Ionotropic Glutamate Receptors Research Articles

Inhibitory Role of L-theanine, a Structural Analogue of Glutamate, Against GluR5 Kainate Receptor and its Prospective Utility Against Excitotoxicity.

Overactivation of receptors that respond to excitatory neurotransmitters can result in various harmful outcomes, such as the inability to properly modulate calcium levels, generation of free radicals, initiation of the mitochondrial permeability transition, and subsequent secondary damage caused by excitotoxicity. A non-proteinogenic amino acid of tea, L-theanine, is structurally related to glutamate, the major stimulatory neurotransmitter in the brain. Previous reports have emphasised its ability to bind with glutamate receptors. An in-depth understanding of the binding compatibility between ionotropic glutamate receptors and L-theanine is a compelling necessity. In this molecular docking study, the antagonistic effect of L-theanine and its possible therapeutic benefit in GluR5 kainate receptor inhibition has been evaluated and compared to the familiar AMPA and kainite receptor antagonists, cyanoquinoxaline (CNQX) and dinitroquinoxaline (DNQX), using Molegro Virtual Docker 7.0.0. The capacity of L-theanine to cohere with the GluR5 receptor was revealed to be higher than that of glutamate, although it could not surpass the high binding tendency of competitive antagonists CNQX and DNQX. Nonetheless, the drug-likeness score and the blood-brain barrier traversing potential of L-theanine were higher than CNQX and DNQX. The study provides an inference to the advantage of L-theanine, which can be a safe and effective alternative natural therapy for rescuing neuronal death due to excitotoxicity.

The gating properties of Drosophila NMJ glutamate receptors and their dependence on Neto.

The Drosophila neuromuscular junction (NMJ) is a powerful genetic system that has revealed numerous conserved mechanisms for synapse development and homeostasis. The fly NMJ uses glutamate as the excitatory neurotransmitter and relies on kainate-type glutamate receptors and their auxiliary protein Neto for synapse assembly and function. However, despite decades of study, the reconstitution of NMJ glutamate receptors using heterologous systems has been achieved only recently, and there are no reports on the gating properties for the recombinant receptors. Here, using outside-out, patch clamp recordings and fast ligand application, we examine for the first time the biophysical properties of native type-A and type-B NMJ receptors in complexes with either Neto-α or Neto-β and compare them with recombinant receptors expressed in HEK293T cells. We found that type-A and type-B receptors have strikingly different gating properties that are further modulated by Neto-α and Neto-β. We captured single-channel events and revealed major differences between type-A and type-B receptors and also between Neto splice variants. Surprisingly, we found that deactivation is extremely fast and that the decay of synaptic currents resembles the rate of ionotropic glutamate receptor (iGluR) desensitization. The functional analyses of recombinant iGluRs that we report here should greatly facilitate the interpretation of compound in vivo phenotypes of mutant animals. KEY POINTS: We report the reconstitution of Drosophila neuromuscular junction ionotropic glutamate receptors (iGluRs) with Neto splice forms. Using outside-out patches and fast ligand application, we examine the deactivation and desensitization of the four iGluR/Neto complexes found in vivo. Expression of functional channels is absolutely dependent on Neto. Single-channel recordings revealed different lifetimes for different receptor complexes. The decay of synaptic currents is controlled by desensitization.

Trapping of spermine, Kukoamine A, and polyamine toxin blockers in GluK2 kainate receptor channels

Kainate receptors (KARs) are a subtype of ionotropic glutamate receptor (iGluR) channels, a superfamily of ligand-gated ion channels which mediate the majority of excitatory neurotransmission in the central nervous system. KARs modulate neuronal circuits and plasticity during development and are implicated in neurological disorders, including epilepsy, depression, schizophrenia, anxiety, and autism. Calcium-permeable KARs undergo ion channel block, but the therapeutic potential of channel blockers remains underdeveloped, mainly due to limited structural knowledge. Here, we present closed-state structures of GluK2 KAR homotetramers in complex with ion channel blockers NpTx-8, PhTx-74, Kukoamine A, and spermine. We find that blockers reside inside the GluK2 ion channel pore, intracellular to the closed M3 helix bundle-crossing gate, with their hydrophobic heads filling the central cavity and positively charged polyamine tails spanning the selectivity filter. Molecular dynamics (MD) simulations of our structures illuminate interactions responsible for different affinity and binding poses of the blockers. Our structures elucidate the trapping mechanism of KAR channel block and provide a template for designing new blockers that can selectively target calcium-permeable KARs in neuropathologies.

