Crystal structures of the GluR5 and GluR6 ligand binding cores: molecular mechanisms underlying kainate receptor selectivity

Little is known about the molecular mechanisms underlying differences in the ligand binding properties of AMPA, kainate, and NMDA subtype glutamate receptors. Crystal structures of the GluR5 and GluR6 kainate receptor ligand binding cores in complexes with glutamate, 2S,4R-4-methylglutamate, kainate, and quisqualate have now been solved. The structures reveal that the ligand binding cavities are 40% (GluR5) and 16% (GluR6) larger than for GluR2. The binding of AMPA- and GluR5-selective agonists to GluR6 is prevented by steric occlusion, which also interferes with the high-affinity binding of 2S,4R-4-methylglutamate to AMPA receptors. Strikingly, the extent of domain closure produced by the GluR6 partial agonist kainate is only 3 degrees less than for glutamate and 11 degrees greater than for the GluR2 kainate complex. This, together with extensive interdomain contacts between domains 1 and 2 of GluR5 and GluR6, absent from AMPA receptors, likely contributes to the high stability of GluR5 and GluR6 kainate complexes.     

Identification of Kainate Receptor-Mediated Intracellular Calcium Increases in Cultured Rat Cerebellar Granule Cells

Abstract: The functional expression of the kainate subtype of glutamate receptor (GluR) has been investigated in cultured rat cerebellar granule cells using single cell intracellular calcium ([Ca²⁺]_i) measurements. Both AMPA- and kainate-induced [Ca²⁺]_i increases could be blocked completely by the AMPA receptor-selective antagonist LY300168 (50 µ M ). However, following treatment with concanavalin A, an inhibitor of kainate receptor desensitisation, 30% of cells showed a kainate-induced [Ca²⁺]_i rise of >100 n M in the presence of LY300168. Responses to 30 µ M kainate in the presence of LY300168 were virtually abolished by the AMPA and GluR5 kainate receptor competitive antagonist LY293558 (100 µ M ). These results demonstrate the presence of functional kainate receptors on cultured cerebellar granule cells, and suggest that the GluR5 subtype of kainate receptor plays a significant role in kainate receptor-mediated [Ca²⁺]_i increases.     

3-Substituted phenylalanines as selective AMPA- and kainate receptor ligands

On the basis of X-ray structures of ionotropic glutamate receptor constructs in complex with amino acid-based AMPA and kainate receptor antagonists, a series of rigid as well as flexible biaromatic alanine derivatives carrying selected hydrogen bond acceptors and donors have been synthesized in order to investigate the structural determinants for receptor selectivity between AMPA and the GluR5 subtype of kainate receptors. Compounds selective for either GluR5 or AMPA receptors were identified. One particular substituent position appeared to be of special importance for control of ligand selectivity. Using molecular modeling the observed structure–activity relationships at AMPA and GluR5 receptors were deduced.     

Expression and localization of ionotropic glutamate receptor subunits in the goldfish retina—an in situ hybridization and immunocytochemical study

The expression and distribution of AMPA, kainate and NMDA glutamate receptor subunits was studied in the goldfish retina. For the immunocytochemical localization of the AMPA receptor antisera against GluR2, GluR2/3 and GluR4 were used, and for in situ hybridization rat specific probes for GluR1 and GluR2 and goldfish specific probes for GluR3 and GluR4 were used. The localization of the low affinity kainate receptor and NMDA receptor was studied using antisera against GluR5-7 and NR1. All AMPA receptor subtypes were demonstrated to be present in the goldfish retina both by immunocytochemistry and in situ hybridization. In situ hybridization revealed expression of all AMPA receptors subunit at the inner border of the INL. Only GluR3 was also strongly expressed in the outer border of the INL. Some of the ganglion cells displayed a strong signal for GluR1, GluR3 and GluR4. GluR1-immunoreactivity was present in subsets of bipolar, amacrine, and ganglion cells. GluR2 and GluR2/3-immunoreactivity was mainly localized in the outer plexiform layer. GluR2 and GluR2/3-immunoreactivity are associated with the photoreceptor synaptic terminals. GluR4-immunoreactivity is present on Müller cells in the inner retina and on dendrites of bipolar cells in the OPL, whereas GluR5-7-immunoreactivity was prominently present on horizontal cell axon terminals. Finally, NR1-immunoreactivity was confined to amacrine cells, the inner plexiform layer and ganglion cells. This study shows that there is a strong heterogeneity of glutamate receptor subunit expression in the various layers of the retina. Of the AMPA receptor subunits GluR3 seems to be expressed the most widely in all layers with strong glutamatergic synaptic interactions whereas all the other subunits seem to have a more restricted expressed pattern.     

