Search

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 Ca 2+ 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 Ca 2+ 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., (Copyright © 2023 the authors.)

Kainate receptors (KARs) are glutamate-gated cation channels with diverse roles in the central nervous system. Bi-allelic loss of function of the KAR-encoding gene GRIK2 causes a nonsyndromic neurodevelopmental disorder (NDD) with intellectual disability and developmental delay as core features. The extent to which mono-allelic variants in GRIK2 also underlie NDDs is less understood because only a single individual has been reported previously. Here, we describe an additional eleven individuals with heterozygous de novo variants in GRIK2 causative for neurodevelopmental deficits that include intellectual disability. Five children harbored recurrent de novo variants (three encoding p.Thr660Lys and two p.Thr660Arg), and four children and one adult were homozygous for a previously reported variant (c.1969G>A [p.Ala657Thr]). Individuals with shared variants had some overlapping behavioral and neurological dysfunction, suggesting that the GRIK2 variants are likely pathogenic. Analogous mutations introduced into recombinant GluK2 KAR subunits at sites within the M3 transmembrane domain (encoding p.Ala657Thr, p.Thr660Lys, and p.Thr660Arg) and the M3-S2 linker domain (encoding p.Ile668Thr) had complex effects on functional properties and membrane localization of homomeric and heteromeric KARs. Both p.Thr660Lys and p.Thr660Arg mutant KARs exhibited markedly slowed gating kinetics, similar to p.Ala657Thr-containing receptors. Moreover, we observed emerging genotype-phenotype correlations, including the presence of severe epilepsy in individuals with the p.Thr660Lys variant and hypomyelination in individuals with either the p.Thr660Lys or p.Thr660Arg variant. Collectively, these results demonstrate that human GRIK2 variants predicted to alter channel function are causative for early childhood development disorders and further emphasize the importance of clarifying the role of KARs in early nervous system development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Key Points: Ionotropic glutamate receptor (iGluR) subunits are N-glycosylated at 4-12 sites, and Golgi processing produces mature receptors that contain high-mannose, hybrid and complex oligosaccharides. N-glycosylation is crucial for receptor biogenesis, influences receptor trafficking and provides a binding site for carbohydrate binding proteins. Glycan moieties are large, polar and occasionally charged, and they are attached at sites along iGluRs that position them for involvement in the structural changes underlying gating. Altering glycan content on kainate receptors (KARs), a subfamily of iGluRs, changes functional properties of the receptor, such as desensitization, recovery from desensitization and deactivation. We report the first observation that the charged trisaccharide HNK-1 is conjugated to native KARs, and we find that it substantially alters recombinant KAR functional properties. Our results show that the molecular composition of N-glycans can influence KAR biophysical properties, revealing a potential mechanism for fine-tuning the function of these receptors., Abstract: Ionotropic glutamate receptors (iGluRs) are tetrameric proteins with between four and 12 consensus sites for N-glycosylation on each subunit, which potentially allows for a high degree of structural diversity conferred by this post-translational modification. N-glycosylation is required for proper folding of iGluRs in mammalian cells, although the impact of oligosaccharides on the function of successfully folded receptors is less clear. Glycan moieties are large, polar, occasionally charged and mediate many protein-protein interactions throughout the nervous system. Additionally, they are attached at sites along iGluR subunits that position them for involvement in the structural changes underlying gating. In the present study, we show that altering glycan content on kainate receptors (KARs) changes the functional properties of the receptors in a manner dependent on the identity of both the modified sugars and the subunit composition of the receptor to which they are attached. We also report that native KARs carry the complex capping oligosaccharide human natural killer-1. Glycosylation patterns probably differ between cell types, across development or with pathologies, and thus our findings reveal a potential mechanism for context-specific fine-tuning of KAR function through diversity in glycan structure., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