BHLHE40-mediated transcriptional activation of GRIN2D in gastric cancer is involved in metabolic reprogramming.

Gastric cancer (GC) is the third leading cause of death in developed countries. The reprogramming of energy metabolism represents a hallmark of cancer, particularly amplified dependence on aerobic glycolysis. Here, we aimed to illustrate the functional role of glutamate ionotropic receptor N-methyl-D-aspartate type subunit 2D (GRIN2D) in the regulation of glycolysis in GC and the mechanisms involved. Differentially expressed genes were analyzed using the GEO and GEPIA databases, followed by prognostic value prediction using the Kaplan-Meier Plotter database. The effect of GRIN2D knockdown on the malignant behavior and glycolysis of GC cells was explored. GRIN2D expression was upregulated in GC cells and promoted the malignant behavior of GC cells by activating glycolysis. Class E basic helix-loop-helix protein 40 (BHLHE40) was overexpressed in GC cells and mediated transcriptional activation of GRIN2D. The anti-tumor effects of BHLHE40 knockdown on GC cells in vitro and in vivo were reversed by GRIN2D overexpression. Knockdown of GRIN2D or BHLHE40 downregulated the expression of mRNA of electron transport chain subunits and phosphorylation of p38 MARK and inhibited calcium efflux in GC cells. Overexpression of GRIN2D promoted calcium efflux, phosphorylation of p38 MARK protein, and proliferation of GES1 cells. Altogether, the findings derived from this study suggest that BHLHE40 knockdown suppresses the growth, mobility, and glycolysis of GC cells by inhibiting GRIN2D transcription and disrupting the BHLHE40/GRIN2D axis may be an attractive therapeutic strategy for GC.

CNSC-39. INTEGRATED CLINICAL GENETIC ANALYSIS REVEALS NEUROTRANSMITTER RECEPTOR DYSREGULATION IN MENINGIOMAS CAUSING SEIZURE

Abstract Molecular correlates of seizures in meningioma remain unexplored. Previously, we identified MenG molecular grouping, wherein MenG C meningiomas recur more than MenG A or B. We therefore sought to identify molecular and clinical drivers of seizures in meningioma. We identified 144 primary supratentorial meningiomas, excluding those with pre-existing epilepsy. Whole exome sequencing assessed somatic mutation burden. Bulk RNA-sequencing identified differentially expressed genes and gene ontologies (GO). Immunohistochemistry confirmed protein levels. No differences in canonical gene mutation burden (NF2, TRAF7, AKT1, KLF4, SMO, SMARC) was found between seizure and non-seizure groups. MenG C tumors had higher seizure incidence compared to MenG A or B (p=0.03). Seizure causing meningiomas, though extra-axial, exhibited dysregulated neuronal signaling genes. GABAA receptor subunit genes GABRA3 and GABRG1 were downregulated in tumors causing seizure (Log2FC=-1.67, padj=0.014; Log2FC=-2.24, padj=0.009). Immunohistochemistry confirmed expression of GABRB2 subunit, the first documentation of GABA receptor expression in meningiomas. Neuropeptide Y Receptor Y1 (NPY1R) is downregulated in seizure causing meningiomas (Log2FC=-1.64, padj=0.002). NPY1R loss correlates is observed in mesial temporal lobe epilepsy and post-electroconvulsive shock mouse brains. In GO analysis, the terms “GABA signaling pathway,” “neuropeptide signaling,” “excitatory postsynaptic membrane potential,” and “ionotropic glutamate pathway,” were downregulated in meningiomas with seizure (padj<0.05). We assessed which alterations within MenG C tumors caused seizures by identifying genes associated with both seizure presentation and MenG C classification. Glutamate ionotropic receptor AMPA type subunit 1 (GRIA1) is downregulated in MenG C tumors (Log2FC=-2.29, padj=6.02E-07) and seizure causing tumors (Log2FC=-1.75, padj=0.03). Clinically, patients experiencing pre-operative seizures more frequently had cerebral edema, non-homogenous enhancement, intratumoral calcification, higher MIB-1, atypical grade 1, reduced GCS, and ER presentation; they were less likely to have incidental or headache presentation (p<0.05 for all). MenG classification predicts seizure presentation whereas somatic mutation status does not. Meningiomas causing seizures show pathologic neurotransmitter receptor dysregulation.

