3 Kozak sequence variant rs148754219 affects the risk of schizophrenia and alcohol dependence as well as bipolar disorder[END_REF], as well as cancer development at the periphery [START_REF] Kunz | Oncogenes in melanoma: an update[END_REF][START_REF] Yi | The miR-487b-3p/GRM3/TGFbeta signaling axis is an important regulator of colon cancer tumorigenesis[END_REF]. When compared to other mGlu receptor subtypes, mGlu3 receptor often displays a strong basal activity in transfected cells, therefore limiting their full functional and pharmacological characterization.

The present study aimed to elucidate the structural and molecular basis of the high basal activity of the mGlu3 receptor detected in heterologous system, a surprising property given its high structural similarity with the mGlu2 receptor that does not display the same behavior. We demonstrate the existence of a unique allosteric interaction between glutamate and Cl -ions in the mGlu3 receptor. This strengthened interaction network increases the stability of the glutamate-induced active state of the extracellular domain of the receptor, making the mGlu3 receptor highly responsive to low ambient glutamate concentrations Finally, we propose that this mechanism allows an accurate detection of sub-micromolar extracellular glutamate concentrations.

MATERIAL AND METHODS

Cell culture and transfection

HEK293 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Life Technologies, Cergy Pontoise, France), supplemented with 10% of Fetal Bovine Serum (FBS) and were transiently transfected with rat mGluRs receptors by electroporation or reverse lipofectamine 2000 protocol as previously described [START_REF] Doumazane | Illuminating the activation mechanisms and allosteric properties of metabotropic glutamate receptors[END_REF]. Receptors were also co-transfected with the glutamate transporter EAAC1 to minimize influence of extracellular glutamate and if needed, chimeric Gi/Gq G-protein (GqTOP) to allow the coupling to the phospholipase C pathway for second messenger experiments. Cells were seeded in a polyornithine coated 96-well plate at the density of 150 000 cells/well. 24h (48h for chimeras) after transfection, medium was changed by Glutamax TM (Life Technologies, Cergy Pontoise, France) to reduce extracellular glutamate concentration 2 hours before experiments.

Directed mutagenesis and chimeras constructs

mGlu3 mutants receptors were obtained using the Quick-Change strategy (Stratagene, La Jolla, CA, USA).

All mutations were made in the HA and HA SNAP encoding plasmids and were verified by sequencing. Total and cell surface expression was quantified by ELISA using anti-HA primary antibody coupled to peroxidase (3F10, Roche, Basel, Switzerland) as previously described [START_REF] Goudet | Heptahelical domain of metabotropic glutamate receptor 5 behaves like rhodopsinlike receptors[END_REF]. For chimeras receptors, a restriction site was introduced after the Venus Flytrap (VFT) encoding sequence in HA SNAP mGlu2 and mGlu3 plasmids using the Quick-Change strategy. Then, digested VFT sequences were sub-cloned into mGlu2 or mGlu3 plasmids according chimera design and final constructs were also verified by sequencing. Chimeras expression levels were quantified by fluorescence at 620 nm on Lumi4-Tb labeled receptors as described elsewhere [START_REF] Doumazane | Illuminating the activation mechanisms and allosteric properties of metabotropic glutamate receptors[END_REF].

Chloride buffers composition

All chemical products were purchased from Sigma-Aldrich (L'Isle d'Abeau, France). Reference high Cl - buffer used for calcium mobilization, TR-FRET mGlu sensor and smFRET assays contained 154.6 mM of Cl - ions and included NaCl 150 mM, KCl 2.6 mM, MgSO4 1.18 mM, D-glucose 10 mM, HEPES 10 mM, CaCl2 1 mM and 0.5% (w/v) BSA. To reach 200 mM Cl -concentration, NaCl was increased. For buffers with lower Cl -concentration, NaCl and KCl were progressively replaced by gluconate equivalents (NaC6H11O7 and KC6H11O7 respectively). pH was adjusted at 7.4. It was previously shown that gluconate does not impair cell signaling [START_REF] Tora | Allosteric modulation of metabotropic glutamate receptors by chloride ions[END_REF].

Second messengers assays

Inositol monophosphate production was determined using the IP-One HTRF (Homogenous Time Resolved Fluorescence) kit (Cisbio Bioassays, Codolet, France) according to the manufacturer's recommendations [START_REF] Trinquet | D-myo-inositol 1-phosphate as a surrogate of D-myo-inositol 1,4,5-tris phosphate to monitor G protein-coupled receptor activation[END_REF]. For the calcium mobilization assay, HA tagged mGluRs expressing cells were seeded in black-walled, clear bottom 96-well plates. High (154.6 mM) and low (2 mM) Cl -buffers supplemented with 4 mM probenecid were used. 24h after transfection, cells were loaded with 1 μM of calcium sensitive fluorescent dye Fluo 4-AM (Life Technologies, Cergy Pontoise, France) diluted in fresh high Cl -buffer, for 1h at 37°C. After washing, cells were maintained in 2 or 154.6 mM Cl -buffers prior glutamate addition.

Intracellular calcium release was determined using Flexstation ® 3 (Molecular Devices, Sunnyvale, CA, USA).

Fluorescence was detected for 60 seconds at 485 nm excitation and 525 nm emission. In order to assess gluconate side effects on cell signaling, Ca 2+ mobilization was also measured after stimulation of an AVP sensitive endogenous receptor on non-transfected cells, which was used as an internal control in each experiment, as previously described [START_REF] Tora | Allosteric modulation of metabotropic glutamate receptors by chloride ions[END_REF] .

TR-FRET mGlu sensor assay

SNAP tagged mGluRs were used for this assay. Cells were seeded in black-walled, black bottom 96-well plates. 24 hours (48 hours for chimeras) after transfection, receptors were covalently labeled with 100 nM of SNAP-Lumi4-Tb (donor) and 60 nM of SNAP-Green (acceptor) diluted in 154.6 mM high Cl -buffer. Donor and acceptor concentrations were chosen in order to label surface receptors equivalently, with both 1 donor and 1 acceptor molecule. After 1 hour at 37°C, cells were washed in appropriate Cl -buffers and drugs were added. TR-FRET measurements were collected at 520 nm using PHERAstar F5 microplate reader as previously described [START_REF] Doumazane | Illuminating the activation mechanisms and allosteric properties of metabotropic glutamate receptors[END_REF].

