Differential activation of individual subunits in heteromeric 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.     

Role of Glutamate Receptors and Glutamate Transporters in the Regulation of the Glutamate-Glutamine Cycle in the Awake Rat

In the present study we investigate the effects of a specific glutamate reuptake blocker, L-trans-pyrrolidine-3,4-dicarboxylic acid (PDC), on extracellular concentrations of glutamine and glutamate in the striatum of the freely moving rat. Intracerebral infusions of PDC (1, 2 and 4 mM) produced a dose-related increase in extracellular concentrations of glutamate and a dose-related decrease in extracellular concentrations of glutamine. These increases in extracellular glutamate and decreases in extracellular glutamine were significantly correlated. To investigate the involvement of ionotropic glutamate receptors in the decreases of extracellular glutamine produced by PDC, N-methyl-D-aspartate (NMDA) receptor antagonist and -amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor antagonist were used. Perfusion of the NMDA receptor antagonist blocked the decrease of extracellular glutamine but had no effect on the increase of extracellular glutamate, both produced by PDC. Perfusion of the AMPA/kainate receptor antagonist attenuated the increase of extracellular glutamate and not only blocked the decrease of extracellular glutamine but also produced a significant increase of extracellular glutamine. The results reported in this study suggest that both NMDA and AMPA/kainate glutamatergic receptors are involved in the regulation of extracellular glutamine.     

A glutamate receptor channel with high affinity for domoate and kainate.

The non-NMDA family of glutamate receptors comprises a growing number of structurally related subunits (GluR-A to -D or -1 to -4; GluR-5, -6; KA-1). GluR-A to -D appear to constitute the major AMPA receptor subtypes but the functional and pharmacological characteristics of the other subunits are unresolved. Using a mammalian expression system we demonstrate here that homomeric GluR-5 receptors exhibit properties of a high affinity domoate (KD approximately 2 nM) and kainate (KD approximately 70 nM) binding site. For these receptors, the rank order of ligands competing with [3H]kainate binding was domoate much greater than quisqualate approximately glutamate much greater than AMPA approximately CNQX. The respective receptor channels were gated in decreasing order of sensitivity by domoate, kainate, glutamate and AMPA. In contrast to recombinantly expressed GluR-A to -D channels, currents elicited at GluR-5 receptor desensitize channels to all agonists. This property is characteristic of currents in peripheral neurons on sensory ganglia. These findings suggest the existence of at least two distinct types of non-NMDA receptor channels, both gated by AMPA and kainate, but differing in pharmacology and current properties.     

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.     

Ligand binding is a critical requirement for plasma membrane expression of heteromeric kainate receptors

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.     

Functional expression from cloned cDNAs of glutamate receptor species responsive to kainate and quisqualate.

The complete amino acid sequences of two mouse glutamate receptor subunits (GluR1 and GluR2) have been deduced by cloning and sequencing the cDNAs. Xenopus oocytes injected with mRNA derived from the GluR1 cDNA exhibit current responses both to kainate and to quisqualate as well as to glutamate, whereas oocytes injected with mRNA derived from the GluR2 cDNA show little response. Injection of oocytes with both the mRNAs produces current responses larger than those induced by the GluR1-specific mRNA and the dose-response relations indicate a positively cooperative interaction between the two subunits. These results suggest that kainate and quisqualate can activate a common glutamate receptor subtype and that glutamate-gated ionic channels are hetero-oligomers of different subunits.     

Agonist binding to the GluK5 subunit is sufficient for functional surface expression of heteromeric GluK2/GluK5 kainate receptors

Trafficking of ionotropic glutamate receptors to the plasma membrane commonly requires occupation of the agonist binding sites. This quality control check does not typically involve receptor activation, as binding by competitive antagonists or to non-functional channels may also permit surface expression. The tetrameric kainate receptors can be assembled from five different subunits (GluK1–GluK5). While the “low-affinity” GluK1-3 subunits are able to produce functional homomeric receptors, the “high-affinity” GluK4 and GluK5 subunits require co-assembly with GluK1, 2, or 3 for surface expression. These two different types of subunits have distinct functional roles in the receptor. Therefore, we examined the relative importance of occupancy of the agonist site of the GluK2 or GluK5 subunit for surface expression of heteromeric receptors. We created subunits with a mutation within the S2 ligand-binding domain which decreased agonist affinity. Mutations at this site reduced functional surface expression of homomeric GluK2 receptors, but surface expression of these receptors could be increased with either a competitive antagonist or co-assembly with wild-type GluK5. In contrast, mutations in the GluK5 subunit reduced the production of functional heteromeric receptors at the membrane, and could not be rescued with either an antagonist or wild-type GluK2. These findings indicate that ligand binding to only the GluK5 subunit is both necessary and sufficient to allow trafficking of recombinant GluK2/K5 heteromers to the cell membrane, but that occupancy of the GluK2 site alone is not. Our results suggest a distinct role for the GluK5 subunit in regulating surface expression of heteromeric kainate receptors.     