TMIC-18. ROLE OF NEUROTRANSMITTERS IN GLIOBLASTOMA PATHOPHYSIOLOGY: EMERGING INSIGHTS

Abstract INTRODUCTION Glioblastoma (GBM) are the most common primary brain tumor in adults associated with a dismal patient outcome. While it is well-known that glutamatergic neurons can form functional synapses with glioblastoma cells to enhance tumor growth, lesser is known about the effects of activity of other neuronal types on glioblastoma biology, especially among different glioblastoma molecular subtypes. Here, we examined the effects of various neurotransmitters on the growth of glioblastoma cells characterized as classical, proneural and mesenchymal subtypes. METHODS Patient-derived glioblastoma cell lines were exposed to varying concentrations of different neurotransmitters and assessed for cell viability using CCK-8 assay. Transcriptomic data from TCGA (The Cancer Genome Atlas) database was analyzed for correlation of expression of neurotransmitter receptors (NTRs) with patient survival. Using IVY-GAP database, we compared the expression of NTRs at leading-edge versus tumor core of tumors to assess their possible involvement in cell migration and microenvironmental interactions. RESULTS Consistent with previous findings, we observed an increase in cell viability in all GBM cell lines after being incubated with L-glutamate. The classical subtype has a stronger growth response to L-glutamate compared to the other two subtypes. TCGA data revealed the expression of GRIK4 (glutamate ionotropic receptor kainate type subunit 4) as highest in the classical subtype as well as inversely correlated with worse patient survival in this GBM subtype, as opposed to proneural or mesenchymal patient groups. CCK8 assay also suggested that acetylcholine and dopamine increase glioblastoma cell growth. A subset of cholinergic and glutamatergic receptors (CHRM1, GRM2) was found upregulated in infiltrating/leading-edge regions of glioblastoma tumors compared to the tumor core, suggesting possible involvement in glioblastoma-neuron interactions. CONCLUSION Our findings suggest that GBM subtypes may differentially leverage neuro-modulatory mechanisms to support their growth. Future studies will investigate the differential neuronal responses in GBM subtypes using pharmacological/genetic approaches.

Exploring the Landscape of Pre- and Post-Synaptic Pediatric Disorders with Epilepsy: A Narrative Review on Molecular Mechanisms Involved.

Neurodevelopmental disorders (NDDs) are a group of conditions affecting brain development, with variable degrees of severity and heterogeneous clinical features. They include intellectual disability (ID), autism spectrum disorder (ASD), attention-deficit/hyperactivity disorder (ADHD), often coexisting with epilepsy, extra-neurological comorbidities, and multisystemic involvement. In recent years, next-generation sequencing (NGS) technologies allowed the identification of several gene pathogenic variants etiologically related to these disorders in a large cohort of affected children. These genes encode proteins involved in synaptic homeostasis, such as SNARE proteins, implicated in calcium-triggered pre-synaptic release of neurotransmitters, or channel subunit proteins, such as post-synaptic ionotropic glutamate receptors involved in the brain's fast excitatory neurotransmission. In this narrative review, we dissected emerged molecular mechanisms related to NDDs and epilepsy due to defects in pre- and post-synaptic transmission. We focused on the most recently discovered SNAREopathies and AMPA-related synaptopathies.