Subtype-specific assembly of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunits is mediated by their n-terminal domains.

Glutamate receptors (GluR) are oligomeric protein complexes formed by the assembly of four or perhaps five subunits. The rules that govern the selectivity of this process are not well understood. Here, we expressed combinations of subunits from two related GluR subfamilies in COS7 cells, the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate receptors. By co-immunoprecipitation experiments, we assessed the ability of AMPA receptor subunits to assemble into multimeric complexes. Subunits GluR1-4 associated with indistinguishable efficiency with each other, whereas the kainate receptor subunits GluR6 and 7 showed a much lower degree of association with GluR1. Using chimeric receptors and truncation fragments of subunits, we show that this assembly specificity is determined by N-terminal regions of these subunits and that the most N-terminal domain of GluR2 together with a membrane anchor efficiently associates with GluR1.     

The KA-2 subunit of excitatory amino acid receptors shows widespread expression in brain and forms ion channels with distantly related subunits.

A new ionotropic glutamate receptor subunit termed KA-2, cloned from rat brain cDNA, exhibits high affinity for [3H]kainate (KD approximately 15 nM). KA-2 mRNA is widely expressed in embryonic and adult brain. Homomeric KA-2 expression does not generate agonist-sensitive channels, but currents are observed when KA-2 is coexpressed with GluR5 or GluR6 subunits. Specifically, coexpression of GluR5(R) and KA-2 produces channel activity, whereas homomeric expression of either subunit does not. Currents through heteromeric GluR5(Q)/KA-2 channels show more rapid desensitization and different current-voltage relations when compared with GluR5(Q) currents. GluR6/KA-2 channels are gated by AMPA, which fails to gate homomeric GluR6 receptor channels. These results suggest possible in vivo partnership relations for high affinity kainate receptors.     

A binding site tyrosine shapes desensitization kinetics and agonist potency at GluR2. A mutagenic, kinetic, and crystallographic study

Binding of an agonist to the 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)-propionic acid (AMPA) receptor family of the glutamate receptors (GluRs) results in rapid activation of an ion channel. Continuous application results in a non-desensitizing response for agonists like kainate, whereas most other agonists, such as the endogenous agonist (S)-glutamate, induce desensitization. We demonstrate that a highly conserved tyrosine, forming a wedge between the agonist and the N-terminal part of the bi-lobed ligand-binding site, plays a key role in the receptor kinetics as well as agonist potency and selectivity. The AMPA receptor GluR2, with mutations in Tyr-450, were expressed in Xenopus laevis oocytes and characterized in a two-electrode voltage clamp setup. The mutation GluR2(Y450A) renders the receptor highly kainate selective, and rapid application of kainate to outside-out patches induced strongly desensitizing currents. When Tyr-450 was substituted with the larger tryptophan, the (S)-glutamate desensitization is attenuated with a 10-fold increase in steady-state/peak currents (19% compared with 1.9% at the wild type). Furthermore, the tryptophan mutant was introduced into the GluR2-S1S2J ligand binding core construct and co-crystallized with kainate, and the 2.1-A x-ray structure revealed a slightly more closed ligand binding core as compared with the wild-type complex. Through genetic manipulations combined with structural and electrophysiological analysis, we report that mutations in position 450 invert the potency of two central agonists while concurrently strongly shaping the agonist efficacy and the desensitization kinetics of the AMPA receptor GluR2.     