Data analysis

IP-One, Ca 2+ mobilization and TR-FRET data were analyzed with Prism 6 software (GraphPadSoftware, San Diego, CA, USA). Concentration-responses curve parameters were derived using a four-parameter nonlinear regression equation. For clarity, potency was expressed as absolute (positive) logarithms (pEC50) and nH (Hill slope) describes the steepness of the curve. In Cl -experiments, data from Ca 2+ mobilization were normalized by the WT receptor maximum response obtained in 2 mM Cl -buffer and Emax represent the maximum response obtained at saturating agonist concentration. In TR-FRET mGlu sensor assay, base and maximum of the curve were normalized by high and low FRET ratio of the WT receptor in 2 mM Cl -buffer to define respectively 100% and 0% of the signal. For chimeras experiments, data were normalized by mGlu2 receptor maximum window (top and bottom of mGlu2 receptor curve). Data shown in the figures represent the mean ± S.E.M. of at least three experiments performed at least in duplicates. Statistical differences were determined by One-way ANOVA with Dunnett's and Sidak's post-test.

smFRET experiments

For smFRET experiments, human SNAP tagged mGluRs extracellular domains (ECD) fused to a GPI anchor in the C-terminus (HA SNAP mGlu3 Del570 GPI) were transiently co-transfected with glutamate transporter EAAC1 in HEK293 TSA cells by reverse Lipofectamine 200 protocol (Life Technologies, Cergy Pontoise, France), in 6-well plates. After 24 hours expression, receptors were covalently labeled with 100 nM Benzyl-Guanine-Cy3B (donor) and 1 µM Benzyl-Guanine-d2 (acceptor) (Cisbio Bioassays, Codolet, France) diluted in fresh 154.6 mM high Cl -buffer for 1 hour at 37°C. Then several washings with different Cl -buffers were performed prior to ECD cleavage using 0.25 U.ml -1 Bacillus cereus Phosphatidylinositol-specific Phospholipase C (Life Technologies, Cergy Pontoise, France), at 37°C for 30 minutes. ECD were purified using Zeba TM spin desalting columns (Life Technologies, Cergy Pontoise, France) and stored at -20°C supplemented with 50% glycerol and 0.1 mg.ml -1 BSA. For smFRET measurements, samples were diluted to picomolar concentration and loaded into 384-well plates (Greiner Bio One). Data acquisition was performed on our home-built confocal MFD-PIE (Multiparameter Fluorescence Detection -Pulsed Interleaved Excitation) microscope [START_REF] Olofsson | Pulsed interleaved excitation fluorescence spectroscopy with a supercontinuum source[END_REF] as previously described [START_REF] Olofsson | Fine tuning of sub-millisecond conformational dynamics controls metabotropic glutamate receptors agonist efficacy[END_REF]. Data analysis was performed using the Software Package for Multiparameter Fluorescence Spectroscopy, Full Correlation and Multiparameter Fluorescence Imaging developed in C.A.M. Seidel lab (http://www.mpc.uniduesseldorf.de/seidel/) [START_REF] Widengren | Singlemolecule detection and identification of multiple species by multiparameter fluorescence detection[END_REF]. Pulsed interleaved excitation allowed us to construct the 2Dhistogram of the proximity ratio EPR (i.e. FRET efficiency) and stoichiometry ratio S, where single-labeled species and/or free fluorophores can be excluded, and only double-labeled species are selected for further analysis. Apparent EPR histograms were analyzed using Origin ® 8. At least three measurements were performed for each sample and data shown in the figures represent the mean ± S.E.M. Statistical differences were determined by One-way ANOVA with Sidak's post-test.

Binding assay

For ligand binding assay, membranes from mGluRs transfected HEK293 cells were prepared. After 48 hours expression, cells were washed with cold PBS, scratched and collected by centrifugation at 4°C. Then membranes were prepared by successive homogenization (on ice) and centrifugation (1000 rpm at 4°C) steps.

After a final centrifugation at 19 500 rpm for 2 hours at 4°C, membranes were resuspended in sensor buffer (high Cl -), quantified by BCA method and stored at -80°C. For binding assay, 15-20 μg of mGlu2 or mGlu3 membranes were incubated respectively with 5 or 3 nM [ 3 H]-LY341495 (corresponding to Kd of this radioligand) and various concentrations of cold glutamate for 1h at room temperature. Membranes were then collected on filters and washed 3 times with a 0.9% NaCl solution. Filters were placed in tubes containing a liquid scintillation mixture solution, and radioactivity was measured with a Packard beta counter (Perkin Elmer). Unspecific binding was determined in presence of 100 μM LY379268. Data were analyzed with Prism 6 software (GraphPadSoftware, San Diego, CA, USA) to determine Ki values.

Glutamate quantification

Medium from plated cells transiently expressing mGluRs with or without EAAC1 transporter was sampled for glutamate analysis at different experiment times: after 24 hours expression, cells were washed one time and let 15 min in sensor assay buffer (high Cl -); then 2 hours or 5 hours after medium change by Glutamax TM .

Glutamax TM medium, IP-One and high Cl -Ca + /sensor assay buffers were also tested. Samples were filtered on 0.2-0.8 μm filters prior to analysis.

Glutamate quantification was performed by a reversed phase narrow bore liquid chromatography assay with fluorescence detection after precolumn derivatization with o-phtalaldehyde and β-mercaptoethanol, as previously described [START_REF] Van Hemelrijck | Determination of amino acids associated with cerebral ischaemia in rat brain microdialysates using narrowbore liquid chromatography and fluorescence detection[END_REF]. The compounds were eluted by gradient elution with a mobile phase that was delivered at a rate of 0.17 mL/min. The gradient elution was performed using mobile phase A (0.025 M sodium phosphate solution pH 9 and 1% tetrahydrofuran) and mobile phase B (methanol:water 90:10). The temperature of the autosampler tray was set at 4°C and the injection volume was 10 µL on a C18 narrow bore column (5 µm particle size, 250 x 2 mm, Capcell Pak MG®, Shiseido). Detection was performed with a RF 10A XL fluorescence detector (Shimadzu) at excitation and emission wavelengths of 340 and 450 nm, respectively. Data acquisition was carried out with the integration computer program Clarity (DataApex, Antec, Zoeterwoude, the Netherlands). All analyses were performed at least in duplicate.

Structural and sequence analysis

Crystal structures of mGlu2 (PDB 4XAQ) and mGlu3 receptors (PDB 5CNK, 2E4U) were used to compare their VFT domains using the Discovery Studio 4.0 software (BIOVIA -A Dassault Systèmes brand -5005 Wateridge Vista Drive, San Diego, CA 92121 USA). For multiple sequence alignment, we used Discovery Studio's tool Align123 based on the ClustalW program [START_REF] Thompson | CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice[END_REF]. Sequences were obtained from Uniprot website (http://www.uniprot.org/), using the gene names for the queries, and only the sequence of the extra-cellular domain (ECD) was kept for the alignment. Output alignment was then further optimized.

RESULTS

The glutamate binding domain is involved in the high basal activity of the mGlu3 receptor

First, we compared the glutamate-induced production of second messengers by the different mGluRs.

Receptors were transiently transfected in HEK293 cells and their glutamate-induced signaling responses were evaluated by measuring inositol monophosphate (IP1) production (Fig. 1A). To allow the Gi-coupled group-II and -III mGluRs to activate the phosphoinositides pathway, receptors were co-transfected with a Gi/Gq chimeric G-protein. We found that the basal IP1 production was very high in mGlu3-expressing cells, in contrast to cells expressing other mGluR subtypes (p<0.001), such that glutamate addition did not induce a significant increase of IP1 production.

To understand the structural origin of this phenomenon, we compared the activity of the mGlu3 receptor to its closest relative mGlu2 and built mGlu2/3 receptor chimeras by inverting their VFT domains. We then measured their activity using both functional (IP1 production) and conformational mGluR sensors (Fig. 1B-C, supplemental figure 1 and supplemental table 1). These latter are based on VFT conformational changes occurring during receptor activation. SNAP-tags are inserted at the N-termini of each mGluR subunit and are covalently labeled with fluorescent dyes. Variation in distance between the fluorophores can be measured using Lanthanide Resonance Energy Transfer (LRET), using a Time-Resolved Förster Energy Transfer approach (TR-FRET) [START_REF] Doumazane | Illuminating the activation mechanisms and allosteric properties of metabotropic glutamate receptors[END_REF][START_REF] Scholler | HTS-compatible FRET-based conformational sensors clarify membrane receptor activation[END_REF]. Agonist binding induces closure of mGluRs VFT domains, leading to a reorientation of the protomers increasing the distance between the Ntermini and so, resulting to a large decrease in the TR-FRET signals (Fig. 1D).