Peripheral sensory neurons in the dorsal root ganglia (DRG) are the initial transducers of sensory stimuli, including painful stimuli, from the periphery to central sensory and pain-processing centers. Small- to medium-diameter non-peptidergic neurons in the neonatal DRG express functional kainate receptors (KARs), one of three subfamilies of ionotropic glutamate receptors, as well as the putative KAR auxiliary subunit Neuropilin- and tolloid-like 2 (Neto2). Neto2 alters recombinant KAR function markedly but has yet to be confirmed as an auxiliary subunit that assembles with and alters the function of endogenous KARs. KARs in neonatal DRG require the GluK1 subunit as a necessary constituent, but it is unclear to what extent other KAR subunits contribute to the function and proposed roles of KARs in sensory ganglia, which include promotion of neurite outgrowth and modulation of glutamate release at the DRG-dorsal horn synapse. In addition, KARs containing the GluK1 subunit are implicated in modes of persistent but not acute pain signaling. We show here that the Neto2 protein is highly expressed in neonatal DRG and modifies KAR gating in DRG neurons in a developmentally regulated fashion in mice. Although normally at very low levels in adult DRG neurons, Neto2 protein expression can be upregulated via MEK/ERK signaling and after sciatic nerve crush and Neto2 -/- neurons from adult mice have stunted neurite outgrowth. These data confirm that Neto2 is a bona fide KAR auxiliary subunit that is an important constituent of KARs early in sensory neuron development and suggest that Neto2 assembly is critical to KAR modulation of DRG neuron process outgrowth. SIGNIFICANCE STATEMENT Pain-transducing peripheral sensory neurons of the dorsal root ganglia (DRG) express kainate receptors (KARs), a subfamily of glutamate receptors that modulate neurite outgrowth and regulate glutamate release at the DRG-dorsal horn synapse. The putative KAR auxiliary subunit Neuropilin- and tolloid-like 2 (Neto2) is also expressed in DRG. We show here that it is a developmentally downregulated but dynamic component of KARs in these neurons, that it contributes to regulated neurite regrowth in adult neurons, and that it is increased in adult mice after nerve injury. Our data confirm Neto2 as a KAR auxiliary subunit and expand our knowledge of the molecular composition of KARs in nociceptive neurons, a key piece in understanding the mechanistic contribution of KAR signaling to pain-processing circuits., (Copyright © 2017 the authors 0270-6474/17/373352-12$15.00/0.)

Kainate receptors are members of the glutamate receptor family that regulate synaptic function in the brain. They modulate synaptic transmission and the excitability of neurons; however, their contributions to neural circuits that underlie behavior are unclear. To understand the net impact of kainate receptor signaling, we generated knockout mice in which all five kainate receptor subunits were ablated (5ko). These mice displayed compulsive and perseverative behaviors, including over-grooming, as well as motor problems, indicative of alterations in striatal circuits. There were deficits in corticostriatal input to spiny projection neurons (SPNs) in the dorsal striatum and correlated reductions in spine density. The behavioral alterations were not present in mice only lacking the primary receptor subunit expressed in adult striatum (GluK2 KO), suggesting that signaling through multiple receptor types is required for proper striatal function. This demonstrates that alterations in striatal function dominate the behavioral phenotype in mice without kainate receptors., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Key Points: Kainate receptors (KARs) are ionotropic glutamate receptors (iGluRs) that modulate synaptic transmission and intrinsic neuronal excitability. KARs associate with the auxiliary proteins neuropilin- and tolloid-like 1 and 2 (Neto1 and Neto2), which act as allosteric modulators of receptor function impacting all biophysical properties of these receptors studied to date. M3-S2 linkers play a critical role in KAR gating; we found that individual residues in these linkers bidirectionally influence Neto2 modulation of KAR desensitization in an agonist specific manner. We also identify the D1 dimer interface as a novel site of Neto2 modulation and functionally correlate the actions of Neto2 modulation of desensitization with modulation of cation sensitivity. We identify these domains as determinants of Neto2 modulation. Thus, our work contributes to the understanding of auxiliary subunit modulation of KAR function and could aid the development of KAR-specific modulators to alter receptor function., Abstract: Kainate receptors (KARs) are important modulators of synaptic transmission and intrinsic neuronal excitability in the CNS. Their activity is shaped by the auxiliary proteins Neto1 and Neto2, which impact KAR gating in a receptor subunit- and Neto isoform-specific manner. The structural basis for Neto modulation of KAR gating is unknown. Here we identify the M3-S2 gating linker as a critical determinant contributing to Neto2 modulation of KARs. M3-S2 linkers control both the valence and magnitude of Neto2 modulation of homomeric GluK2 receptors. Furthermore, a single mutation in this domain abolishes Neto2 modulation of heteromeric receptor desensitization. Additionally, we found that cation sensitivity of KAR gating is altered by Neto2 association, suggesting that stability of the D1 dimer interface in the ligand-binding domain (LBD) is an important determinant of Neto2 actions. Moreover, modulation of cation sensitivity was eliminated by mutations in the M3-S2 linkers, thereby correlating the action of Neto2 at these structurally discrete sites on receptor subunits. These results demonstrate that the KAR M3-S2 linkers and LBD dimer interface are critical determinants for Neto2 modulation of receptor function and identify these domains as potential sites of action for the targeted development of KAR-specific modulators that alter the function of auxiliary proteins in native receptors., (© 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.)

Here we report the bioactivity-guided isolation of novel galectins from the marine sponge Cinachyrella sp., collected from Iriomote Island, Japan. The lectin proteins, which we refer to as the Cinachyrella galectins (CchGs), were identified as the active principles in an aqueous sponge extract that modulated the function of mammalian ionotropic glutamate receptors. Aggregation of rabbit erythrocytes by CchGs was competed most effectively by galactosides but not mannose, a profile characteristic of members of the galectin family of oligosaccharide-binding proteins. The lectin activity was remarkably stable, with only a modest loss in hemagglutination after exposure of the protein to 100°C for 1 h, and showed little sensitivity to calcium concentration. CchG-1 and -2 appeared as 16 and 18 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively, whereas matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry indicated broad ion clusters centered at 16,216 and 16,423, respectively. The amino acid sequences of the CchGs were deduced using a combination of Edman degradation and cDNA cloning and revealed that the proteins were distant orthologs of animal prototype galectins and that multiple isolectins comprised the CchGs. One of the isolectins was expressed as a recombinant protein and exhibited physico-chemical and biological properties comparable with those of the natural lectins. The biochemical properties of the CchGs as well as their unexpected activity on mammalian excitatory amino acid receptors suggest that further analysis of these new members of the galectin family will yield further glycobiological and neurophysiological insights.

