The kainate receptor GluK2 mediates cold sensing in mice.

Thermosensors expressed in peripheral somatosensory neurons sense a wide range of environmental temperatures. While thermosensors detecting cool, warm and hot temperatures have all been extensively characterized, little is known about those sensing cold temperatures. Though several candidate cold sensors have been proposed, none has been demonstrated to mediate cold sensing in somatosensory neurons in vivo, leaving a knowledge gap in thermosensation. Here we characterized mice lacking the kainate-type glutamate receptor GluK2, a mammalian homolog of the Caenorhabditis elegans cold sensor GLR-3. While GluK2 knockout mice respond normally to heat and mechanical stimuli, they exhibit a specific deficit in sensing cold but not cool temperatures. Further analysis supports a key role for GluK2 in sensing cold temperatures in somatosensory DRG neurons in the periphery. Our results reveal that GluK2-a glutamate-sensing chemoreceptor mediating synaptic transmission in the central nervous system-is co-opted as a cold-sensing thermoreceptor in the periphery.

Tritium and deuterium labelling of a kainate receptor antagonist and evaluation as a radioligand.

Kainate receptors play a crucial role in mediating synaptic transmission within the central nervous system. However, the lack of selective pharmacological tool compounds for the GluK3 subunit represents a significant challenge in studying these receptors. Recently presented compound 1 stands out as a potent antagonist of GluK3 receptors, exhibiting nanomolar affinity at GluK3 receptors and strongly inhibiting glutamate-induced currents at homomeric GluK1 and GluK3 receptors in HEK293 cells with Kb values of 65 and 39 nM, respectively. This study presents the synthesis of two potent GluK3-preferring iodine derivatives of compound 1, serving as precursors for radiolabelling. Furthermore, we demonstrate the optimisation of dehalogenation conditions using hydrogen and deuterium, resulting in [2H]-1, and demonstrate the efficient synthesis of the radioligand [3H]-1 with a specific activity of 1.48 TBq/mmol (40.1 Ci/mmol). Radioligand binding studies conducted with [3H]-1 as a radiotracer at GluK1, GluK2, and GluK3 receptors expressed in Sf9 and rat P2 membranes demonstrated its potential applicability for selectively studying native GluK3 receptors in the presence of GluK1 and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-blocking ligands.

The positive allosteric modulator BPAM344 and L-glutamate introduce an active-like structure of the ligand-binding domain of GluK2.

Kainate receptors belong to the family of ionotropic glutamate receptors and contribute to the majority of fast excitatory neurotransmission. Consequently, they also play a role in brain diseases. Therefore, understanding how these receptors can be modulated is of importance. Our study provides a crystal structure of the dimeric ligand-binding domain of the kainate receptor GluK2 in complex with L-glutamate and the small-molecule positive allosteric modulator, BPAM344, in an active-like conformation. The role of Thr535 and Gln786 in modulating GluK2 by BPAM344 was investigated using a calcium-sensitive fluorescence-based assay on transiently transfected cells expressing GluK2 and mutants hereof. This study may aid in the design of compounds targeting kainate receptors, expanding their potential as targets for the treatment of brain diseases.

Behavioral analysis of kainate receptor KO mice and the role of GluK3 subunit in anxiety.

Kainate receptors (KARs) are one of the ionotropic glutamate receptors in the central nervous system (CNS) comprised of five subunits, GluK1-GluK5. There is a growing interest in the association between KARs and psychiatric disorders, and there have been several studies investigating the behavioral phenotypes of KAR deficient mice, however, the difference in the genetic background has been found to affect phenotype in multiple mouse models of human diseases. Here, we examined GluK1-5 single KO mice in a pure C57BL/6N background and identified that GluK3 KO mice specifically express anxiolytic-like behavior with an alteration in dopamine D2 receptor (D2R)-induced anxiety, and reduced D2R expression in the striatum. Biochemical studies in the mouse cortex confirmed that GluK3 subunits do not assemble with GluK4 and GluK5 subunits, that can be activated by lower concentration of agonists. Overall, we found that GluK3-containing KARs function to express anxiety, which may represent promising anti-anxiety medication targets.