Evidence for hybrid NMDA/kainate receptors from protein reconstitution studies and expression of vertebrate CNS RNAs in xenopus oocytes

1. A review is presented of recent advances in glutamate receptor research with particular emphasis on studies which show that some glutamate receptors in the central nervous systems (CNS) of Xenopus and rat contain a mixture of $\text{N-}\text{methyl}\text{-}\text{D}\text{-}\text{aspartate-sensitive}$ and kainate-sensitive subunits.2. Protein isolated from Xenopus CNS using a domoic acid affinity column exhibits complex pharmacological properties. It binds both [³H]kainate and [³H]glycine: the binding of the latter is strychnine-insensitive.3. When reconstituted into lipid bilayers, channels gated by kainate and NMDA can be elicited and the properties of these channels are similar to those gated by kainate receptors and NMDA receptors, respectively, in studies of vertebrate central neurones in situ.4. The protein can be fractionated into two components; one of which is sensitive only to kainate and AMPA, the other exhibiting sensitivity to both kainate and NMDA.5. When RNA isolated from Xenopus and rat CNS is injected into Xenopus oocytes, responses to kainate and NMDA can be seen within 2–3 days. The responses to co-application of these agonists support the contention that some of the glutamate receptors expressed in oocytes contain both kainate-sensitive and NMDA-sensitive subunits.     

Regulation of Kainate Receptor Subunit mRNA by Stress and Corticosteroids in the Rat Hippocampus

Kainate receptors are a class of ionotropic glutamate receptors that have a role in the modulation of glutamate release and synaptic plasticity in the hippocampal formation. Previous studies have implicated corticosteroids in the regulation of these receptors and recent clinical work has shown that polymorphisms in kainate receptor subunit genes are associated with susceptibility to major depression and response to anti-depressant treatment. In the present study we sought to examine the effects of chronic stress and corticosteroid treatments upon the expression of the mRNA of kainate receptor subunits GluR5-7 and KA1-2. Our results show that, after 7 days, adrenalectomy results in increased expression of hippocampal KA1, GluR6 and GluR7 mRNAs, an effect which is reversed by treatment with corticosterone in the case of KA1 and GluR7 and by aldosterone treatment in the case of GluR6. 21 days of chronic restraint stress (CRS) elevated the expression of the KA1 subunit, but had no effect on the expression of the other subunits. Similarly, 21 days of treatment with a moderate dose of corticosterone also increased KA1 mRNA in the dentate gyrus, whereas a high corticosterone dose has no effect. Our results suggest an interaction between hippocampal kainate receptor composition and the hypothalamic-pituitary-adrenal (HPA) axis and show a selective chronic stress induced modulation of the KA1 subunit in the dentate gyrus and CA3 that has implications for stress-induced adaptive structural plasticity.     

Agonist-activated cobalt uptake identifies divalent cation-permeable kainate receptors on neurons and glial cells.

Activation of kainate receptors causes Co2+ influx into neurons, type-2 astrocytes, and O-2A progenitor cells. Agonist-activated Co2+ uptake can be performed using cultured cells or fresh tissue slices. Based on the pattern of response to kainate, glutamate, and quisqualate, three functionally different kainate-activated ion channels (K1, K2, and K3) can be discriminated. Co2+ uptake through the K1 receptor was only activated by kainate. Both kainate and glutamate activated Co2+ uptake through the K2 receptor. Co2+ uptake through the K3 receptor was activated by all three ligands: kainate, glutamate, and quisqualate. Co2+ uptake occurred through a nonselective cation entry pathway permeable to Co2+, Ca2+, and Mn2+. The agonist-dependent activation of divalent cation influx through different kainate receptors could be correlated with expression of certain kainate receptor subunit combinations. These results are indicative of kainate receptors that may contribute to excitatory amino acid-mediated neurotoxicity.     

Functional analysis of Caenorhabditis elegans glutamate receptor subunits by domain transplantation

Glutamate receptors are not only abundant and important mediators of fast excitatory synaptic transmission in vertebrates, but they also serve a similar function in invertebrates such as Drosophila and the nematode Caenorhabditis elegans. In C. elegans, an animal with only 302 neurons, 10 different glutamate receptor subunits have been identified and cloned. To study the ion channel properties of these receptor subunits, we recorded glutamate-gated currents from Xenopus oocytes that expressed either C. elegans glutamate receptor subunits or chimeric rat/C. elegans glutamate receptor subunits. The chimeras were constructed between the C. elegans glutamate receptor pore domains and either the rat kainate receptor subunit GluR6, the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunit GluR1, or the N-methyl-d-aspartate (NMDA) receptor subunit NMDAR1-1a. Although native subunits were nonfunctional, 9 of 10 ion pores were found to conduct current upon transplantation into rat receptor subunits. A provisional classification of the C. elegans glutamate receptor subunits was attempted based on functionality of the chimeras. C. elegans glutamate receptor ion pores, at a position homologous to a highly conserved site critical for ion permeation properties in vertebrate glutamate receptor pores, contain amino acids not found in vertebrate glutamate receptors. We show that the pore-constricting Q/R site, which in vertebrate receptors determines calcium permeability and rectification properties of the ion channel, in C. elegans can be occupied by other amino acids, including, surprisingly, lysine and proline, without loss of these properties.     