Ionotropic glutamate receptor subunit distribution on hypoglossal motoneuronal pools in the rat

The expression of ionotropic glutamate receptor subunits in the motoneuronal pools of the hypoglossal nucleus was studied using specific antibodies against subunits of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), kainate and N-methyl-D-aspartate (NMDA) subtypes. The highest numbers of intensely immunolabelled motoneurons were found in the dorsal tier and caudoventromedial part of the hypoglossal nucleus with all antibodies except that against the GluR1 AMPA subunit. Labelling for the GluR1 subunit was weak except for caudally located groups of motoneurons which innervate tongue muscles related to respiratory activity. By contrast, most motoneurons were intensely immunostained with antibodies against GluR2/3 and GluR4 subunits of the AMPA subtype. The low staining observed using an antibody specific for the GluR2 subunit (which prevents Ca²⁺-entry through AMPA channels) strongly suggests that AMPA receptors in hypoglossal motoneurons are Ca²⁺-permeable. Immunolabelling for the GluR5/6/7 kainate receptor subunits was found in many motoneuronal somata as well as in thin axon-like profiles and puncta that resembled synaptic boutons. Most motoneurons were intensely immunostained for the NMDA receptor subunit NR1. These results show that the hypoglossal nucleus contains five heterogeneous pools of motoneurons which innervate functionally defined groups of tongue muscles. The uneven expression of the different receptor subunits analysed here could reflect diverse phenotypic properties of hypoglossal motoneurons which might be expected to generate different patterns of motor responses under different physiological or pathological conditions.     

Glutamate-induced cobalt uptake elicited by kainate receptors in rat taste bud cells

Glutamate-induced cobalt uptake reveals non-N-methyl-D-aspartate (non-NMDA) glutamate receptors (GluRs) in rat taste bud cells. However, it is not known which type of non-NMDA glutamate receptors is involved. We used a cobalt staining technique combined with pharmacological tests for kainate or alpha-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) receptors and/or immunohistochemistry against subunits of GluRs to examine the presence of non-NMDA receptors in rat foliate tastebud cells. Cobalt uptake into taste cells was elicited by treating taste buds with glutamate, kainate or SYM 2081, a kainate receptor agonist. Treating taste buds with AMPA or fluorowillardiine did not stimulate significant cobalt uptake. Moreover, 6-cyano-7-nitro-quinoxaline-2, 3-dione significantly reduced cobalt staining elicited by glutamate or kainate receptor agonists, but SYM 2206, an AMPA receptor antagonist, did not. Immunohistochemistry against subunits of GluRs reveals GluR6 and KA1-like immunoreactivity. Moreover, most glutamate-induced cobalt-stained cells showed GluR6 and KA1-like immunoreactivity. These results suggest that glutamate-induced cobalt uptake in taste cells occurs mainly via kainate type GluRs.     

Roles of ionotropic glutamate receptors in early developing neurons derived from the P19 mouse cell line

We cultured a P19 mouse teratocarcinoma cell line and induced its neuronal differentiation to study the function of ionotropic glutamate receptors (GluRs) in early neuronal development. Immunocytochemical studies showed 85% neuronal population at 5 days in vitro (DIV) with microtubule-associated protein 2-positive staining. Thirty percent and 50% of the cells expressed the alpha-amino-3-hydroxy-5-methyl-4-isopropinonate (AMPA) receptor subunit, GluR2/3, and the kainate (kainic acid; KA) receptor subunit, GluR5/6/7, respectively. In Western blot analysis, the temporal expression of GluR2/3 began to appear at 3 DIV, whereas GluR5/6/7 was already expressed in the undifferentiated cells. P19-derived neurons began to respond to glutamate, AMPA and KA, but not to the metabotropic GluR agonist trans-1-aminocyclopentane-1,3-decarboxylic acid, by 5 DIV in terms of increases in intracellular calcium and phospholipase C-mediated poly-phosphoinositide turnover. Furthermore, KA reduced cell death of P19-derived neurons in both atmospheric and hypobaric conditions in a phospholipase C-dependent manner. The common AMPA/KA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, but not the AMPA receptor antagonist, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium, profoundly increased hypobaric insult-induced neurotoxicity. In a flow cytometry study, the nerve growth factor-mediated antiapoptotic effect was facilitated by AMPA, with an induction of TrkA, but not p75(NTR) expression. Therefore, AMPA and KA receptors might mediate neurotrophic functions to facilitate neurotrophic factor signaling to protect neurons against hypoxic insult in early neuronal development.     