Exploring thienothiadiazine dioxides as isosteric analogues of benzo- and pyridothiadiazine dioxides in the search of new AMPA and kainate receptor positive allosteric modulators.

The synthesis and biological evaluation on AMPA and kainate receptors of new examples of 3,4-dihydro-2H-1,2,4-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxides is described. The introduction of a cyclopropyl chain instead of an ethyl chain at the 4-position of the thiadiazine ring was found to dramatically improve the potentiator activity on AMPA receptors, with compound 32 (BPAM395) expressing in vitro activity on AMPARs (EC2x = 0.24 µM) close to that of the reference 4-cyclopropyl-substituted benzothiadiazine dioxide 10 (BPAM344). Interestingly, the 4-allyl-substituted thienothiadiazine dioxide 27 (BPAM307) emerged as the most promising compound on kainate receptors being a more effective potentiator than the 4-cyclopropyl-substituted thienothiadiazine dioxide 32 and supporting the view that the 4-allyl substitution of the thiadiazine ring could be more favorable than the 4-cyclopropyl substitution to induce marked activity on kainate receptors versus AMPA receptors. The thieno-analogue 36 (BPAM279) of the clinically tested S18986 (11) was selected for in vivo evaluation in mice as a cognitive enhancer due to a safer profile than 32 after massive per os drug administration. Compound 36 was found to increase the cognition performance in mice at low doses (1 mg/kg) per os suggesting that the compound was well absorbed after oral administration and able to reach the central nervous system. Finally, compound 32 was selected for co-crystallization with the GluA2-LBD (L504Y,N775S) and glutamate to examine the binding mode of thienothiadiazine dioxides within the allosteric binding site of the AMPA receptor. At the allosteric site, this compound established similar interactions as the previously reported BTD-type AMPA receptor modulators.

Enhanced Membrane Incorporation of H289Y Mutant GluK1 Receptors from the Audiogenic Seizure-Prone GASH/Sal Model: Functional and Morphological Impacts on Xenopus Oocytes.

Epilepsy is a neurological disorder characterized by abnormal neuronal excitability, with glutamate playing a key role as the predominant excitatory neurotransmitter involved in seizures. Animal models of epilepsy are crucial in advancing epilepsy research by faithfully replicating the diverse symptoms of this disorder. In particular, the GASH/Sal (genetically audiogenic seizure-prone hamster from Salamanca) model exhibits seizures resembling human generalized tonic-clonic convulsions. A single nucleotide polymorphism (SNP; C9586732T, p.His289Tyr) in the Grik1 gene (which encodes the kainate receptor GluK1) has been previously identified in this strain. The H289Y mutation affects the amino-terminal domain of GluK1, which is related to the subunit assembly and trafficking. We used confocal microscopy in Xenopus oocytes to investigate how the H289Y mutation, compared to the wild type (WT), affects the expression and cell-surface trafficking of GluK1 receptors. Additionally, we employed the two-electrode voltage-clamp technique to examine the functional effects of the H289Y mutation. Our results indicate that this mutation increases the expression and incorporation of GluK1 receptors into an oocyte's membrane, enhancing kainate-evoked currents, without affecting their functional properties. Although further research is needed to fully understand the molecular mechanisms responsible for this epilepsy, the H289Y mutation in GluK1 may be part of the molecular basis underlying the seizure-prone circuitry in the GASH/Sal model.

A Pathogenic Missense Mutation in Kainate Receptors Elevates Dendritic Excitability and Synaptic Integration through Dysregulation of SK Channels.