In contrast to what was observed with the mGlu2 receptor, glutamate concentration-response curves from mGlu3 receptor-expressing cells were nearly flat in both the IP1 and TR-FRET assays (Fig. 1B-C). In vehicle conditions, the low FRET ratio measured for mGlu3 receptors suggested most receptors were mainly in an active conformation, despite the lack of glutamate addition (Fig. 1C, supplemental table 1). This result, combined with high IP1 levels in the same condition, highlights the high basal activity of mGlu3 receptors.

When mGlu2 VFT was fused to mGlu3 TMD (VFT2/TMD3 chimera), this basal activity was abolished and the receptor could be activated by glutamate similarly to the mGlu2 receptor. In contrast, the fusion of mGlu3 VFT with mGlu2 TMD (VFT3/TMD2 chimera) showed an enhanced basal activity and a nearly flat glutamate concentration-response curve, similar to the mGlu3 receptor (Fig. 1B-C, supplemental table 1). Taken together, these findings revealed that mGlu3 VFT domain plays a key role in the high basal activity of the receptor.

Glutamate is required for the mGlu3 receptor high basal activity

To explore the potential involvement of glutamate in the high basal activity of mGlu3 receptors, we first measured conformational changes in presence or absence of the competitive antagonist LY341495 (Fig. 2A).

Interestingly, in presence of LY341495, the FRET ratio is significantly increased (p<0.001) in comparison to the ratio measured in the vehicle condition. These results suggest either that LY341495 is an inverse agonist, or that possibly it competes with ambient glutamate present in the assay medium, preventing its action on the receptor.

We then designed a mutant mGlu3 receptor less sensitive to glutamate. To that aim, we mutated the threonine in position 174, a key residue involved in the binding of the amino acid moiety of glutamate [START_REF] Acher | Amino acid recognition by Venus flytrap domains is encoded in an 8-residue motif[END_REF], into an alanine. We then measured the conformational changes of mGlu3 T174A in comparison to the WT mGlu3 receptor. As expected, the mGlu3 T174A construct was not activated by glutamate, except at high concentrations (1 mM) where a small decrease in the FRET ratio was measured.

Interestingly, contrary to WT receptor, the mGlu3 T174A mutant did not display any basal activity (Fig. 2B).

Decreasing receptor affinity for glutamate strongly reduces its basal activity, further supporting a key role of ambient glutamate provided by the cells in such an assay.

We quantified the ambient glutamate concentrations in cell media and buffers used for our experiments (table 1). The glutamate concentration in cell medium is strongly affected by incubation time after transfection and co-expression of high-affinity glutamate transporter EAAC1. In optimized experimental conditions (i.e. EAAC1 co-expression and 2 hours incubation in a glutamate-free cell medium before experiments, see Material and Methods section for details), a residual glutamate concentration of 0.59 ± 0.03 μM was measured.

To correlate this result to glutamate affinity, we performed binding experiments on mGlu2 and mGlu3 receptors expressing membranes (supplemental Fig. 2). Radiolabeled LY341495 displacement curves indicated that the mGlu3 receptor has a higher affinity for glutamate (sub-micromolar range, 0.90 ± 0.31 μM) compared to its homolog mGlu2 receptor (ten micromolar range, 10.95 ± 6.92 μM). Of note, the glutamate Ki value of the mGlu3 receptor calculated from binding experiments correlated to the extrapolated potency derived from conformational sensor experiments in Fig. 3 (0.88 vs. 0.90 μM) (supplemental table 2). According to the glutamate concentration in the assay, we can deduce that about 40% of the receptors are occupied in these conditions.

Altogether, these results are highlighting that high basal activity likely of the mGlu3 receptor results mainly from receptor activation by ambient glutamate.

Extracellular chloride modulates both basal and agonist-induced mGlu3 receptor activity

We have previously reported the positive allosteric modulation of mGluRs' activity by Cl - [START_REF] Tora | Allosteric modulation of metabotropic glutamate receptors by chloride ions[END_REF], with the mGlu2 receptor being the less sensitive to extracellular Cl -as compared to other mGluRs. In that study, the effect of Cl -on mGlu3 receptor was not considered. However, another study proposed that Cl - ions could directly activate mGlu3 receptor [START_REF] Diraddo | Chloride is an Agonist of Group II and III Metabotropic Glutamate Receptors[END_REF]. In the light of these findings, we thus examined the effect of extracellular Cl -on mGlu3 receptor basal and agonist-induced activity.

First, the receptor activity was measured both by functional (intracellular Ca 2+ mobilization) and conformational assays (TR-FRET mGlu sensor assay) in two different buffers containing either a physiological (154.6 mM, referred to "high Cl -") or a low Cl -concentration (2 mM, referred to "low Cl -"), where Cl -ions were replaced by gluconate equivalents to maintain osmolarity. In the functional assay, the glutamate concentration-response curve of mGlu3 was nearly flat in high Cl -buffer, whereas a robust dose-dependent activation of mGlu3 receptors by glutamate can be observed in the low Cl -buffer (Supplemental Fig. 3 supplemental table 2). In the conformational assay, a concentration-dependent change of FRET signal was observed in both high and low Cl -buffers (Fig. 3A). However, in the basal condition, the TR-FRET ratio in the high Cl -buffer is approximately half of the ratio observed in the low Cl -buffer (Fig. 3A,supplemental table 2).

Positive cooperativity between Cl -and glutamate is further highlighted in figure 3B. Increasing extracellular Cl -concentration potentiated both receptor basal activity and glutamate potency, in a concentration-dependent manner. To quantify the Cl -effect, we plotted FRET ratios from Fig. 3B to define a Cl -concentration-response curve either in vehicle condition or in presence of 30 μM of glutamate (the latter corresponds to a concentration engendering 80 % of the maximal response (i.e. EC80), in high Cl -) (Fig. 3C).

At EC80, Cl -potency was estimated to 30.98 mM, indicating a strong positive modulation on mGlu3 receptors.

The Hill slope value was 1.11 ± 0.24, suggesting Cl -effect may occur predominantly at one binding site.

Interestingly, in absence of glutamate addition, Cl -concentration-response curve was not well defined, but Cl - potency could be extrapolated to 470.10 mM, a concentration far above the physiological range which is considered to be around 120 mM [START_REF] Jiang | Cl-and Na+ homeostasis during anoxia in rat hypoglossal neurons: intracellular and extracellular in vitro studies[END_REF]. Taken together, these findings indicate mGlu3 receptor is highly modulated by Cl -, in contrast to its homolog mGlu2 (Fig. 3D).

We then measured conformational changes in presence or absence of the competitive antagonist LY341495 in low and high Cl -buffers (Fig. 3E). Interestingly, contrary to what is observed in high Cl -buffer (Fig. 2A), LY341495 did not change the FRET ratio in low Cl -buffer as compared to the vehicle condition.

These results suggest that reducing the mGlu3 receptor affinity for glutamate by depleting extracellular Cl - dramatically decreases its basal activity, in line with the results obtained with the insensitive mutant mGlu3 T174A receptor.

Taken together, these results highlight the link between extracellular Cl -concentration, glutamate and the mGlu3 receptor's basal activity. They suggest that the high basal activity of the mGlu3 receptor may result from Cl -potentiation of the activity induced by residual glutamate.