Testosterone amplifies excitotoxic damage of cultured oligodendrocytes

An overactivation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptors has been implicated in the pathophysiology of oligodendrocyte damage in demyelinating disorders of the CNS. We decided to examine the effect of testosterone on excitotoxic death of oligodendrocytes because a gender difference exists in the incidence and disease course of multiple sclerosis. Short-term pure cultures of oligodendrocytes (4 days in vitro) were exposed to a brief pulse with kainate or AMPA + cyclothiazide for the induction of excitotoxicity. Exposure to testosterone enantate was slightly toxic per se and amplified both AMPA and kainate toxicity. Testosterone treatment induced all gene targets of p53, and amplified the induction of these genes induced by kainate. The effect of testosterone was mediated by the activation of androgen receptors and was resistant to the aromatase inhibitors, dl-aminoglutethimide and 4-hydroxyandrost-4-ene-3,17-dione. Testosterone treatment also potentiated the stimulation of 45Ca2+ influx induced by AMPA + cyclothiazide or kainate without changing the expression of the glutamate receptor (GluR) 1, -2/3, and -4 subunits of AMPA receptors or the GluR6/7 subunits of kainate receptors. We conclude that testosterone amplifies excitotoxic damage of oligodendrocytes acting at an early step of the death cascade triggered by AMPA/kainate receptors.     

Calcium influx through subunits GluR1/GluR3 of kainate/AMPA receptor channels is regulated by cAMP dependent protein kinase.

Excitatory synaptic transmission in the central nervous system (CNS) is mediated by three major classes of glutamate receptors, namely the ionotropic NMDA (N-Methyl-D-Aspartate) and KA/AMPA (kainate/alpha-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid) receptors and the metabotropic receptor type. Among the ionotropic receptors, NMDA receptors are thought to mediate their physiological response mainly through the influx of extracellular calcium, while KA/AMPA receptor channels are mainly thought to carry the influx of monovalent cations. Recently, we have challenged this view by showing that cloned KA/AMPA receptor subunits GluR1 and GluR3 form ion channels which are permeable to calcium. We now directly demonstrate large increases in intracellular calcium concentrations induced by calcium fluxes through KA/AMPA receptor channels in solutions with physiological calcium concentrations. Calcium fluxes were observed through glutamate receptor channels composed of the subunits GluR1 and GluR3, which are both abundantly present in various types of central neurones. The calcium influx was fluorometrically monitored in Xenopus oocytes injected with the calcium indicator dye fura-2. Bath application of the membrane permeable analogue of adenosine cyclic monophosphate (cAMP) potentiated the current and also the flux of calcium through open KA/AMPA receptor channels. Further pharmacological experiments suggested that this effect was mediated by the activation of protein kinase A. Our results provide a molecular interpretation for the function of calcium permeable KA/AMPA receptor channels in neurones and identify two of the subunits of the KA/AMPA receptor channel which are regulated by the cAMP dependent second messenger system.     

Metabotropic glutamate and dopamine receptors co-regulate AMPA receptor activity through PKA in cultured chick retinal neurones: effect on GluR4 phosphorylation and surface expression

Glutamate receptor phosphorylation has been implicated in several forms of modulation of synaptic transmission. It has been reported that protein kinase A (PKA) can phosphorylate the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit GluR4 on Ser842, both in vitro and in vivo. Here, we studied the regulation of GluR4 phosphorylation and intracellular trafficking by PKA and by metabotropic receptors coupled to adenylyl cyclase (AC), in cultured chick retinal amacrine-like neurones, which are enriched in GluR4. The regulation of AMPA receptor activity by PKA and by metabotropic AC-coupled receptors was also investigated by measuring the [Ca2+]i response to kainate in Na(+)-free medium. Stimulation of AC with forskolin (FSK), or using the selective agonist of dopamine D1 receptors (+/-)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol (SKF38393), increased the [Ca2+]i response to kainate, GluR4 phosphorylation at Ser842 and GluR4 surface expression. Pre-incubation of the cells with (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV), an agonist of group II metabotropic glutamate receptors (mGluR), which are coupled to inhibition of AC, inhibited the effect of FSK and of SKF38393 on AMPA receptor activity, GluR4 phosphorylation and expression at the plasma membrane. These results indicate that there is a functional cross-talk between dopamine D1 receptors and group II mGluR in the regulation of GluR4 phosphorylation and AMPA receptor activity. Our data show that GluR4 phosphorylation at Ser842 by PKA, and its recruitment to the plasma membrane upon phosphorylation, is regulated by metabotropic receptors.     