Numerous rare variants that cause neurodevelopmental disorders (NDDs) occur within genes encoding synaptic proteins, including ionotropic glutamate receptors. However, in many cases, it remains unclear how damaging missense variants affect brain function. We determined the physiological consequences of an NDD causing missense mutation in the GRIK2 kainate receptor (KAR) gene, that results in a single amino acid change p.Ala657Thr in the GluK2 receptor subunit. We engineered this mutation in the mouse Grik2 gene, yielding a GluK2(A657T) mouse, and studied mice of both sexes to determine how hippocampal neuronal function is disrupted. Synaptic KAR currents in hippocampal CA3 pyramidal neurons from heterozygous A657T mice exhibited slow decay kinetics, consistent with incorporation of the mutant subunit into functional receptors. Unexpectedly, CA3 neurons demonstrated elevated action potential spiking because of downregulation of the small-conductance Ca2+ activated K+ channel (SK), which mediates the post-spike afterhyperpolarization. The reduction in SK activity resulted in increased CA3 dendritic excitability, increased EPSP-spike coupling, and lowered the threshold for the induction of LTP of the associational-commissural synapses in CA3 neurons. Pharmacological inhibition of SK channels in WT mice increased dendritic excitability and EPSP-spike coupling, mimicking the phenotype in A657T mice and suggesting a causative role for attenuated SK activity in aberrant excitability observed in the mutant mice. These findings demonstrate that a disease-associated missense mutation in GRIK2 leads to altered signaling through neuronal KARs, pleiotropic effects on neuronal and dendritic excitability, and implicate these processes in neuropathology in patients with genetic NDDs.SIGNIFICANCE STATEMENT Damaging mutations in genes encoding synaptic proteins have been identified in various neurodevelopmental disorders, but the functional consequences at the cellular and circuit level remain elusive. By generating a novel knock-in mutant mouse, this study examined the role of a pathogenic mutation in the GluK2 kainate receptor (KAR) subunit, a subclass of ionotropic glutamate receptors. Analyses of hippocampal CA3 pyramidal neurons determined elevated action potential firing because of an increase in dendritic excitability. Increased dendritic excitability was attributable to reduced activity of a Ca2+ activated K+ channel. These results indicate that a pathogenic KAR mutation results in dysregulation of dendritic K+ channels, which leads to an increase in synaptic integration and backpropagation of action potentials into distal dendrites.

Presynaptic Kainate Receptors onto Somatostatin Interneurons Are Recruited by Activity throughout Development and Contribute to Cortical Sensory Adaptation.

Somatostatin (SST) interneurons produce delayed inhibition because of the short-term facilitation of their excitatory inputs created by the expression of metabotropic glutamate receptor 7 (mGluR7) and presynaptic GluK2-containing kainate receptors (GluK2-KARs). Using mice of both sexes, we find that as synaptic facilitation at layer (L)2/3 SST cell inputs increases during the first few postnatal weeks, so does GluK2-KAR expression. Removal of sensory input by whisker trimming does not affect mGluR7 but prevents the emergence of presynaptic GluK2-KARs, which can be restored by allowing whisker regrowth or by acute calmodulin activation. Conversely, late trimming or acute inhibition of Ca2+/calmodulin-dependent protein kinase II is sufficient to reduce GluK2-KAR activity. This developmental and activity-dependent regulation also produces a specific reduction of L4 GluK2-KARs that advances in parallel with the maturation of sensory processing in L2/3. Finally, we find that removal of both GluK2-KARs and mGluR7 from the synapse eliminates short-term facilitation and reduces sensory adaptation to repetitive stimuli, first in L4 of somatosensory cortex, then later in development in L2/3. The dynamic regulation of presynaptic GluK2-KARs potentially allows for flexible scaling of late inhibition and sensory adaptation.SIGNIFICANCE STATEMENT Excitatory synapses onto somatostatin (SST) interneurons express presynaptic, calcium-permeable kainate receptors containing the GluK2 subunit (GluK2-KARs), activated by high-frequency activity. In this study we find that their presence on L2/3 SST synapses in the barrel cortex is not based on a hardwired genetic program but instead is regulated by sensory activity, in contrast to that of mGluR7. Thus, in addition to standard synaptic potentiation and depression mechanisms, excitatory synapses onto SST neurons undergo an activity-dependent presynaptic modulation that uses GluK2-KARs. Further, we present evidence that loss of the frequency-dependent synaptic components (both GluK2-KARs and mGluR7 via Elfn1 deletion) contributes to a decrease in the sensory adaptation commonly seen on repetitive stimulus presentation.