Evaluation of allosteric NMDA receptor modulation by GluN2A-selective antagonists using pharmacological equilibrium modeling.

NMDA-type ionotropic glutamate receptors are critically involved in excitatory neurotransmission and their dysfunction is implicated in many brain disorders. Allosteric modulators with selectivity for specific NMDA receptor subtypes are therefore attractive as therapeutic agents, and sustained drug discovery efforts have resulted in a wide range of new allosteric modulators. However, evaluation of allosteric NMDA receptor modulators is limited by the lack of operational ligand-receptor models to describe modulator binding dissociation constants (KB) and effects on agonist binding affinity (α) and efficacy (β). Here, we describe a pharmacological equilibrium model that encapsulates activation and modulation of NMDA receptors, and we apply this model to afford deeper understanding of GluN2A-selective negative allosteric modulators (NAMs), TCN-201, MPX-004, and MPX-007. We exploit slow NAM unbinding to examine receptors at hemi-equilibrium when fully occupied by agonists and modulators to demonstrate that TCN-201 display weaker binding and negative modulation of glycine binding affinity (KB = 42 nM, α = 0.0032) compared to MPX-004 (KB = 9.3 nM, α = 0.0018) and MPX-007 (KB = 1.1 nM, α = 0.00053). MPX-004 increases agonist efficacy (β = 1.19), whereas TCN-201 (β = 0.76) and MPX-007 (β = 0.82) reduce agonist efficacy. These values describing allosteric modulation of diheteromeric GluN1/2A receptors with two modulator binding sites are unchanged in triheteromeric GluN1/2A/2B receptors with a single binding site. This evaluation of NMDA receptor modulation reveals differences between ligand analogs that shape their utility as pharmacological tool compounds and facilitates the design of new modulators with therapeutic potential. Significance Statement Detailed understanding of allosteric NMDA receptor modulation requires pharmacological methods to quantify modulator binding affinity and the strengths of modulation of agonist binding and efficacy. We describe a generic ligand-receptor model for allosteric NMDA receptor modulation and use this model for the characterization of GluN2A-selective NAMs. The model enables quantitative evaluation of a broad range of NMDA receptor modulators and provides opportunities to optimize these modulators by embellishing the interpretation of their structure-activity relationships.

Structural studies of ion channels: achievements, problems and perspectives

The superfamily of membrane proteins known as P-loop channels encompasses potassium, sodium, and calcium channels, as well as TRP channels and ionotropic glutamate receptors. An increasing number of crystal and cryo-EM structures are uncovering both general and specific features of these channels. Fundamental folding principles, the arrangement of structural segments, key residues that influence ionic selectivity, gating, and binding sites for toxins and medically relevant ligands have now been firmly established. The advent of AlphaFold2 (AF2) models represents another significant step in computationally predicting protein structures. Comparison of experimental P-loop channel structures with their corresponding AF2 models shows consistent folding patterns in experimentally resolved regions. Despite this remarkable progress, many crucial structural details, particularly important for predicting the outcomes of mutations and designing new medically relevant ligands, remain unresolved. Certain methodological challenges currently hinder the direct assessment of such details. Until the next methodological breakthrough occurs, a promising approach to analyzing ion channel structures in greater depth involves integrating various experimental and theoretical methods.