Kainate receptors exhibit a highly compartmentalized distribution within the brain; however, the molecular and cellular mechanisms that coordinate their expression at neuronal sites of action are poorly characterized. Here we report that the GluK1 and GluK2 kainate receptor subunits interact with the spectrin-actin binding scaffolding protein 4.1N through a membrane-proximal domain in the C-terminal tail. We found that this interaction is important for the forward trafficking of GluK2a receptors, their distribution in the neuronal plasma membrane, and regulation of receptor endocytosis. The association between GluK2a receptors and 4.1N was regulated by both palmitoylation and protein kinase C (PKC) phosphorylation of the receptor subunit. Palmitoylation of the GluK2a subunit promoted 4.1N association, and palmitoylation-deficient receptors exhibited reduced neuronal surface expression and compromised endocytosis. Conversely, PKC activation decreased 4.1N interaction with GluK2/3-containing kainate receptors in acute brain slices, an effect that was reversed after inhibition of PKC. Our data and previous studies therefore demonstrate that these two post-translational modifications have opposing effects on 4.1N association with GluK2 kainate and GluA1 AMPA receptors. The convergence of the signaling pathways regulating 4.1N protein association could thus result in the selective removal of AMPA receptors from the plasma membrane while simultaneously promoting the insertion and stabilization of kainate receptors, which may be important for tuning neuronal excitability and synaptic plasticity.

Receptors and channels that underlie nociceptive signaling constitute potential sites of intervention for treatment of chronic pain states. The kainate receptor family of glutamate-gated ion channels represents one such candidate set of molecules. They have a prominent role in modulation of excitatory signaling between sensory and spinal cord neurons. Kainate receptors are also expressed throughout central pain neuraxis, where their functional contributions to neural integration are less clearly defined. Pharmacological inhibition or genetic ablation of kainate receptor activity reduces pain behaviors in a number of animal models of chronic pain, and small clinical trials have been conducted using several orthosteric antagonists. This review will cover kainate receptor function and participation in pain signaling as well as the pharmacological studies supporting further consideration as potential targets for therapeutic development.

Kainate receptors are a family of ionotropic glutamate receptors whose physiological roles differ from those of other subtypes of glutamate receptors in that they predominantly serve as modulators, rather than mediators, of synaptic transmission. Neuronal kainate receptors exhibit unusually slow kinetic properties that have been difficult to reconcile with the behaviour of recombinant kainate receptors. Recently, however, the neuropilin and tolloid-like 1 (NETO1) and NETO2 proteins were identified as auxiliary kainate receptor subunits that shape both the biophysical properties and synaptic localization of these receptors.

Auxiliary proteins modify the biophysical function and pharmacological properties of ionotropic glutamate receptors and likely are important components of receptor signaling complexes in vivo. The neuropilin and tolloid-like proteins (NETO) 1 and NETO2, two closely related CUB domain-containing integral membrane proteins, were identified recently as auxiliary proteins that slowed GluK2a kainate receptor current kinetics without impacting receptor membrane localization. Here we demonstrate that NETO2 profoundly slows the desensitization rate of GluK1 kainate receptors, promotes plasma membrane localization of transfected receptors in heterologous cells and rat hippocampal neurons, and targets GluK1-containing receptors to synapses. Conversely, the closely related protein NETO1 increases the rate of GluK1 receptor desensitization. Incorporation of NETO proteins into kainate receptor-signaling complexes therefore extends the temporal range of receptor gating by over an order of magnitude. The presence of these auxiliary proteins could underlie some of the unusual aspects of kainate receptor function in the mammalian CNS.