Depressive-like Behaviors Induced by mGluR5 Reduction in 6xTg in Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is one representative dementia characterized by the accumulation of amyloid beta (Aß) plaques and neurofibrillary tangles (NFTs) in the brain, resulting in cognitive decline and memory loss. AD is associated with neuropsychiatric symptoms, including major depressive disorder (MDD). Recent studies showed a reduction in mGluR5 expression in the brains of stress-induced mice models and individuals with MDD compared to controls. In our study, we identified depressive-like behavior and memory impairment in a mouse model of AD, specifically in the 6xTg model with tau and Aß pathologies. In addition, we investigated the expression of mGluR5 in the brains of 6xTg mice using micro-positron emission tomography (micro-PET) imaging, histological analysis, and Western blot analysis, and we observed a decrease in mGluR5 levels in the brains of 6xTg mice compared to wild-type (WT) mice. Additionally, we identified alterations in the ERK/AKT/GSK-3ß signaling pathway in the brains of 6xTg mice. Notably, we identified a significant negative correlation between depressive-like behavior and the protein level of mGluR5 in 6xTg mice. Additionally, we also found a significant positive correlation between depressive-like behavior and AD pathologies, including phosphorylated tau and Aß. These findings suggested that abnormal mGluR5 expression and AD-related pathologies were involved in depressive-like behavior in the 6xTg mouse model. Further research is warranted to elucidate the underlying mechanisms and explore potential therapeutic targets in the intersection of AD and depressive-like symptoms.

Progressive development of synchronous activity in the hippocampal neuronal network is modulated by GluK1 kainate receptors.

Kainate receptors are potent modulators of circuit excitability and have been repeatedly implicated in pathophysiological synchronization of limbic networks. While the role of aberrant GluK2 subunit containing KARs in generation of epileptiform hypersynchronous activity is well described, the contribution of other KAR subtypes, including GluK1 subunit containing KARs remain less well understood. To investigate the contribution of GluK1 KARs in developmental and pathological synchronization of the hippocampal neural network, we used multielectrode array recordings on organotypic hippocampal slices that display first multi-unit activity and later spontaneous population discharges resembling ictal-like epileptiform activity (IEA). Chronic blockage of GluK1 activity using selective antagonist ACET or lentivirally delivered shRNA significantly delayed developmental synchronization of the hippocampal CA3 network and generation of IEA. GluK1 overexpression, on the other hand, had no significant effect on occurrence of IEA, but enhanced the size of the neuron population participating in the population discharges. Correlation analysis indicated that local knockdown of GluK1 locally in the CA3 neurons reduced their functional connectivity, while GluK1 overexpression increased the connectivity to both CA1 and DG. These data suggest that GluK1 KARs regulate functional connectivity between the excitatory neurons, possibly via morphological changes in glutamatergic circuit, affecting synchronization of neuronal populations. The significant effects of GluK1 manipulations on network activity call for further research on GluK1 KAR as potential targets for antiepileptic treatments, particularly during the early postnatal development when GluK1 KARs are strongly expressed in the limbic neural networks.

Structure-Activity Relationship and Solubility Studies of N1-Substituted Quinoxaline-2,3-diones as Kainate Receptor Antagonists.

Kainate receptors are a class of ionotropic glutamate receptors that respond to the excitatory neurotransmitter glutamate in the central nervous system and play an important role in the development of neurodegenerative disorders and the regulation of synaptic function. In the current study, we investigated the structure- activity relationship of the series of quinoxaline-2,3-diones substituted at N1, 6, and 7 positions, as ligands of kainate homomeric receptors GluK1-3 and GluK5. Pharmacological characterization showed that all derivatives obtained exhibited micromolar affinity at GluK3 receptors with Ki values in the range 0.1-4.4 µM range. The antagonistic properties of the selected analogues: N-(7-fluoro-6-iodo-2,3-dioxo-3,4-dihydroquinoxalin-1(2H)-yl)-3-sulfamoylbenzamide, N-(7-(1H-imidazol-1-yl)-6-iodo-2,3-dioxo-3,4-dihydroquinoxalin-1(2H)-yl)-3-sulfamoylbenzamide and N-(7-(1H-imidazol-1-yl)-2,3-dioxo-6-(phenylethynyl)-3,4-dihydroquinoxalin-1(2H)-yl)-3-sulfamoylbenzamide at GluK3 receptors, were confirmed by an intracellular calcium imaging assay. To correlate in vitro affinity data with structural features of the synthesized compounds and to understand the impact of the substituent in N1 position on ability to form additional protein-ligand interactions, molecular modeling and docking studies were carried out. Experimental solubility studies using UV spectroscopy detection have shown that 7-imidazolyl-6-iodo analogues with a sulfamoylbenzamide moiety at the N1 position are the best soluble compounds in the series, with molar solubility in TRISS buffer at pH 9 more than 3-fold higher compared to NBQX, a known AMPA/kainate antagonist.