New insight into the molecular etiopathogenesis of konzo: Cyanate could be a plausible neurotoxin contributing to konzo, contrary to thiocyanate

IntroductionChronic cassava-derived cyanide poisoning is associated with the appearance of konzo, a tropical spastic paraparesis due to selective upper motor neuron damage. Whether the disease is caused by a direct action of cyanide or its metabolites is still an open question. This preliminary study assessed the neurotoxic effects of thiocyanate (SCN) and cyanate (OCN), two cyanide metabolites hypothesized to be plausible toxic agents in konzo. MethodsCultured mouse neuroblastoma (Neuro-2A) and human neuroblastoma (SH-SY5Y) cell lines were incubated (24, 48, and 72hours) in sodium OCN or sodium SCN in a disease-relevant concentration range. Cell viability, caspase (3, 8, and 9) activities, and reactive oxygen species (ROS) generation were evaluated using appropriate assay kits. Additionally, electrophysiological responses induced by OCN and SCN in primary spinal cord neurons (from Sprague Dawley rats) were assessed by whole-cell patch-clamp techniques. ResultsBoth OCN and SCN were toxic in a dose-dependent way, even if SCN toxicity appeared at very high concentrations (30mM, corresponding to more than 100-fold higher than normal plasmatic levels), contrary to OCN (0.3-3mM). OCN was markedly more toxic in a poor culture medium (MEM; IC50 = 3.2mM) compared to a glucose- and amino acid-rich medium (DMEM; IC50=7.6mM). OCN treatment increased the ROS generation by 8.9 folds, as well as the Caspase-3, Caspase-8, and Caspase-9 activities by 3.2, 2.5, and 2.6 folds, respectively. Finally, OCN (and SCN to a lesser extent) induced depolarizing currents in primary spinal cord neurons, through an activation of ionotropic glutamate receptors. ConclusionOur results suggest OCN as the most plausible neurotoxic agent involved in konzo, while SCN toxicity could be questioned at such high concentrations. Also, they support apoptosis, oxidative stress, and excitotoxicity as probable mechanisms of OCN neurotoxicity.

Pubertal- and Stress-Dependent Changes in Cellular Activation and Expression of Excitatory Amino Acid Receptor Subunits in the Paraventricular Nucleus of the Hypothalamus in Male and Female Rats

Introduction: Pubertal maturation is marked by significant changes in stress-induced hormonal responses mediated by the hypothalamic-pituitary-adrenal (HPA) axis, with prepubertal male and female rats often exhibiting greater HPA reactivity compared to adult males and females. Though the implications of these changes are unclear, elevated stress responsiveness might contribute to the stress-related vulnerabilities often associated with puberty. Methods: The current experiments sought to determine whether differences in cellular activation, as measured by FOS immunohistochemistry, or excitatory ionotropic glutamate receptor subunit expression, as measured by qRT-PCR, in the paraventricular nucleus (PVN) were associated with these noted pubertal shifts in stress reactivity in male and female rats. As the PVN is the key nucleus responsible for activating the hormonal stress response, we predicted greater cellular activation and higher expression levels of glutamate receptor subunits in the PVN of prepubertal males and females compared to their adult counterparts. Results: Our FOS data revealed that while prepubertal males showed greater stress-induced activation in the PVN than adult males, prepubertal females showed less activation than adult females. Moreover, many of the NMDA, AMPA, and kainate receptor subunits measured, including Grin1, Grin2b, Gria1, Gria2, Grik1, and Grik2, had higher expression levels in adults, particularly in males. Conclusions: Though not supporting our initial predictions, these data do indicate that age and stress influence the activation of the PVN and the expression of glutamate receptor subunits important in its function. These data also suggest that the effects of age and stress are different in males and females. Though still far from a clear understanding of what mechanism(s) mediate pubertal shift in stress reactivity, these data add to our growing understanding of how age, stress, and sex influence HPA function.

Mechanisms of Action Underlying Conductance-Modifying Positive Allosteric Modulators of the NMDA Receptor.