The ionotropic glutamate receptor subunit, GluK1 (GluR5), is expressed in many regions of the nervous system related to sensory transmission. Recently, a selective ligand for the GluK1 receptor, MSVIII-19 (8,9-dideoxy-neodysiherbaine), was synthesized as a derivative of dysiherbaine, a toxin isolated from the marine sponge Lendenfeldia chondrodes. MSVIII-19 potently desensitizes GluK1 receptors without channel activation, rendering it useful as a functional antagonist. Given the high selectivity for GluK1 and the proposed role for this glutamate receptor in nociception, we sought to test the analgesic potential of MSVIII-19 in a series of models of inflammatory, neuropathic, and visceral pain in mice. MSVIII-19 delivered intrathecally dose-dependently reduced formalin-induced spontaneous behaviors and reduced thermal hypersensitivity 3 hours after formalin injection and 24 hours after complete Freund's adjuvant-induced inflammation, but had no effect on mechanical sensitivity in the same models. Intrathecal MSVIII-19 significantly reduced both thermal hyperalgesia and mechanical hypersensitivity in the chronic constriction injury model of neuropathic pain, but had no effect in the acetic acid model of visceral pain. Peripheral administration of MSVIII-19 had no analgesic efficacy in any of these models. Finally, intrathecal MSVIII-19 did not alter responses in Tail-flick tests or performance on the accelerating RotaRod. These data suggest that spinal administration of MSVIII-19 reverses hypersensitivity in several models of pain in mice, supporting the clinical potential of GluK1 antagonists for the management of pain., (Copyright © 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

Two decades have passed since the first report of the cloning of a kainate-type glutamate receptor (KAR) subunit. The intervening years have seen a rapid growth in our understanding of the biophysical properties and function of KARs in the brain. This research has led to an appreciation that KARs play very distinct roles at synapses relative to other members of the glutamate-gated ion channel receptor family, despite structural and functional commonalities. The surprisingly diverse and complex nature of KAR signaling underlies their unique impact upon neuronal networks through their direct and indirect effects on synaptic transmission, and their prominent role in regulating cell excitability. This review pieces together highlights from the two decades of research subsequent to the cloning of the first subunit, and provides an overview of our current understanding of the role of KARs in the CNS and their potential importance to neurological and neuropsychiatric disorders., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Kainate receptor antagonists have potential as therapeutic agents in a number of neuropathologies. Synthetic modification of the convulsant marine toxin neodysiherbaine A (NDH) previously yielded molecules with a diverse set of pharmacological actions on kainate receptors. Here we characterize three new synthetic analogs of NDH that contain substituents at the C10 position in the pyran ring of the marine toxin. The analogs exhibited high-affinity binding to the GluK1 (GluR5) subunit and lower affinity binding to GluK2 (GluR6) and GluK3 (GluR7) subunits in radioligand displacement assays with recombinant kainate and AMPA receptors. As well, the natural toxin NDH exhibited approximately 100-fold selectivity for GluK2 over GluK3 subunits, which was attributable to the C8 hydroxyl group in NDH. We used molecular dynamic simulations to determine the specific interactions between NDH and residues within the ligand-binding domains of these two kainate receptor subunits that contribute to the divergent apparent affinities for the compound. These data demonstrate that interactions with the GluK1 subunit are preserved in analogs with substitutions at C10 in NDH and further reveal the determinants of selectivity and pharmacological activity of molecules acting on kainate receptor subunits, which could aid in design of additional compounds that target these receptors., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Ionotropic glutamate receptors (iGluRs) are enticing targets for pharmaceutical research; however, the search for selective ligands is a laborious experimental process. Here we introduce a purely computational procedure as an approach to evaluate ligand-iGluR pharmacology. The ligands are docked into the closed ligand-binding domain and during the molecular dynamics (MD) simulation the bi-lobed interface either opens (partial agonist/antagonist) or stays closed (agonist) according to the properties of the ligand. The procedure is tested with closely related set of analogs of the marine toxin dysiherbaine bound to GluK1 kainate receptor. The modeling is set against the abundant binding data and electrophysiological analyses to test reproducibility and predictive value of the procedure. The MD simulations produce detailed binding modes for analogs, which in turn are used to define structure-activity relationships. The simulations suggest correctly that majority of the analogs induce full domain closure (agonists) but also distinguish exceptions generated by partial agonists and antagonists. Moreover, we report ligand-induced opening of the GluK1 ligand-binding domain in free MD simulations. The strong correlation between in silico analysis and the experimental data imply that MD simulations can be utilized as a predictive tool for iGluR pharmacology and functional classification of ligands., (2009 Elsevier Ltd. All rights reserved.)

Kainate receptors signal through both ionotropic and metabotropic pathways. The high-affinity subunits, GluK4 and GluK5, are unique among the five receptor subunits, as they do not form homomeric receptors but modify the properties of heteromeric assemblies. Disruption of the Grik4 gene locus resulted in a significant reduction in synaptic kainate receptor currents. Moreover, ablation of GluK4 and GluK5 caused complete loss of synaptic ionotropic kainate receptor function. The principal subunits were distributed away from postsynaptic densities and presynaptic active zones. There was also a profound alteration in the activation properties of the remaining kainate receptors. Despite this, kainate receptor-mediated inhibition of the slow afterhyperpolarization current (I(sAHP)), which is dependent on metabotropic pathways, was intact in GluK4/GluK5 knockout mice. These results uncover a previously unknown obligatory role for the high-affinity subunits for ionotropic kainate receptor function and further demonstrate that kainate receptor participation in metabotropic signaling pathways does not require their classic role as ion channels.