Interneuronal GluK1 kainate receptors control maturation of GABAergic transmission and network synchrony in the hippocampus.

Kainate type glutamate receptors (KARs) are strongly expressed in GABAergic interneurons and have the capability of modulating their functions via ionotropic and G-protein coupled mechanisms. GABAergic interneurons are critical for generation of coordinated network activity in both neonatal and adult brain, yet the role of interneuronal KARs in network synchronization remains unclear. Here, we show that GABAergic neurotransmission and spontaneous network activity is perturbed in the hippocampus of neonatal mice lacking GluK1 KARs selectively in GABAergic neurons. Endogenous activity of interneuronal GluK1 KARs maintains the frequency and duration of spontaneous neonatal network bursts and restrains their propagation through the hippocampal network. In adult male mice, the absence of GluK1 in GABAergic neurons led to stronger hippocampal gamma oscillations and enhanced theta-gamma cross frequency coupling, coinciding with faster spatial relearning in the Barnes maze. In females, loss of interneuronal GluK1 resulted in shorter sharp wave ripple oscillations and slightly impaired abilities in flexible sequencing task. In addition, ablation of interneuronal GluK1 resulted in lower general activity and novel object avoidance, while causing only minor anxiety phenotype. These data indicate a critical role for GluK1 containing KARs in GABAergic interneurons in regulation of physiological network dynamics in the hippocampus at different stages of development.

Positive and negative allosteric modulation of GluK2 kainate receptors by BPAM344 and antiepileptic perampanel.

Kainate receptors (KARs) are a subtype of ionotropic glutamate receptors that control synaptic transmission in the central nervous system and are implicated in neurological, psychiatric, and neurodevelopmental disorders. Understanding the regulation of KAR function by small molecules is essential for exploring these receptors as drug targets. Here, we present cryoelectron microscopy (cryo-EM) structures of KAR GluK2 in complex with the positive allosteric modulator BPAM344, competitive antagonist DNQX, and negative allosteric modulator, antiepileptic drug perampanel. Our structures show that two BPAM344 molecules bind per ligand-binding domain dimer interface. In the absence of an agonist or in the presence of DNQX, BPAM344 stabilizes GluK2 in the closed state. The closed state is also stabilized by perampanel, which binds to the ion channel extracellular collar sites located in two out of four GluK2 subunits. The molecular mechanisms of positive and negative allosteric modulation of KAR provide a guide for developing new therapeutic strategies.

Focusing on the Emerging Role of Kainate Receptors in the Dorsal Cochlear Nucleus (DCN) and Cerebellum.

Mammals have a dorsal cochlear nucleus (DCN), which is thought to be a cerebellum-like structure with similar features in terms of structure and microcircuitry to the cerebellum. Both the DCN and cerebellum perform their functions depending on synaptic and neuronal networks mediated by various glutamate receptors. Kainate receptors (KARs) are one class of the glutamate receptor family and are strongly expressed in the hippocampus, the cerebellum, and cerebellum-like structures. The cellular distribution and the potential role of KARs in the hippocampus have been extensively investigated. However, the cellular distribution and the potential role of KARs in cerebellum-like structures, including the DCN and cerebellum, are poorly understood. In this review, we summarize the similarity between the DCN and cerebellum at the levels of structure, circuitry, and cell type as well as the investigations referring to the expression patterns of KARs in the DCN and cerebellum according to previous studies. Recent studies on the role of KARs have shown that KARs mediate a bidirectional modulatory effect at parallel fiber (PF)-Purkinje cell (PC) synapses in the cerebellum, implying insights into their roles in cerebellum-like structures, including the DCN, that remain to be explored in the coming years.