N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that mediate a slow, Ca2+-permeable component of excitatory neurotransmission. Modulation of NMDAR function has the potential for disease modification as NMDAR dysfunction has been implicated in neurodevelopment, neuropsychiatric, neurologic, and neurodegenerative disorders. We recently described the thieno[2,3-day]pyrimidin-4-one (EU1622) class of positive allosteric modulators, including several potent and efficacious analogs. Here we have used electrophysiological recordings from Xenopus oocytes, human embryonic kidney cells, and cultured cerebellar and cortical neurons to determine the mechanisms of action of a representative member of this class of modulator. EU1622-240 enhances current response to saturating agonist (doubling response amplitude at 0.2-0.5 μM), slows the deactivation time course following rapid removal of glutamate, increases open probability, enhances coagonist potency, and reduces single-channel conductance. We also show that EU1622-240 facilitates NMDAR activation when only glutamate or glycine is bound. EU1622-240-bound NMDARs channels activated by a single agonist (glutamate or glycine) open to a unique conductance level with different pore properties and Mg2+ sensitivity, in contrast to channels arising from activation of NMDARs with both coagonists bound. These data demonstrate that previously hypothesized distinct gating steps can be controlled by glutamate and glycine binding and shows that the 1622-series modulators enable glutamate- or glycine-bound NMDARs to generate open conformations with different pore properties. The properties of this class of allosteric modulators present intriguing therapeutic opportunities for the modulation of circuit function. SIGNIFICANCE STATEMENT: NMDA receptors are expressed throughout the central nervous system and are permeable to calcium. EU1622-240 increases open probability and agonist potency while reducing single-channel conductance and prolonging the deactivation time course. EU1622-240 allows NMDA receptor activation by the binding of one coagonist (glycine or glutamate), which produces channels with distinct properties. Evaluation of this modulator provides insight into gating mechanisms and may lead to the development of new therapeutic strategies.

Transcriptomic analysis of sexually dimorphic cypris larvae of the rhizocephalan barnacle Peltogasterella gracilis

Peltogasterella gracilis (Cirripedia: Rhizocephala), a crustacean parasite of hermit crabs, displays genotypic sex determination. Its larvae are planktonic, and female larvae settle on the host. Subsequently, the females control the host's behavior by spreading a root-like structure called “the interna” within the host's body, and form sacs containing eggs called “the externae” outside the host's body. On the other hand, male larvae settle on immature externae and become dwarf males. The cypris larvae of P. gracilis show sexual dimorphism in size and morphology. However, there is no understanding of the molecular mechanisms underlying the sexual dimorphism observed at the larval stage. Here, we conducted a transcriptome analysis and compared the expression of genes in male and female cyprids to better understand their sexual differentiation and settlement processes. A total of 2870 differentially expressed transcripts, comprising 456 female- and 2414 male-biased transcripts were identified. Among the male-biased ones, ionotropic glutamate receptor-, heat shock protein-, acetylcholine-, and homeobox-, cuticle-related transcripts were included. Additionally, 29 gene ontology terms were associated with the sex-specific traits. The present study improves our understanding of sex determination, sexual differentiation, and settlement processes of rhizocephalans.

Developmental Spike Timing-Dependent Long-Term Depression Requires Astrocyte d-Serine at L2/3-L2/3 Synapses of the Mouse Somatosensory Cortex.

Spike timing-dependent plasticity (STDP) is a learning rule important for synaptic refinement and for learning and memory during development. While different forms of presynaptic t-LTD have been deeply investigated, little is known about the mechanisms of somatosensory cortex postsynaptic t-LTD. In the present work, we investigated the requirements and mechanisms for induction of developmental spike timing-dependent long-term depression (t-LTD) at L2/3-L2/3 synapses in the juvenile mouse somatosensory cortex. We found that postnatal day (P) 13-21 mice of either sex show t-LTD at L2/3-L2/3 synapses induced by pairing single presynaptic activity with single postsynaptic action potentials at low stimulation frequency (0.2 Hz) that is expressed postsynaptically and requires the activation of ionotropic postsynaptic NMDA-type glutamate receptors containing GluN2B subunits. In addition, it requires postsynaptic Ca2+, Ca2+ release from internal stores, calcineurin, postsynaptic endocannabinoid synthesis, activation of CB1 receptors, and astrocytic signaling to release the gliotransmitter d-serine to activate postsynaptic NMDARs to induce t-LTD. These results show direct evidence of the mechanism involved in developmental postsynaptic t-LTD at L2/3-L2/3 synapses, revealing a central role of astrocytes and their release of d-serine in its induction.