The prevailing structural model for ligand activation of ionotropic glutamate receptors posits that agonist efficacy arises from the stability and magnitude of induced domain closure in the ligand-binding core structure. Here we describe an exception to the correlation between ligand efficacy and domain closure. A weakly efficacious partial agonist of very low potency for homomeric iGluR5 kainate receptors, 8,9-dideoxyneodysiherbaine (MSVIII-19), induced a fully closed iGluR5 ligand-binding core. The degree of relative domain closure, approximately 30 degrees , was similar to that we resolved with the structurally related high affinity agonist dysiherbaine and to that of l-glutamate. The pharmacological activity of MSVIII-19 was confirmed in patch clamp recordings from transfected HEK293 cells, where MSVIII-19 predominantly inhibits iGluR5-2a, with little activation apparent at a high concentration (1 mm) of MSVIII-19 (<1% of mean glutamate-evoked currents). To determine the efficacy of the ligand quantitatively, we constructed concentration-response relationships for MSVIII-19 following potentiation of steady-state currents with concanavalin A (EC(50) = 3.6 microm) and on the nondesensitizing receptor mutant iGluR5-2b(Y506C/L768C) (EC(50) = 8.1 microm). MSVIII-19 exhibited a maximum of 16% of full agonist efficacy, as measured in parallel recordings with glutamate. Molecular dynamics simulations and electrophysiological recordings confirm that the specificity of MSVIII-19 for iGluR5 is partly attributable to interdomain hydrogen bond residues Glu(441) and Ser(721) in the iGluR5-S1S2 structure. The weaker interactions of MSVIII-19 with iGluR5 compared with dysiherbaine, together with altered stability of the interdomain interaction, may be responsible for the apparent uncoupling of domain closure and channel opening in this kainate receptor subunit.

Intracellular glutamate binding within the endoplasmic reticulum (ER) is thought to be necessary for plasma membrane expression of ionotropic glutamate receptors. Here we determined the importance of glutamate binding to folding and assembly of soluble ligand-binding domains (LBDs), as well as full-length receptors, by comparing the secretion of a soluble GluR6-S1S2 protein versus the plasma membrane localization of GluR6 kainate receptors following mutagenesis of the LBD. The mutations were designed to either eliminate glutamate binding, thereby trapping the bilobate LBD in an "open" conformation, or "lock" the LBD in a closed conformation with an engineered interdomain disulfide bridge. Analysis of plasma membrane localization, medium secretion of soluble LBD proteins, and measures of folding efficiency suggested that loss of glutamate binding affinity significantly impacted subunit protein folding and assembly. In contrast, receptors with conformationally restricted LBDs also exhibited decreased PM expression and altered oligomeric receptor assembly but did not exhibit any deficits in subunit folding. Secretion of the closed LBD protein was enhanced compared with wild-type GluR6-S1S2. Our results suggest that glutamate acts as a chaperone molecule for appropriate folding of nascent receptors and that relaxation of LBDs from fully closed states during oligomerization represents a critical transition that necessarily engages other determinants within receptor dimers. Glutamate receptor LBDs therefore must access multiple conformations for efficient biogenesis.

Marine-derived small molecules and peptides have played a central role in elaborating pharmacological specificities and neuronal functions of mammalian ionotropic glutamate receptors (iGluRs), the primary mediators of excitatory synaptic transmission in the central nervous system (CNS). As well, the pathological sequelae elicited by one class of compounds (the kainoids) constitute a widely-used animal model for human mesial temporal lobe epilepsy (mTLE). New and existing molecules could prove useful as lead compounds for the development of therapeutics for neuropathologies that have aberrant glutamatergic signaling as a central component. In this chapter we discuss natural source origins and pharmacological activities of those marine compounds that target ionotropic glutamate receptors.

Antagonists for kainate receptors (KARs), a family of glutamategated ion channels, are efficacious in a number of animal models of neuropathologies, including epilepsy, migraine pain, and anxiety. To produce molecules with novel selectivities for kainate receptors, we generated three sets of analogs related to the natural marine convulsant neodysiherbaine (neoDH), and we characterized their pharmacological profiles. Radioligand displacement assays with recombinant alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and KARs demonstrated that functional groups at two positions on the neoDH molecule are critical pharmacological determinants; only binding to the glutamate receptor (GluR)5-2a subunit was relatively insensitive to structural modifications of the critical functional groups. NeoDH analogs in which the l-glutamate congener was disrupted by epimerization retained low affinity for GluR5-2a and GluR6a KAR subunits. Most of the analogs showed agonist activity in electrophysiological recordings from human embryonic kidney-T/17 cells expressing GluR5-2a KARs, similar to the natural convulsant neoDH. In contrast, 2,4-epi-neoDH inhibited glutamate currents evoked from both GluR5-2a and GluR6a receptor-expressing cells. Therefore, this compound represents the first compound to exhibit functional antagonist activity on GluR5-2a and GluR6a KAR subunits without concurrent activity on AMPA receptor subunits. Finally, binding affinity of the synthetic ligands for the GluR5-2a subunit closely correlated with their seizurogenic potency, strongly supporting a role for receptors containing this subunit in the convulsant reaction to KAR agonists. The analogs described here offer further insight into structural determinants of ligand selectivity for KARs and potentially represent useful pharmacological tools for studying the role of KARs in synaptic physiology and pathology.