Presenilin and APP Regulate Synaptic Kainate Receptors.

Kainate receptors (KARs) form a family of ionotropic glutamate receptors that regulate the activity of neuronal networks by both presynaptic and postsynaptic mechanisms. Their implication in pathologies is well documented for epilepsy. The higher prevalence of epileptic symptoms in Alzheimer's disease (AD) patients questions the role of KARs in AD. Here we investigated whether the synaptic expression and function of KARs was impaired in mouse models of AD. We addressed this question by immunostaining and electrophysiology at synapses between mossy fibers and CA3 pyramidal cells, in which KARs are abundant and play a prominent physiological role. We observed a decrease of the immunostaining for GluK2 in the stratum lucidum in CA3, and of the amplitude and decay time of synaptic currents mediated by GluK2-containing KARs in an amyloid mouse model (APP/PS1) of AD. Interestingly, a similar phenotype was observed in CA3 pyramidal cells in male and female mice with a genetic deletion of either presenilin or APP/APLP2 as well as in organotypic cultures treated with γ-secretase inhibitors. Finally, the GluK2 protein interacts with full-length and C-terminal fragments of APP. Overall, our data suggest that APP stabilizes KARs at synapses, possibly through a transsynaptic mechanism, and this interaction is under the control the γ-secretase proteolytic activity of presenilin.SIGNIFICANCE STATEMENT Synaptic impairment correlates strongly with cognitive deficits in Alzheimer's disease (AD). In this context, many studies have addressed the dysregulation of AMPA and NMDA ionotropic glutamate receptors. Kainate receptors (KARs), which form the third family of iGluRs, represent an underestimated actor in the regulation of neuronal circuits and have not yet been examined in the context of AD. Here we provide evidence that synaptic KARs are markedly impaired in a mouse model of AD. Additional experiments indicate that the γ-secretase activity of presenilin acting on the amyloid precursor protein controls synaptic expression of KAR. This study clearly indicates that KARs should be taken into consideration whenever addressing synaptic dysfunction and related cognitive deficits in the context of AD.

Structure, Function, and Regulation of the Kainate Receptor.

Neural communication and modulation are complex processes. Ionotropic glutamate receptors (iGluRs) significantly contribute to mediating the fast-excitatory branch of neurotransmission in the mammalian brain. Kainate receptors (KARs), a subfamily of the iGluRs, act as modulators of the neuronal circuitry by playing important roles at both the post- and presynaptic sites of specific neurons. The functional tetrameric receptors are formed by two different gene families, low agonist affinity (GluK1-GluK3) and high agonist affinity (GluK4-GluK5) subunits. These receptors garnered attention in the past three decades, and since then, much work has been done to understand their localization, interactome, physiological functions, and regulation. Cloning of the receptor subunits (GluK1-GluK5) in the early 1990s led to recombinant expression of kainate receptors in heterologous systems. This facilitated understanding of the functional differences between subunit combinations, splice variants, trafficking, and drug discovery. Structural studies of individual domains and recent full-length homomeric and heteromeric kainate receptors have revealed unique functional mechanisms, which have answered several long-standing questions in the field of kainate receptor biology. In this chapter, we review the current understanding of kainate receptors and associated disorders.

Discovery of the First Highly Selective Antagonist of the GluK3 Kainate Receptor Subtype.

Kainate receptors belong to the family of glutamate receptors ion channels, which are responsible for the majority of rapid excitatory synaptic transmission in the central nervous system. The therapeutic potential of kainate receptors is still poorly understood, which is also due to the lack of potent and subunit-selective pharmacological tools. In search of selective ligands for the GluK3 kainate receptor subtype, a series of quinoxaline-2,3-dione analogues was synthesized and pharmacologically characterized at selected recombinant ionotropic glutamate receptors. Among them, compound 28 was found to be a competitive GluK3 antagonist with submicromolar affinity and unprecedented high binding selectivity, showing a 400-fold preference for GluK3 over other homomeric receptors GluK1, GluK2, GluK5 and GluA2. Furthermore, in functional assays performed for selected metabotropic glutamate receptor subtypes, 28 did not show agonist or antagonist activity. The molecular determinants underlying the observed affinity profile of 28 were analyzed using molecular docking and molecular dynamics simulations performed for individual GluK1 and GluK3 ligand-binding domains.