Chloride ions favor agonist-induced conformational change of the mGlu3 receptor

To better understand the consequence of Cl -modulation on the receptor, we further analyzed the glutamateinduced conformational changes at the single molecule (smFRET) level in comparison to the ensemble level (TR-FRET) (Fig. 4).

Compared to ensemble FRET, smFRET allows the measurement of sub-millisecond conformational dynamics, thus providing a more accurate analysis of receptor activation mechanisms [START_REF] Olofsson | Fine tuning of sub-millisecond conformational dynamics controls metabotropic glutamate receptors agonist efficacy[END_REF].

We used isolated SNAP-tagged mGlu3 extracellular domains (ECD) for smFRET experiments, as previously described for mGlu2 receptors [START_REF] Olofsson | Fine tuning of sub-millisecond conformational dynamics controls metabotropic glutamate receptors agonist efficacy[END_REF]. This glycosylphosphatidyl inositol (GPI)-fused construct is cleaved by phospholipase C (PLC) in order to produce soluble ECDs, labeled with smFRET compatible fluorophores (Fig. 4B, upper panel). Like full-length receptors, soluble ECDs oscillate between an active and an inactive state. This conformational equilibrium is affected by ligand binding, therefore shifting the apparent FRET efficiency (EPR) of the main population in smFRET experiment on diffusing molecules (Fig. 4B, lower panel). Moreover, the use of highly diluted solutions of purified ECDs allows working with solutions containing very low ambient glutamate concentrations.

We tested the effect of Cl -concentrations (2, 75 and 154.6 mM) on basal conditions (vehicle) or following glutamate addition on WT mGlu3 receptor or on mGlu3 T174A mutant, which is less sensitive to glutamate.

Both TR-FRET and smFRET data confirmed the effect of Cl -on the equilibrium shift of the WT receptor dimer toward the active state, as seen both on basal activity (vehicle condition) and glutamate-induced FRET changes. In contrast, we observed that mGlu3 T174A is not activated by glutamate, as expected, nor by Cl - (Fig. 4A andC, supplemental Fig. 4). Therefore, the Cl -ion appears as a strong positive allosteric modulator of the mGlu3 receptor but did not display agonism by itself. Taking together, these results further confirm that the basal activity of mGlu3 is due to the receptor's high affinity for glutamate and Cl -potentiation of glutamate binding.

A unique "chloride lock" stabilizes the active state of the mGlu3 receptor

To delineate a molecular basis of the mGlu3 receptor's Cl --related basal activity, we examined the crystal structures of mGlu3 and mGlu2 VFTs (PDB 5CNK and 4XAQ, respectively), recently solved in their dimer active closed conformation (Acc) binding the potent agonist LY354740 and glutamate (Glu) respectively (Monn et al., 2015a;Monn et al., 2015b) (Fig. 5).

A first difference resides in the number of Cl -binding sites in mGlu2 and mGlu3 VFTs. Several halide sites can be observed in mGlu3 VFT (Figure 5A-B). Among them, two sites correspond to functional Cl -binding sites that have been described in the VFT of most mGluRs, contrary to the mGlu2 receptor, which possesses only one of these functional sites [START_REF] Tora | Allosteric modulation of metabotropic glutamate receptors by chloride ions[END_REF]. The first Cl -site (Site 1) is located within VFT lobe 1, in a binding pocket adjacent to the glutamate binding site, which was previously predicted [START_REF] Acher | A critical pocket close to the glutamate binding site of mGlu receptors opens new possibilities for agonist design[END_REF] and demonstrated to be a Cl -binding site conserved in all mGluRs (Supplemental Fig. 6A) [START_REF] Diraddo | Chloride is an Agonist of Group II and III Metabotropic Glutamate Receptors[END_REF][START_REF] Tora | Allosteric modulation of metabotropic glutamate receptors by chloride ions[END_REF]. The second site (Site 2) is created by the interface of both VFT lobes, when the receptor is in a closed active conformation and shows more divergence within the mGluR family. This binding site is absent in mGlu2 where the S152 that is critical for Cl -binding in site 2 is replaced by an aspartate residue [START_REF] Tora | Allosteric modulation of metabotropic glutamate receptors by chloride ions[END_REF].

Another major difference between mGlu2 and mGlu3 VFT appears at α-helix 7 and loop 7 (Fig. 5A-B and supplemental Fig. 6). Whereas loop 7 adopts a straight conformation in mGlu2, it is longer and curled in mGlu3 because of α-helix 7 unfolding. This occurrence results in a critical difference at the dimer interface between the Acc protomers, which allows several additional interactions with the facing monomer. A closer look at the mGlu3 structures suggests that Cl -bound at site 2 could coordinate this network of interactions between the two lobes, tightening the closed VFT. In particular, Cl -may bridge S152 (lobe 1, loop 3) and R277 (lobe 2, loop 8) in the agonist-induced closed VFT conformation (Fig. 5C-D right panel). Due to the ionic nature of the chloride, the interaction between S152 and R277 is stronger compared to that with a neutral water molecule [START_REF] Rozas | Hydrogen bonds and Coulombic interactions: a theoretical study[END_REF] (Fig. 5D central and right panel). Thereby, Cl -may act as a "Cl -lock" underlying the high agonist affinity by stabilizing the glutamate-induced closed conformation of mGlu3 VFT (Fig. 5 and6).

To verify our hypothesis, we mutated mGlu3 S152 that seems to be crucial in Cl -binding into an aspartate (as in mGlu2) or an alanine and measured receptor activation by TR-FRET assay and signaling activity by Ca 2+ mobilization (Fig. 5E, supplemental Fig. 6B-D and supplemental table 2). Glutamate potencies of mGlu3 S152A and mGlu3 S152D were not changed compared to the WT receptor in high Cl -buffer (supplemental Fig. 6B). However, both S152A and S152D mutations abolished the receptor's basal activity. Indeed, FRET ratios measured in high Cl -were similar to the WT receptor in low Cl -(Fig. 7C). Ca 2+ mobilization data indicated signaling activity was rescued for both mutants in high Cl -buffer, in comparison to the WT (supplemental Fig. 6C). Regarding Cl -sensitivity, the mGlu3 S152D mutant exhibited a reduced Cl - sensitivity, similar to the mGlu2 receptor (supplemental table 2). Mutations did not change expression of the receptor (supplemental Fig. 6D). Taken together, these data confirmed the involvement of Cl -binding site 2 in mGlu3 receptor's activity and unravel the key role of S152 on ion stabilization. At a molecular level, these data may be interpreted in terms of stability of the active conformation of the receptor. With the mutants S152D and S152A of the mGlu3 receptor, the most stable network is similar to that at low Cl -concentrations and the high basal activity is thus not observed, resembling to what happens at mGlu2 receptors.

DISCUSSION

In this study, we investigated the molecular mechanisms underlying the atypical high-basal activity observed in cells expressing the mGlu3 receptor. Our main finding shows that it is not corresponding to a constitutive activity of the mGlu3 receptor in the absence of bound agonist. Instead, it results mostly from a Cl --mediated amplified response to low ambient glutamate concentrations, such as those measured in cell media. This strong positive allosteric modulation of glutamate by Cl -ions allows the mGlu3 receptor to sense and efficiently react to sub-micromolar concentrations of glutamate, making it the most sensitive member of mGluR family.