Hippocampal tau-induced GRIN3A deficiency in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by significant alterations in hippocampal function and structure, but the molecular mechanisms underlying the hippocampal region remain elusive. We integrated multiple transcriptome datasets including human or rat hippocampus (GSE173955, GSE129051, GSE84422) to identify candidate genes. Subsequent analyses including gene ontology analysis and protein-protein interaction mapping were performed to identify key genes and pathways. We found that glutamate ionotropic receptor NMDA-type subunit 3A (GRIN3A) and glutamate metabotropic receptor 8 (GRM8), which are related to the glutamatergic system, were the top two annotated genes and directly related to MAPT, which encodes a tau protein. Since there is no direct evidence of interaction between tauopathy and these genes in AD, further transcriptomic data(GSE125957, GSE56772) from tau transgenic mice and experimental validations through primary rat hippocampal neurons and induced pluripotent stem cell (iPSC)-derived brain organoids were performed. Interestingly, we identified that decreased NR3A (encoded by GRIN3A) and mGluR8 (encoded by GRM8) are correlated with tauopathy and loss of postsynaptic function in AD. Taken together, our results identified a novel tauopathy biomarker GRIN3A in AD. Furthermore, our findings suggest that an integrated approach combining public databases and diverse experimental validations can contribute to the advancement of precision medicine therapies.

Bi-directional allosteric pathway in NMDA receptor activation and modulation

N-methyl-D-aspartate (NMDA) receptors are ionotropic glutamate receptors involved in learning and memory. NMDA receptors primarily comprise two GluN1 and two GluN2 subunits. The GluN2 subunit dictates biophysical receptor properties, including the extent of receptor activation and desensitization. GluN2A- and GluN2D-containing receptors represent two functional extremes. To uncover the conformational basis of their functional divergence, we utilize single-molecule fluorescence resonance energy transfer to probe the extracellular domains of these receptor subtypes under resting and ligand-bound conditions. We find that the conformational profile of the GluN2 amino-terminal domain correlates with the disparate functions of GluN2A- and GluN2D-containing receptors. Changes at the pre-transmembrane segments inversely correlate with those observed at the amino-terminal domain, confirming direct allosteric communication between these domains. Additionally, binding of a positive allosteric modulator at the transmembrane domain shifts the conformational profile of the amino-terminal domain towards the active state, revealing a bidirectional allosteric pathway between extracellular and transmembrane domains.

Exploring the role of AMPA receptor auxiliary proteins in synaptic functions and diseases.

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) ionotropic glutamate receptors (AMPARs) mediate rapid excitatory synaptic transmission in the mammalian brain, primarily driven by the neurotransmitter glutamate. The modulation of AMPAR activity, particularly calcium-permeable AMPARs (CP-AMPARs), is crucially influenced by various auxiliary subunits. These subunits are integral membrane proteins that bind to the receptor's core and modify its functional properties, including ion channel kinetics and receptor trafficking. This review comprehensively catalogs all known AMPAR auxiliary proteins, providing vital insights into the biochemical mechanisms governing synaptic modulation and the specific impact of CP-AMPARs compared to their calcium-impermeable AMPA receptor (CI-AMPARs). Understanding the complex interplay between AMPARs and their auxiliary subunits in different brain regions is essential for elucidating their roles in cognitive functions such as learning and memory. Importantly, alterations in these auxiliary proteins' expression, function or interactions have been implicated in various neurological disorders. Aberrant signaling through CP-AMPARs, in particular, is associated with severe synaptic dysfunctions across neurodevelopmental, neurodegenerative and psychiatric conditions. Targeting the distinct properties of AMPAR-auxiliary subunit complexes, especially those involving CP-AMPARs, could disclose new therapeutic strategies, potentially allowing for more precise interventions in treating complex neuronal disorders.

The diversity of AMPA receptor inhibition mechanisms among amidine-containing compounds.