Kainate receptors (KARs) are neuronal proteins that exhibit a highly polarized distribution in the mammalian CNS. Assembly, intracellular trafficking, and synaptic targeting of KARs and other ionotropic glutamate receptors are processes controlled, in part, by various determinants within the constituent subunit proteins themselves. Here, we demonstrate that the linker region between the M3 and S2 domains, which in current structural models is thought to transduce ligand-binding energy into channel opening, additionally has an essential role in receptor biogenesis. Our results show that this gating-associated domain is engaged at two distinct critical stages of KAR biogenesis: first, during the transition from dimeric to tetrameric assembly states and, second, at a postassembly trafficking checkpoint within the endoplasmic reticulum. Alteration of a basic residue, arginine 663, altered the desensitization properties of the GluR6 kainate receptor in response to glutamate application, and these changes were weakly correlated with intracellular retention of the mutant receptors. Elimination of the positive charge also significantly attenuated oligomerization and stability of the intracellular subunit protein. Furthermore, charge swapping with an adjacent residue, glutamate 662, normalized the receptor physiological behavior and reversed the deficits in assembly and degradation, but only partially restored plasma membrane expression of the receptors. These results reveal a new role for this linker domain in glutamate receptor biogenesis and contribute to understanding the cellular controls of receptor assembly and trafficking, which will be important for relating receptor stoichiometry to their neuronal targeting and function.

Dysiherbaine (1) and its congener neodysiherbaine A (2) are naturally occurring excitatory amino acids with selective and potent agonistic activity for ionotropic glutamate receptors. We describe herein the total synthesis of 2 and its structural analogues 3-8. Advanced key intermediate 16 was employed as a branching point to assemble a series of these analogues 3-8 with respect to the C8 and C9 functionalities, which would not have been accessible through manipulations of the natural product itself. The synthesis of key intermediate 16 features (i) stereocontrolled C-glycosylation to set the C6 stereocenter, (ii) concise synthesis of the bicyclic ether skeleton through chemo- and stereoselective dihydroxylation of the exo-olefin and stereoselective epoxidation of the endo-olefin, followed by epoxide ring opening/5-exo ring closure, and (iii) catalytic asymmetric hydrogenation of enamide ester to construct the amino acid appendage. A preliminary biological evaluation of analogues for their in vivo toxicity against mice and binding affinity for glutamate receptors showed that both the type and stereochemistry of the C8 and C9 functional groups affected the subtype selectivity of dysiherbaine analogues for members of the kainic acid receptor family.

Assembly and trafficking of neurotransmitter receptors are processes contingent upon interactions between intracellular chaperone systems and discrete determinants in the receptor proteins. Kainate receptor subunits, which form ionotropic glutamate receptors with diverse roles in the central nervous system, contain a variety of trafficking determinants that promote either membrane expression or intracellular sequestration. In this report, we identify the coatomer protein complex I (COPI) vesicle coat as a critical mechanism for retention of the kainate receptor subunit KA2 in the endoplasmic reticulum. COPI subunits immunoprecipitated with KA2 subunits from both cerebellum and COS-7 cells, and beta-COP protein interacted directly with immobilized KA2 peptides containing the arginine-rich retention/retrieval determinant. Association between COPI proteins and KA2 subunits was significantly reduced upon alanine substitution of this signal in the cytoplasmic tail of KA2. Temperature-sensitive degradation of COPI complex proteins was correlated with an increase in plasma membrane localization of the homologous KA2 receptor. Assembly of heteromeric GluR6a/KA2 receptors markedly reduced association of KA2 and COPI. Finally, the reduction in COPI binding was correlated with an increased association with 14-3-3 proteins, which mediate forward trafficking of other integral signaling proteins. These interactions therefore represent a critical early checkpoint for biosynthesis of functional KARs.