Ion dependence of mGluRs activity has been previously reported, notably to Ca 2+ and Cl - [START_REF] Kuang | Ion dependence of ligand binding to metabotropic glutamate receptors[END_REF]. While mGlu1 and mGlu3 receptors were demonstrated to be sensitive to Ca 2+ , Cl -modulation was reported for all mGluRs, with a lesser extent for the mGlu2 receptor [START_REF] Diraddo | A real-time method for measuring cAMP production modulated by Galphai/o-coupled metabotropic glutamate receptors[END_REF][START_REF] Jiang | Extracellular calcium modulates actions of orthosteric and allosteric ligands on metabotropic glutamate receptor 1alpha[END_REF][START_REF] Tora | Allosteric modulation of metabotropic glutamate receptors by chloride ions[END_REF][START_REF] Vafabakhsh | Conformational dynamics of a class C G-proteincoupled receptor[END_REF]. Of note, the cooperativity between an amino acid ligand and ions to stabilize the active conformation of the receptor is also observed in another receptor from the GPCR class C, the Ca 2+ sensing receptor. This receptor is activated concomitantly both by Ca 2+ ions and an L-amino acid, such as L-Phe, that binds in a pocket corresponding to the glutamate binding pocket in mGluRs [START_REF] Zhang | Identification of an L-phenylalanine binding site enhancing the cooperative responses of the calcium-sensing receptor to calcium[END_REF]. Here, we showed that the mGlu3 receptor is particularly strongly potentiated by Cl -ions as compared to other mGluRs. This strong positive cooperativity between Cl -ions and glutamate occurs through a unique strengthened interaction network between the two lobes of the extracellular domain of the mGlu3 receptor. This network acts as a "Cl --lock" that dramatically reinforces glutamate binding and function.

Previously, both Ca 2+ and Cl -ions were proposed to display agonist activity on the mGlu3 receptor [START_REF] Diraddo | Chloride is an Agonist of Group II and III Metabotropic Glutamate Receptors[END_REF][START_REF] Vafabakhsh | Conformational dynamics of a class C G-proteincoupled receptor[END_REF]. However, in our experiments, Cl -per se does not display direct agonism, as revealed by the glutamate insensitive mGlu3 mutant T174A, which is not activated by Cl -. We propose that what has been interpreted previously as Cl -direct agonism in very sensitive assays may correspond in fact to the potentiation by Cl -ions of the activity induced by residual ambient glutamate. Indeed, cells are constitutively releasing glutamate in extracellular media. We have recorded concentrations ranging from 0.6 to more than 5 µM. These glutamate concentrations are in line with those measured in another recent publication [START_REF] Doornbos | Constitutive activity of the metabotropic glutamate receptor 2 explored with a whole-cell label-free biosensor[END_REF]. Thus, Cl -ions are pure positive allosteric modulators of the mGlu3 receptor.

It is unlikely to observe the occurrence of rapid Cl-concentration changes compatible with dynamic modulation of mGlu3 receptor activity. Short-term variations occur during transport or synaptic activities where rapid Cl -fluxes through GABA or glycine-gated ion channels modify Cl-concentrations [START_REF] Staley | Ionic mechanisms of neuronal excitation by inhibitory GABAA receptors[END_REF]. We have previously evaluated the discrete variations of Cl -concentrations in the synaptic cleft during GABAergic events in physiological or pathological conditions [START_REF] Tora | Allosteric modulation of metabotropic glutamate receptors by chloride ions[END_REF]. We found that multiple synaptic events can modify synaptic Cl -concentrations up to 25 mM. Long-term changes in Cl -concentrations can be observed during maturation [START_REF] Ben-Ari | GABA: a pioneer transmitter that excites immature neurons and generates primitive oscillations[END_REF], modifications of extracellular matrix (ECM) and pathologies [START_REF] De Koninck | Altered chloride homeostasis in neurological disorders: a new target[END_REF][START_REF] Kaila | Cation-chloride cotransporters in neuronal development, plasticity and disease[END_REF]. Interestingly, fast extracellular Cl-changes due to synaptic activity and slow changes could be additive. Thus, in particular conditions, such as anoxia where variations of Cl-exceeding 30 mM can be observed [START_REF] Jiang | Cl-and Na+ homeostasis during anoxia in rat hypoglossal neurons: intracellular and extracellular in vitro studies[END_REF], Cl-variations could reach up to 55 mM. However, given the potency of Cl -on the mGlu3 receptor (~30 mM) as compared to the physiological extracellular Cl - concentration in mature CNS (~120 mM), the potentiation of mGlu3 receptor activity by Cl -ions is probably mostly constitutive. Other members of the mGluR family could be dynamically regulated by Cl -, such as the presynaptic mGlu4 receptor [START_REF] Tora | Allosteric modulation of metabotropic glutamate receptors by chloride ions[END_REF]. Indeed, unlike the mGlu3 receptor, mGlu4 presents characteristics that makes it more susceptible to be actively regulated by discrete Cl-variations. First, this receptor is expressed in GABAergic synapses where rapid Cl-variations occur following opening of GABAA receptors. Second, the potency of Cl -on mGlu4 receptoris close to the physiological Cl-concentration (~80 mM) and presents a steep relationship between Cl-and glutamate-induced activity (with an NHill ~6). This means that small changes in Cl-concentration yield to large changes in response to glutamate. On the other hand, dramatic changes in Cl-homoeostatis occur during CNS maturation [START_REF] Ben-Ari | GABA: a pioneer transmitter that excites immature neurons and generates primitive oscillations[END_REF]. We can hypothesize that mGlu3 receptors should shift from a "dynamic" mode of glutamate response during the early stages to the highly-glutamate sensitive and sustained response mode described in the present study in mature stages. This should affect its biological functions in early stages, such as the crucial control of mGlu5-mediated neuroprotection described in Di [START_REF] Menna | Functional partnership between mGlu3 and mGlu5 metabotropic glutamate receptors in the central nervous system[END_REF]. One can hypothesize that when extracellular Cl -concentrations are low in premature stages, mGlu3 receptor should be able to signal and regulate the release of neurotrophic factors from astrocytes, given its high Cl -sensitivity. However, in the absence of specific data this remains purely speculative and further investigations will be needed to evaluate the impact of Cl -on mGluR function in the premature brain. In the mature brain, while it is unlikely to observe the occurrence of Cl -concentrations changes compatible with a significant modulation of mGlu3 receptor activity, a Cl-mediated rapid adaptation of the regulatory role of some mGluRs, such as mGlu4 receptors present on GABAergic terminals, could be observed.

Although the overall structures of mGlu2 and mGlu3 VFTs are highly similar, two major differences can be observed: (i) the number of functional Cl -binding sites, one in mGlu2 versus two in mGlu3 receptors and (ii) the loop 7 in lobe 2 is longer in mGlu3 than in the mGlu2 receptor. These two particularities of the mGlu3 receptor contribute to the building-up of a unique "Cl -lock" that greatly stabilizes the glutamate-induced closed conformation of its VFT. Indeed, as compared to mGlu2, the presence of Cl -in site 2 in mGlu3 receptor is coordinating a network of interactions between the two lobes, involving notably the twisted loop 7, that is tightening the closed VFT. As a result, the atypical high-basal activity of the mGlu3 receptor is not observed in the cognate mGlu2 receptor in which the Cl -lock is absent. The residue S152 seems to play a crucial role in Cl -binding and bridging between the lobes. This amino acid was previously reported to be involved in mGlu3 Ca 2+ -based basal activity [START_REF] Kubo | Structural basis for a Ca2+-sensing function of the metabotropic glutamate receptors[END_REF][START_REF] Vafabakhsh | Conformational dynamics of a class C G-proteincoupled receptor[END_REF], but no Ca 2+ ion was found in crystal structures at this location. Regarding literature, Ca 2+ seems to play a role in mGlu3 receptor activity, but its binding(s) site(s) as well as its contribution to the receptor's basal activity remains unclear. In contrast, in the mGlu2 receptor, the corresponding serine residue is mutated into an aspartate (D146), which disrupts Cl - binding at site 2, explaining divergence with mGlu3 receptors towards both Cl -sensitivity and basal activity.