Amidine-containing compounds are primarily known as antiprotozoal agents (pentamidine, diminazene, furamidine) or as serine protease inhibitors (nafamostat, sepimostat, camostat, gabexate). DAPI is widely recognized as a fluorescent DNA stain. Recently, it has been shown that these compounds also act as NMDA receptor inhibitors. In this study, we examined the activity of these compounds and analyzed the mechanisms of action in relation to another important class of ionotropic glutamate receptors-calcium-permeable AMPA receptors (CP-AMPARs) and calcium-impermeable AMPA receptors (CI-AMPARs) - using the whole-cell patch-clamp method on isolated male Wistar rat brain neurons. Gabexate and camostat were found to be inactive. Other compounds preferentially inhibited calcium-permeable AMPA receptors with IC50 values of 30-60µM. DAPI and furamidine were also active against CI-AMPARs with IC50s of 50-60μM, while others showed poor activity. All active compounds acted as channel blockers, which are able for permeating into the cytoplasm on both CP- and CI-AMPARs. Specifically, sepimostat showed trapping in the closed CP-AMPAR channel. Furamidine and DAPI demonstrated a voltage-independent action on CI-AMPARs, indicating binding to an additional superficial site. While the majority of compounds inhibited glutamate-activated steady-state currents as well as kainate-activated currents on CI-AMPARs, pentamidine significantly potentiated glutamate-induced steady-state responses. The potentiating effect of pentamidine resembles the action of the positive allosteric modulator cyclothiazide although the exact binding site remains unclear. Thus, this study, together with our previous research on NMDA receptors, provides a comprehensive overview of this novel group of ionotropic glutamate receptors inhibitors with a complex pharmacological profile, remarkable diversity of effects and mechanisms of action.

Postsynaptic Density Proteins and Their Role in the Trafficking of Group I Metabotropic Glutamate Receptors.

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system that regulates multiple different forms of synaptic plasticity, including learning and memory. Glutamate transduces its signal by activating ionotropic glutamate receptors and metabotropic glutamate receptors (mGluRs). Group I mGluRs belong to the G protein-coupled receptor (GPCR) family. Regulation of cell surface expression and trafficking of the glutamate receptors represents an important mechanism that assures proper transmission of information at the synapses. There is growing evidence implicating dysregulated glutamate receptor trafficking in the pathophysiology of several neuropsychiatric disorders. The postsynaptic density (PSD) region consists of many specialized proteins which are assembled beneath the postsynaptic membrane of dendritic spines. Many of these proteins interact with group I mGluRs and have essential roles in group I mGluR-mediated synaptic function and plasticity. This review provides up-to-date information on the molecular determinants regulating cell surface expression and trafficking of group I mGluRs and discusses the role of few of these PSD proteins in these processes. As substantial evidences link mGluR dysfunction and maladaptive functioning of many PSD proteins to the pathophysiology of various neuropsychiatric disorders, understanding the role of the PSD proteins in group I mGluR trafficking may provide opportunities for the development of novel therapeutics in multiple neuropsychiatric disorders.

Cross-species single-cell spatial transcriptomic atlases of the cerebellar cortex.

The molecular and cellular organization of the primate cerebellum remains poorly characterized. We obtained single-cell spatial transcriptomic atlases of macaque, marmoset, and mouse cerebella and identified primate-specific cell subtypes, including Purkinje cells and molecular-layer interneurons, that show different expression of the glutamate ionotropic receptor Delta type subunit 2 (GRID2) gene. Distinct gene expression profiles were found in anterior, posterior, and vestibular regions in all species, whereas region-selective gene expression was predominantly observed in the granular layer of primates and in the Purkinje layer of mice. Gene expression gradients in the cerebellar cortex matched well with functional connectivity gradients revealed with awake functional magnetic resonance imaging, with more lobule-specific differences between primates and mice than between two primate species. These comprehensive atlases and comparative analyses provide the basis for understanding cerebellar evolution and function.