Dysiherbaine (DH) and related molecules are high-affinity, subunit-selective kainate receptor (KAR) ligands originally isolated from a marine sponge. To elucidate why DH, an agonist, and MSVIII-19, a competitive antagonist, bind selectively to glutamate receptor (GluR) 5 but not to the KA2 KAR subunit, we used molecular dynamics simulations to generate binding models that were tested experimentally in radioligand binding and electrophysiological assays. Three candidate sites, Val685, Leu735, and Ser741 in GluR5, corresponding to Ile669, Phe719, and Met725 in KA2, were predicted to underlie the distinct binding profiles of the marine toxins. Single or multiple reciprocal mutations introduced into the receptor subunits produced a variety of effects on binding affinity. Most notably, mutation of Met725 to serine in KA2 increased the affinity of DH by 350-fold; in contrast, mutation of one or more of the residues in GluR5 did not markedly alter DH binding. MSVIII-19 affinity for the KA2 subunit was significantly increased in multiple site mutants, and reciprocal mutations in the GluR5 subunit produced substantial (700-fold) reductions in MSVIII-19 affinity. Physiological characterization of the double- and triple-mutant subunits demonstrated altered functional behavior consistent with the changes in binding affinity. The results provide experimental support for the importance of these three ligand binding domain (LBD) residues and suggest steric hindrance in the KA2 subunit LBD is largely responsible for the very low affinity for the two compounds. In this study, we identified the molecular basis for subunit selectivity of these marine-derived molecules on KARs, which could facilitate the rational design of selective ligands with distinct pharmacological profiles.

Intracellular trafficking of ionotropic glutamate receptors is controlled by multiple discrete determinants in receptor subunits. Most such determinants have been localized to the cytoplasmic carboxyl-terminal domain, but other domains in the subunit proteins can play roles in modulating receptor surface expression. Here we demonstrate that formation of an intact glutamate binding site also acts as an additional quality-control check for surface expression of homomeric and heteromeric kainate receptors. A key ligand-binding residue in the KA2 subunit, threonine 675, was mutated to either alanine or glutamate, which eliminated affinity for the receptor ligands kainate and glutamate. We found that plasma membrane expression of heteromeric GluR6/KA2(T675A) or GluR6/KA2(T675E) kainate receptors was markedly reduced compared with wild-type GluR6/KA2 receptors in transfected HEK 293 and COS-7 cells and in cultured neurons. Surface expression of homomeric KA2 receptors lacking a retention/retrieval determinant (KA2-R/A) was also reduced upon mutation of Thr-675 and elimination of the ligand binding site. KA2 Thr-675 mutant subunits were able to co-assemble with GluR5 and GluR6 subunits and were degraded at the same rate as wild-type KA2 subunit protein. These results suggest that glutamate binding and associated conformational changes are prerequisites for forward trafficking of intracellular kainate receptors following multimeric assembly.

Kainate receptor-mediated components of postsynaptic currents at hippocampal mossy fiber synapses have markedly slower kinetics than currents arising from AMPA receptors. Here, we demonstrate that other aspects of kainate and AMPA receptor function at this synapse are distinct; in particular, kainate receptor currents are less sensitive to short- and long-term increases in presynaptic strength. EPSCs arising predominantly from AMPA receptors exhibited well characterized paired-pulse facilitation, frequency facilitation, and NMDA receptor-independent long-term potentiation, whereas isolated kainate receptor synaptic currents (KA-EPSCs) exhibited attenuated facilitation and long-term potentiation. In addition, KA-EPSCs varied in their sensitivity to a low-affinity competitive antagonist, suggestive of a synaptic heterogeneity greater than that of EPSCs comprised predominantly of AMPA receptors. These data suggest that the proportional contribution of AMPA and kainate receptors to ensemble synaptic currents will vary depending on the firing frequency of mossy fiber afferents. These synaptic features may be a mechanism for limiting activation of kainate receptors at mossy fiber synapses, which has been shown to be involved in seizurogenic firing of the CA3 network.

Intracellular trafficking of ionotropic glutamate receptors is regulated predominantly by determinants in the cytoplasmic C-terminal domain of the subunit proteins. Although AMPA receptors are found at the vast majority of excitatory synapses, synaptic kainate receptors exhibit a much more restricted distribution, suggesting that specific mechanisms exist for selective trafficking of these receptor proteins. In this report, we define a critical forward trafficking motif that is necessary for surface expression of the glutamate receptor 6 (GluR6) kainate receptor as well as chimeric proteins containing only the GluR6 C-terminal domain. The trafficking determinant was identified by tracking surface expression of green fluorescent protein-tagged GluR6 receptors with confocal immunofluorescence in COS-7 cells and cultured neurons and patch-clamp electrophysiology in human embryonic kidney 293 cells. Serial truncation and alanine site mutagenesis of the GluR6 subunit C terminus localized the critical motif to a seven amino acid stretch of predominantly basic residues. Alanine mutation of the trafficking motif reduced kainate receptor current amplitudes by >90% and resulted in retention of the mutated receptors in the endoplasmic reticulum. This forward trafficking domain is the first such identified for kainate receptors.

Kainate receptors (KARs) are mediators of excitatory neurotransmission in the mammalian central nervous system, and their efficient targeting and trafficking is critical for normal synaptic function. A key step in the delivery of KARs to the neuronal plasma membrane is the exit of newly assembled receptors from the endoplasmic reticulum (ER). Here we report the identification of a novel ER retention signal in the alternatively spliced C-terminal domain of the GluR5-2b subunit, which controls receptor trafficking in both heterologous cells and neurons. The ER retention motif consists of a critical arginine (Arg-896) and surrounding amino acids, disruption of which promotes ER exit and surface expression of the receptors, as well as altering their physiological properties. The Arg-896-mediated ER retention of GluR5 is regulated by a mutation that mimics phosphorylation of Thr-898, but not by PDZ interactions. Furthermore, two positively charged residues (Arg-900 and Lys-901) in the C terminus were also found to regulate ER export of the receptors. Taken together, our results identify novel trafficking signals in the C-terminal domain of GluR5-2b and demonstrate that alternative splicing is an important mechanism regulating KAR function.