Prior to this study, D146 has been proposed to disrupt the cation-pi interaction and also the Cl -binding site 1, by engendering a flip of R271 (so called "arginine flip"), thus postulating there would be no Cl -binding sites in mGlu2 [START_REF] Diraddo | Chloride is an Agonist of Group II and III Metabotropic Glutamate Receptors[END_REF]. However, in recently released mGlu2 VFT crystal structure (Monn et al., 2015b), the cation-pi interaction is intact and only one Cl -ion is found in site 1, which is consistent with our previous study and the present data [START_REF] Tora | Allosteric modulation of metabotropic glutamate receptors by chloride ions[END_REF]. In addition, it is worth to mention that another Cl -ion is localized near the cation-pi in this structure, but at a different site from site 2, as it is found in only one protomer and therefore may be a crystallization artefact. Additional inter-lobe connections may also participate to the closed VFT stabilization, such as R249 (lobe 2, loop 7), which can bind to G51 (lobe 1, loop 1) in mGlu3 receptors because of the unfolding of the α-helix 7 and a new conformation of loop 7 (Fig. 5C and D right panels). The H-bond between R249 and G51 carbonyl is weaker than the salt bridge with D102 that occurs in mGlu2 (Fig. 5C and D left panels), nevertheless this loss of stability is compensated by new interactions at the dimer interface between loop 7 and helix 6 of the other monomer (See Fig. 6 and supplemental Fig. 5). This interaction network could possibly explain why the two mGlu3 VFT monomers are closer than those of mGlu2.

One can speculate that this proximity may further increase the stability of the active dimer resulting in the basal activity of the receptor at low glutamate concentrations. However, further studies will be necessary to determine more precisely the dimer interface and its involvement in receptor activation.

The specific pathophysiological roles of the mGlu3 receptor are still poorly understood. Until recently, these studies have been hampered by the lack of selective tools discriminating between the cognate mGlu2 and mGlu3 receptors. Indeed, the orthosteric binding sites of these two receptors are 100% identical, making the identification of selective orthosteric ligands highly challenging. Thus, the first ligands available were selective for the group II mGluRs over other groups, but failed to discriminate between mGlu2 and mGlu3 receptors [START_REF] Celanire | Novel metabotropic glutamate receptor 2/3 antagonists and their therapeutic applications: a patent review (2005present)[END_REF]. Still, mGlu2/3 ligands have been very helpful to highlight the involvement and the therapeutic potential of these receptors in different CNS disorders, notably in anxiety, schizophrenia, addiction, depression and chronic pain. Recently, several selective mGlu3 negative allosteric modulators [START_REF] Engers | Discovery of a Selective and CNS Penetrant Negative Allosteric Modulator of Metabotropic Glutamate Receptor Subtype 3 with Antidepressant and Anxiolytic Activity in Rodents[END_REF] or orthosteric ligands [START_REF] Monn | Synthesis and Pharmacological Characterization of C4beta-Amide-Substituted 2-Aminobicyclo[3.1.0]hexane-2,6dicarboxylates. Identification of (1 S,2 S[END_REF]Monn et al., 2015b) have been identified. The selectivity of these newly discovered orthosteric ligands lies behind their interaction with amino acids residing at the periphery of the glutamate binding site of the mGlu3 receptor [START_REF] Monn | Synthesis and Pharmacological Characterization of C4beta-Amide-Substituted 2-Aminobicyclo[3.1.0]hexane-2,6dicarboxylates. Identification of (1 S,2 S[END_REF]. Indeed the crystal structure of LY2794193, a selective mGlu3 agonist, bound to mGlu3 VFT (PDB ID 6B7H) reveals that its methoxyphenyl substituent pushes away R277 but does not prevent the stabilization of the active dimer with the curled loop7 (Supplemental Fig. 7) [START_REF] Monn | Synthesis and Pharmacological Characterization of C4beta-Amide-Substituted 2-Aminobicyclo[3.1.0]hexane-2,6dicarboxylates. Identification of (1 S,2 S[END_REF]. On the other hand, a similar perturbation of R271 in mGlu2 decreases significantly the receptor activation. Thus, it appears that targeting the Cl -binding sites of mGlu3 receptor provide an interesting way to overcome the difficulty of designing selective orthosteric drugs.

Interestingly, this approach is also valid for other mGluRs. For example, the first mGlu4 receptor selective orthosteric agonist named LSP4-2022 is a dualsteric/bitopic ligand targeting both the glutamate-and the Cl - binding pockets of the mGlu4 receptor [START_REF] Goudet | A novel selective metabotropic glutamate receptor 4 agonist reveals new possibilities for developing subtype selective ligands with therapeutic potential[END_REF][START_REF] Selvam | Increased Potency and Selectivity for Group III Metabotropic Glutamate Receptor Agonists Binding at Dual sites[END_REF]. Hopefully, new selective ligands will help to better define the particular roles of mGlu3 receptors.

Interestingly, mGlu2 and mGlu3 receptors differ notably by their cellular distribution. Whereas mGlu2 receptors are mostly neuronal, mGlu3 receptors are expressed both in glia and in neurons. In neurons, mGlu3 receptors are present at the post-synapse and in presynaptic elements [START_REF] Tamaru | Distribution of metabotropic glutamate receptor mGluR3 in the mouse CNS: differential location relative to pre-and postsynaptic sites[END_REF] where their activation reduces synaptic activity and plasticity [START_REF] Harrison | The group II metabotropic glutamate receptor 3 (mGluR3, mGlu3, GRM3): expression, function and involvement in schizophrenia[END_REF]. Interestingly, a functional partnership between neuronal mGlu3 receptors and mGlu5 receptors has recently been described, in which activation of mGlu3 receptors amplifies mGlu5 receptor signaling and shapes the ability of mGlu5 receptors to either amplify or restrain neuronal toxicity [START_REF] Menna | Functional partnership between mGlu3 and mGlu5 metabotropic glutamate receptors in the central nervous system[END_REF]. In astrocytes, mGlu3 receptors have been shown to regulate important functions such as the release of astrocytic glutamate and other factors [START_REF] Aronica | Activation of metabotropic glutamate receptor 3 enhances interleukin (IL)-1beta-stimulated release of IL-6 in cultured human astrocytes[END_REF][START_REF] Corti | The use of knock-out mice unravels distinct roles for mGlu2 and mGlu3 metabotropic glutamate receptors in mechanisms of neurodegeneration/neuroprotection[END_REF][START_REF] Nicoletti | Metabotropic glutamate receptors as drug targets: what's new?[END_REF], the re-uptake of glutamate [START_REF] Aronica | Expression and functional role of mGluR3 and mGluR5 in human astrocytes and glioma cells: opposite regulation of glutamate transporter proteins[END_REF], nitrate homeostasis [START_REF] Wang | mGluR3 Activation Recruits Cytoplasmic TWIK-1 Channels to Membrane that Enhances Ammonium Uptake in Hippocampal Astrocytes[END_REF] and are involved in neuroprotection [START_REF] Battaglia | Activation of mGlu3 metabotropic glutamate receptors enhances GDNF and GLT-1 formation in the spinal cord and rescues motor neurons in the SOD-1 mouse model of amyotrophic lateral sclerosis[END_REF][START_REF] Corti | The use of knock-out mice unravels distinct roles for mGlu2 and mGlu3 metabotropic glutamate receptors in mechanisms of neurodegeneration/neuroprotection[END_REF]. By promoting glutamate sensing in the extracellular space, through the molecular mechanisms described in this study, the mGlu3 receptor could react effectively to concentrations of glutamate below the micromolar range. This might confer mGlu3 receptor the ability to regulate the basal tone of glutamate release from neuronal presynaptic elements, as well as basal release of neuroprotective factors, thereby participating to the fine regulation of glutamate reuptake by glia.