Glutamate can act as a signaling molecule in mouse preimplantation embryos†.

Free amino acids are present in the natural environment of the preimplantation embryo, and their availability can influence early embryo development. Glutamic acid is one of the amino acids with the highest concentrations in female reproductive fluids, and we investigated whether glutamic acid/glutamate can affect preimplantation embryo development by acting through cell membrane receptors. Using reverse transcription-polymerase chain reaction, we detected 15 ionotropic glutamate receptor transcripts and 8 metabotropic glutamate receptor transcripts in mouse ovulated oocytes and/or in vivo developed blastocysts. Using immunohistochemistry, we detected the expression of two α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits, three kainate receptor subunits, and member 5 metabotropic glutamate receptor protein in blastocysts. Extracellular concentrations of glutamic acid starting at 5 mM impaired mouse blastocyst development, and this fact may be of great practical importance since glutamic acid and its salts (mainly monosodium glutamate) are widely used as food additives. Experiments with glutamate receptor agonists (in combination with gene expression analysis) revealed that specific AMPA receptors (formed from glutamate receptor, ionotropic, AMPA3 [GRIA3] and/or glutamate receptor, ionotropic, AMPA4 [GRIA4] subunits), kainate receptors (formed from glutamate receptor, ionotropic, kainate 3 [GRIK3] and glutamate receptor, ionotropic, kainate 4 [GRIK4] or glutamate receptor, ionotropic, kainate 5 [GRIK5] subunits), and member 5 metabotropic glutamate receptor (GRM5) were involved in this effect. The glutamic acid-induced effects were prevented or reduced by pretreatment of blastocysts with AMPA, kainate, and GRM5 receptor antagonists, further confirming the involvement of these receptor types. Our results show that glutamic acid can act as a signaling molecule in preimplantation embryos, exerting its effects through the activation of cell membrane receptors.

Benzothiadiazines derivatives as novel allosteric modulators of kainic acid receptors.

The majority of excitatory neurotransmission in vertebrate CNS is mediated by glutamate binding to different types of receptors. Among them, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and kainite receptors (KAR) are ionotropic receptors playing important pathophysiological roles. A number of small molecules acting as positive allosteric modulators (PAM) of AMPAR have been proposed as drugs for neurological disorders, however, there is no such abundance of ligands capable of modulating KARs activity. We investigated the ability of IDRA21 and of its derivative, compound 2 (c2), to modulate glutamate-evoked currents at native and recombinantly expressed AMPA and KA receptors. By using the patch clamp technique we analyzed the activity of the two compounds in primary cultures of cerebellar granule neurons and in HEK293 cells transiently transfected with KARs and AMPAR subunits. It resulted that both benzothiadiazine derivatives potentiate AMPAR and KAR mediated currents in native and recombinant receptors, c2 being always more potent and efficacious than IDRA21. The potency of both compounds was higher in native receptors than in recombinant receptors. In HEK293 cells transfected with AMPAR subunits, the efficacy of IDRA21 and c2 was much higher in GluA1 than in GluA2 homomeric receptors while their potency did not change. In recombinant KAR, both compounds had a potency in the high micromolar range, while the efficacy reached a maximum in the GluK2 expressing cells. The benzothiadiazine effect, both in native and recombinant receptors, was detected mainly on plateau current, involving a decrease in AMPAR and KAR desensitization. Our study demonstrates for the first time that two positive allosteric modulators of AMPAR, IDRA21 and its derivative, c2, potentiate KAR activity. Furthermore, we highlighted their subunit selectivity that may enable the design of potent and selective PAMs, which could be relevant for the development of new drugs and for a better understanding of KAR functions in the CNS.