Kainate receptors function as mediators of postsynaptic currents and as presynaptic modulators of synaptic transmission at mossy fiber synapses. Despite intense research into the physiological properties of mossy fiber kainate receptors, their subunit composition in the presynaptic and postsynaptic compartments is unclear. Here we describe the distribution of kainate receptor subunits in mossy fiber synapses using subunit-selective antibodies and knock-out mice. We provide morphological evidence for the presynaptic localization of KA1 and KA2 receptor subunits at mossy fiber synapses. Immunogold staining for KA1 and KA2 was commonly seen at synaptic contacts and in vesicular structures. Postsynaptic labeling in dendritic spines was also observed. Although KA1 predominantly showed presynaptic localization, KA2 was concentrated to a greater degree on postsynaptic membranes. Both subunits coimmunoprecipitated from hippocampal membrane extracts with GluR6 but not GluR7 subunits. These results demonstrate that KA1 and KA2 subunits are localized presynaptically and postsynaptically at mossy fiber synapses where they most likely coassemble with GluR6 subunits to form functional heteromeric kainate receptor complexes.

The kainate receptor subunit KA2 does not form functional homomeric channels despite its structural similarity to the functional glutamate receptor 5-7subunits and high agonist binding affinity in in vitro assays. In this study, we first demonstrate that homomeric KA2 receptors fail to reach the plasma membrane and then identify the molecular mechanisms preventing surface expression. Specifically, we show that KA2 subunits form homooligomeric receptors that are confined to the endoplasmic reticulum (ER). We then demonstrate that, in both heterologous expression systems and primary neurons, the intracellular retention of KA2 is not caused by subunit misfolding but, rather, is mediated through discrete protein trafficking signals, including an arginine-rich ER retention/retrieval motif and a di-leucine endocytic sequence in the C terminus of the KA2 subunit. Disruption of these motifs results in ER exit and surface expression of KA2 homomeric receptors that remain nonfunctional. Furthermore, our data suggest that the ER retention/retrieval signal in KA2 is sterically shielded during heteromeric assembly, allowing delivery of functional heteromeric receptors to the plasma membrane. Taken together, our results illustrate novel regulatory mechanisms that control the intracellular trafficking and surface expression of kainate receptors.

Multimeric assemblies of kainate (KA) receptor subunits form glutamate-gated ion channels that mediate EPSCs and function as presynaptic modulators of neurotransmitter release at some central synapses. The KA2 subunit is a likely constituent of many neuronal kainate receptors, because it is widely expressed in most neurons in the CNS. We have studied the effect of genetic ablation of this receptor subunit on synaptic transmission at the mossy-fiber-CA3 pyramidal cell synapse in hippocampal slices, where kainate receptors are localized to both presynaptic and postsynaptic sites. We found that both postsynaptic and presynaptic mossy-fiber kainate receptor function is altered in neurons from KA2-/- mice. The presynaptic facilitatory autoreceptor, which modulates glutamate release from mossy-fiber terminals, had a reduced affinity for exogenous agonists and synaptic glutamate. Although presynaptic facilitation attributable to homosynaptic glutamate release was normal at mossy-fiber synapses in KA2-/- neurons, heterosynaptic kainate receptor-mediated facilitation resulting from the spillover of glutamate from CA3 collateral synapses was absent. Consistent with a decrease in glutamate affinity of the receptor, the half-decay of the postsynaptic kainate-mediated EPSC was shorter in the knock-out mice. These results identify the KA2 subunit as a determinant of kainate receptor function at presynaptic and postsynaptic mossy-fiber kainate receptors.

Neuronal kainate receptors are assembled from subunits with dissimilar specificities for agonists and antagonists. The composite biophysical behavior of heteromeric kainate receptors is determined by intersubunit interactions whose nature is unclear. Here we use dysiherbaine, a selective kainate receptor agonist, to show that GluR5 subunits assembled in heteromeric GluR5/KA-2 kainate receptor complexes can gate current without concomitant activation of their partner KA-2 subunits. A long-lasting interaction between dysiherbaine and GluR5 subunits elicits a tonic current from GluR5/KA-2 receptors; subsequent cooperative gating of KA-2 subunits can be elicited by both agonists, such as glutamate, and some classically defined antagonists, such as CNQX. This study demonstrates that each type of subunit within a heteromeric kainate receptor contributes a distinct conductance upon activation by agonist binding, and therefore provides insight into the biophysical function of ionotropic glutamate receptors.