Given the numerous physiological and pathological actions of glutamate, it is crucial to control acutely extracellular glutamate concentrations in the brain. Indeed glutamate becomes neurotoxic at elevated extracellular concentrations and glutamate dysregulation is associated to many CNS disorders. Different actors ensure the control of glutamate homeostasis. Transporters are maintaining low concentrations of synaptic and extrasynaptic glutamate. While ionotropic glutamate receptors are responsible of the fast-synaptic response to glutamate, mGluRs are responsible for its slow neuromodulatory actions. Group I mGluRs positively modulate responses to glutamate while group II and group III receptors are decreasing neuronal activity by downregulating the release of neurotransmitters. Interestingly, the eight members of the mGluR family display different affinities for glutamate. Three members present submicromolar affinity for glutamate: mGlu1 with 0.3 µM [START_REF] Mutel | Characterization of [(3)H]Quisqualate binding to recombinant rat metabotropic glutamate 1a and 5a receptors and to rat and human brain sections[END_REF][START_REF] Thomsen | A pharmacological characterization of the mGluR1 alpha subtype of the metabotropic glutamate receptor expressed in a cloned baby hamster kidney cell line[END_REF], mGlu5 with 0.5 µM [START_REF] Mutel | Characterization of [(3)H]Quisqualate binding to recombinant rat metabotropic glutamate 1a and 5a receptors and to rat and human brain sections[END_REF] and mGlu3 which Ki values range from 0.041 to 0.9 µM [START_REF] Johnson | -LY341495 as a novel antagonist radioligand for group II metabotropic glutamate (mGlu) receptors: characterization of binding to membranes of mGlu receptor subtype expressing cells[END_REF][START_REF] Laurie | Ligand binding profile of the rat metabotropic glutamate receptor mGluR3 expressed in a transfected cell line[END_REF][START_REF] Schweitzer | Characterization of [(3)H]-LY354740 binding to rat mGlu2 and mGlu3 receptors expressed in CHO cells using semliki forest virus vectors[END_REF] and 0.9 µM in the present study. While the mGlu3 receptor affinity for glutamate is in the same range than the ones of mGlu1 or mGlu5 receptors, its relatively high glutamate affinity combined to its unique Cl -lock favoring the agonist-induced state makes the mGlu3 receptor able to sense and fully respond to submicromolar glutamate concentrations. This could enable mGlu3 receptors to play a role in the regulation of basal conditions with low tone of glutamate. Other members of the family, mGlu2, 4, 6 and 8 receptors present intermediate affinities, in the micromolar range, for glutamate, [START_REF] Cartmell | Characterization of [3H]-(2S,2'R,3'R)-2-(2',3'-dicarboxy-cyclopropyl)glycine ([3H]-DCG IV) binding to metabotropic mGlu2 receptor-transfected cell membranes[END_REF]Eriksen and Thomsen, 1995;[START_REF] Schweitzer | Characterization of [(3)H]-LY354740 binding to rat mGlu2 and mGlu3 receptors expressed in CHO cells using semliki forest virus vectors[END_REF][START_REF] Wright | Binding of[END_REF]. On the opposite side of the spectrum, the mGlu7 receptor is activated by millimolar concentrations of glutamate, with an affinity around 900 µM [START_REF] Wright | Binding of[END_REF]. This receptor is located in the synaptic grid where glutamate is released and can reach occasionally such high concentrations. Contrary to mGlu3 receptor, the role of mGlu7 receptor would then be to react to the highest concentrations of glutamate and may ensure a role of safety valve. This mGluRs large spectrum of glutamate sensitivity associated to their widespread synaptic, non-synaptic and glial distribution allows them to participate to a fine neuroadaptation of the CNS.

In conclusion, we demonstrated that the atypical high-basal activity of the mGlu3 receptor results mostly from its activation by low ambient glutamate concentrations. This is due to a unique interaction network located in the extracellular domain of the mGlu3 receptor. This network acts as "Cl -lock" that is strongly stabilizing the glutamate-induced active conformation of the receptors. This strong allosteric control of the mGlu3 receptor by physiological Cl -ions allows it to sense and respond efficiently to low concentrations of glutamate. Thus, the various mGluR subtypes appear as a family of glutamate sensors able to respond to a wide range of conditions, from high nanomolar to millimolar concentrations of glutamate. Among them, mGlu3 receptor is the most sensitive member of this family, able to mediate sustained responses to low tone of glutamate. between β-strand 2 and α-helix 2) and D146 (loop 3 between β-strand 3 and α-helix 3) in lobe 1 by two salt bridges, mGlu3 receptor (right panel) R249 is not binding to D102 (loop 2), the homologous residues to mGlu2 D95 but to the carbonyl of G51 of loop 1 which is possible because of the twisted loop 7 in the active dimer. This distortion allows several inter-dimer interactions: I252 with A176, R253 and K 246 with E228. (D). The closed mGlu2 VFT is further stabilized by an ionic interaction between D146 (loop 3) and R271 (loop 8 between β-strand 8 and α-helix 8) and a cation-Π interaction between Y144 (loop 3) and R271 in mGlu2 receptor (left panel). In mGlu3 receptor, a similar cation-Π interaction between Y150 and R277 is present in the Resting closed closed dimer conformation, Rcc (PDB structure 2E4U) and in the Acc conformation (central and right panel). However, the distance is too long between S152 and R277 for a direct interaction. It is mediated by a water molecule (red atom, central panel) in the Rcc conformation or by a halide (iodide in 5CNK, violet atom, right panel) ion in the superstable Acc conformation (Supplemental Figure 7). Atom color code: C chain color, N blue, O red, Cl green, I violet. (E) Mutations of S152 in mGlu3 into an alanine (S152A, squares) or aspartate (S152D, triangles) both reduce receptor basal activity without significantly reducing glutamate potency in TR-FRET sensor assay. Each data point corresponds to mean ± SEM of 6 to 11 experiments (see details in supplemental figure 2). Concentration-response curve parameters are listed in supplemental table S2. Glu: glutamate; VEH: vehicle. -: Not Determined.

FIGURES

Figure 1 :

 1 Figure 1: The Venus flytrap domain is required for mGlu3 receptor high basal activity.

Figure 2 :

 2 Figure 2: Glutamate binding is required for mGlu3 receptor high basal activity.

Figure 5 :

 5 Figure 5: Different molecular and structural determinants of glutamate and chloride binding in mGlu2

Figure 6 .