Stability of ligand‐binding domain dimer assembly controls kainate receptor desensitization

AMPA and kainate receptors mediate fast synaptic transmission. AMPA receptor ligand‐binding domains form dimers, which are key functional units controlling ion‐channel activation and desensitization. Dimer stability is inversely related to the rate and extent of desensitization. Kainate and AMPA receptors share common structural elements, but functional measurements suggest that subunit assembly and gating differs between these subtypes. To investigate this, we constructed a library of GluR6 kainate receptor mutants and directly measured changes in kainate receptor dimer stability by analytical ultracentrifugation, which, combined with electrophysiological experiments, revealed an inverse correlation between dimer stability and the rate of desensitization. We solved crystal structures for a series of five GluR6 mutants, to understand the molecular mechanisms for dimer stabilization. We demonstrate that the desensitized state of kainate receptors acts as a deep energy well offsetting the stabilizing effects of dimer interface mutants, and that the deactivation of kainate receptor responses is dominated by entry into desensitized states. Our results show how neurotransmitter receptors with similar structures and gating mechanisms can exhibit strikingly different functional properties.     

Crystal structure of the kainate receptor GluR5 ligand-binding core in complex with (S)-glutamate

The X-ray structure of the ligand-binding core of the kainate receptor GluR5 (GluR5-S1S2) in complex with (S)-glutamate was determined to 1.95 A resolution. The overall GluR5-S1S2 structure comprises two domains and is similar to the related AMPA receptor GluR2-S1S2J. (S)-glutamate binds as in GluR2-S1S2J. Distinct features are observed for Ser741, which stabilizes a highly coordinated network of water molecules and forms an interdomain bridge. The GluR5 complex exhibits a high degree of domain closure (26 degrees) relative to apo GluR2-S1S2J. In addition, GluR5-S1S2 forms a novel dimer interface with a different arrangement of the two protomers compared to GluR2-S1S2J.     

Stereochemistry of glutamate receptor agonist efficacy: engineering a dual-specificity AMPA/kainate receptor

Upon agonist binding, the bilobate ligand-binding domains of the ionotropic glutamate receptors (iGluR) undergo a cleft closure whose magnitude correlates broadly with the efficacy of the agonist. AMPA (alpha-amino-5-methyl-3-hydroxy-4-isoxazolepropionic acid) and kainate are nonphysiological agonists that distinguish between subsets of iGluR. Kainate acts with low efficacy at AMPA receptors. Here we report that the structure-based mutation L651V converts the GluR4 AMPA receptor into a dual-specificity AMPA/kainate receptor fully activated by both agonists. To probe the stereochemical basis of partial agonism, we have also investigated the correlation between agonist efficacy and a series of vibrational and fluorescence spectroscopic signals of agonist binding to the corresponding wild-type and mutant GluR4 ligand-binding domains. Two signals track the extent of channel activation: the maximal change in intrinsic tryptophan fluorescence and the environment of the single non-disulfide bonded C426, which appears to probe the strength of interactions with the ligand alpha-amino group. Both of these signals arise from functional groups that are poised to detect changes in the extent of channel cleft closure and thus provide additional information about the coupling between conformational changes in the ligand-binding domain and activation of the intact receptor.     

Ultrastructural localisation and differential agonist-induced regulation of AMPA and kainate receptors present at the presynaptic active zone and postsynaptic density

Activity-dependent changes in ionotropic glutamate receptors at the postsynaptic membrane are well established and this regulation plays a central role in the expression of synaptic plasticity. However, very little is known about the distributions and regulation of ionotropic receptors at presynaptic sites. To determine if presynaptic receptors are subject to similar regulatory processes we investigated the localisation and modulation of AMPA (GluR1, GluR2, GluR3) and kainate (GluR6/7, KA2) receptor subunits by ultrasynaptic separation and immunoblot analysis of rat brain synaptosomes. All of the subunits were enriched in the postsynaptic fraction but were also present in the presynaptic and non-synaptic synaptosome fractions. AMPA stimulation resulted in a marked decrease in postsynaptic GluR2 and GluR3 subunits, but an increase in GluR6/7. Conversely, GluR2 and GluR3 increased in the presynaptic fraction whereas GluR6/7 decreased. There were no significant changes in any of the compartments for GluR1. NMDA treatment decreased postsynaptic GluR1, GluR2 and GluR6/7 but increased presynaptic levels of these subunits. NMDA treatment did not evoke changes in GluR3 localisation. Our results demonstrate that presynaptic and postsynaptic subunits are regulated in opposite directions by AMPA and NMDA stimulation.