Molecular mechanisms regulating the differential association of kainate receptor subunits with SAP90/PSD-95 and SAP97

Recent studies have demonstrated that kainate receptors are associated with members of the SAP90/PSD-95 family (synapse-associated proteins (SAPs)) in neurons and that SAP90 can cluster and modify the electrophysiological properties of GluR6/KA2 kainate receptors when co-expressed in transfected cells. In vivo, SAP90 tightly binds kainate receptor subunits, while SAP97 is only weakly associated, suggesting that this glutamate receptor differentially associates with SAP90/PSD-95 family members. Here, green fluorescent protein (GFP)-tagged chimeras and deletion mutants of SAP97 and SAP90 were employed to define the molecular mechanism underlying their differential association with kainate receptors. Our results show that a weak interaction between GluR6 and the PDZ1 domain of SAP97 can account for the weak association of GluR6 with the full-length SAP97 observed in vivo. Expression studies in HEK293 cells and in vitro binding studies further show that although the individual Src homology 3 and guanylate kinase domains in SAP97 can interact with the C-terminal tail of KA2 subunit, specific intramolecular interactions in SAP97 (e.g. the SAP97 N terminus (S97N) binding to the Src homology 3 domain) interfere with KA2 binding to the full-length molecule. Because receptor subunits are known to segregate to different parts of the neuron, our results imply that differential association of kainate receptors with SAP family proteins may be one mechanism of subcellular localization.     

α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionic Acid (AMPA) and N-Methyl-d-aspartate (NMDA) Receptors Adopt Different Subunit Arrangements

Ionotropic glutamate receptors are widely distributed in the central nervous system and play a major role in excitatory synaptic transmission. All three ionotropic glutamate subfamilies (i.e. AMPA-type, kainate-type, and NMDA-type) assemble as tetramers of four homologous subunits. There is good evidence that both heteromeric AMPA and kainate receptors have a 2:2 subunit stoichiometry and an alternating subunit arrangement. Recent studies based on presumed structural homology have indicated that NMDA receptors adopt the same arrangement. Here, we use atomic force microscopy imaging of receptor-antibody complexes to show that whereas the GluA1/GluA2 AMPA receptor assembles with an alternating (i.e. 1/2/1/2) subunit arrangement, the GluN1/GluN2A NMDA receptor adopts an adjacent (i.e. 1/1/2/2) arrangement. We conclude that the two types of ionotropic glutamate receptor are built in different ways from their constituent subunits. This surprising finding necessitates a reassessment of the assembly of these important receptors.     

The regulation of glutamate receptor trafficking and function by TARPs and other transmembrane auxiliary subunits

At excitatory synapses in the brain, glutamate released from nerve terminals binds to glutamate receptors to mediate signaling between neurons. Glutamate receptors expressed in heterologous cells show ion channel activity. Recently, native glutamate receptors were shown to contain auxiliary subunits that modulate the trafficking and/or channel properties. The AMPA receptor (AMPAR) can contain TARP and CNIHs as the auxiliary subunits, whereas kainate receptor (KAR) can contain the Neto auxiliary subunit. Each of these auxiliary subunits uniquely modulates the glutamate receptors, and determines properties of native glutamate receptors. A thorough elucidation of the properties of native glutamate receptor complexes is indispensable for the understanding of the molecular machinery that regulates glutamate receptors and excitatory synaptic transmission in the brain.     

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.     

Structure and assembly mechanism for heteromeric kainate receptors

Native glutamate receptor ion channels are tetrameric assemblies containing two or more different subunits. NMDA receptors are obligate heteromers formed by coassembly of two or three divergent gene families. While some AMPA and kainate receptors can form functional homomeric ion channels, the KA1 and KA2 subunits are obligate heteromers which?function only in combination with GluR5-7. The mechanisms controlling glutamate receptor assembly involve an initial step in which the amino terminal domains (ATD) assemble as dimers. Here, we establish by sedimentation velocity that the ATDs of GluR6 and KA2 coassemble as a heterodimer of K(d) 11?nM, 32,000-fold lower than the K(d) for homodimer formation by KA2; we solve crystal structures for the GluR6/KA2 ATD heterodimer and heterotetramer assemblies. Using these structures as a guide, we perform a mutant cycle analysis to probe the energetics of assembly and show that high-affinity ATD interactions are required for biosynthesis of functional heteromeric receptors.     

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.