 6 Figure 6. A unique "chloride lock" stabilizes the glutamate-induced active state of the mGlu3 receptor.

  Figure 3

  Potency (pEC50), maximum response (Emax), basal response (vehicle stimulation) and Hill slope from concentration-response curves obtained with calcium mobilization and TR-FRET sensor assays. Values in red represent the extrapolated parameters from the TR-FRET glutamate concentration-response curve in high Cl - without basal activity. Data are normalized by the maximum or basal response of the WT receptor in low Cl - buffer. Parameters values represent the mean ± SEM from at least three experiments (N) performed at least in duplicates. ΔpEC50 represent the loss of agonist potency due to chloride depletion.

Table 1 [

 1 Glutamate] (μM)

	Plated cells post-transfection (t=0)
	1x wash with sensor buffer -EAAC1	1,33 ± 0,12
	1x wash with sensor buffer + EAAC1	1,39 ± 0,23
	Plated cells before experiments (t=2h)
	in glutamax™ -EAAC1	3,27 ± 0,004
	in glutamax™ + EAAC1	0,59 ± 0,03
	Plated cells before experiments (t=5h)
	in glutamax™ -EAAC1	> 5
	in glutamax™ + EAAC1	1,36 ± 0,01
	Extracellular medium
	Glutamax™	0,07 ± 0,01
	IP-One buffer	0,43 ± 0,03
	Ca 2+ /Sensor buffer	≈ 0

Table 1 : Extracellular media residual glutamate concentrations.

 1 Glutamate concentrations found in different media used in our transient expression system. Data were determined on supernatant from freshly washed plated cells after 24h transfection (t=0), 2 or 5h after medium change by Glutamax™ (before experiments, see material and methods section for details) and on various buffers. Effect of glutamate transporter EAAC1 co-expression was also assessed. Values represent mean ± S.E.M of n=3 experiments. Sensor and Ca 2+ buffers correspond to high Cl -buffer composition.

	Supplemental table 2
	Receptor	Agonist	High Cl -buffer			Low Cl -buffer		N	Chloride sensitivity
		pEC 50	E max		pEC 50	E max			ΔpEC 50
	Calcium mobilization		nH			nH
		(EC 50 μM)	(%WT)		(EC 50 μM)	(%WT)			(EC 50 fold)
	mGlu3				5,27 ± 0,12
		-	-	-		97,99 ± 2,66	2,00 ± 0,28	7	-
	WT				(5,37)
	mGlu3	6,10 ± 0,30			4,73 ± 0,06				-1,37 ± 0,31
		Glutamate	125,44 ± 4,16	1,97 ± 0,51		119,83 ± 7,94	2,51 ± 0,41	5
	S152A	(0,79)			(18,62)				(23,44)
	mGlu3	6,56 ± 0,13			6,14 ± 0,04				-0,44 ± 0,11
			119,68 ± 5,80	1,18 ± 0,16		107,18 ± 2,14	1,46 ± 0,24	5
	S152D	(0,28)			(0,72)				(2,75)
		pEC 50	Base		pEC 50	Base			ΔpEC 50
	TR-FRET sensor		nH			nH
		(EC 50 μM)	(%WT)		(EC 50 μM)	(%WT)			(EC 50 fold)
	mGlu3	5,62 ± 0,11			4,36 ± 0,09				-1,26 ± 0,07
			61,24 ± 6,86	1,24 ± 0,09		99,98 ± 0,02	2,27 ± 0,23	11
	WT	(2,40)			(43,65)				(18,20)
		6,05 ± 0,10
			99.57 ± 2,10	0,71 ± 0,13				Extrapol.
		(0,89)
	mGlu3	5,22 ± 0,15			3,86 ± 0,07				-1,36 ± 0,15
			111,50 ± 12,81	1,94 ± 0,14		147,13 ± 8,22	3,86 ± 0,07	6
	S152A	(6,03)			(138,04)				(22,91)
		Glutamate
	mGlu3	5,30 ± 0,14			5,02 ± 0,11				-0,27 ± 0,05
			105,69 ± 12,34	1,88 ± 0,26		109,77 ± 11,22	1,84 ± 0,32	6
	S152D	(5,01)			(9,55)				(1,86)
	mGlu3
		-	-	-	-	-	-	3	-
	T174A
	mGlu2	4,74 ± 0,19			4,14 ± 0,05				-0,60 ± 0,15
			95,61 ± 2,33	1,99 ± 0,26		100,00 ± 0,00	1,41 ± 0,27	3
	WT	(18,20)			(72,44)				(3,98)

Supplemental table 2: mGlu3 and mGlu2 constructs concentration-response curves parameters in high Cl -and low Cl -buffers.

ACKNOWLEDGMENTS

The authors thank Ebba L. Lagerqvist for critical reading of the manuscript. This work was supported by grants from the Fondation Recherche Médicale (FRM team DEQ20130326522 and DEQ20170336747) to JPP, the Fundació La Marató de TV3 (Ref 110232), Eranet Neuron, the Agence Nationale de la Recherche (ANR-12-NEUR-0003, ANR-13-BSV1-006 to CG, ANR-09-BIOT-018 to JPP and ANR-13-BSV5-0007 to PR) and "Chercheur d'Avenir" grant from the Region Languedoc Roussillon to EM. Cell-based pharmacological assays were performed on the ARPEGE (Pharmacology Screening-Interactome) platform facility at the IGF. JP Pin's research group belongs to the Labex EpiGenMed (ANR-10-LABX-12-01, « Investissements d'avenir » program). The CBS belongs to the France-BioImaging infrastructure supported by the French National Research Agency (ANR-10-INBS-04, « Investments for the future »), the Labex EpiGenMed and is supported by the GIS "IBiSA: Infrastructures en Biologie Sante et Agronomie". AT and LO were supported by PhD fellowships from the Ministère de l'Education Nationale et de la Recherche. AT was supported by the Fondation Recherche Médicale (FRM FDT20140931071). AMC was supported by a PhD fellowship from Labex EpiGenMed and XR by the Beatriu de Pinos program of Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR). AC was supported by ANR-13-BSV1-006 and PS by a PhD fellowship supported by the ANRT and CisBio.

HIGHLIGHTS

• Chloride ions are strong positive allosteric modulators of mGlu3 receptor

• A unique "chloride-lock" network dramatically stabilize the active state of the receptor • This allows the mGlu3 receptor to sense and react to sub-micromolar concentrations of glutamate

AUTHOR CONTRIBUTION

AT, XR, AC, EM, PR, JPP and CG designed research; AT, XR, AC, AVE, FM, LO, PS, HB performed research; EM built the single molecule setup; AT, XR, AC, AVE, IS, FM, EM, FA, PR, JPP, CG analyzed data; AT, XR, AC, IS, EM, FA, JPP, CG wrote the paper.

CONFLICT OF INTEREST

The authors state no conflict of interest. The phenyl substituent of LY2794193 pushes R277 but keeping a cation- interaction instead of with Y150. The curled loop 7 (brown) that stabilizes the active dimer is maintained and activation of mGlu3 occurs. At mGlu2, the analogous R271 is pushed similarly but the disruption of its bindings seems to prevent the stabilization of the active conformation of the receptor. Consequently, mGlu2 activation is notably decreased.

SUPPLEMENTAL TABLES

Supplemental