Glutamate-gated kainate receptors are ubiquitous in the central nervous system of vertebrates, mediate synaptic transmission at the postsynapse and modulate transmitter release at the presynapse
. In ...the brain, the trafficking, gating kinetics and pharmacology of kainate receptors are tightly regulated by neuropilin and tolloid-like (NETO) proteins
. Here we report cryo-electron microscopy structures of homotetrameric GluK2 in complex with NETO2 at inhibited and desensitized states, illustrating variable stoichiometry of GluK2-NETO2 complexes, with one or two NETO2 subunits associating with GluK2. We find that NETO2 accesses only two broad faces of kainate receptors, intermolecularly crosslinking the lower lobe of ATD
, the upper lobe of LBD
and the lower lobe of LBD
, illustrating how NETO2 regulates receptor-gating kinetics. The transmembrane helix of NETO2 is positioned proximal to the selectivity filter and competes with the amphiphilic H1 helix after M4 for interaction with an intracellular cap domain formed by the M1-M2 linkers of the receptor, revealing how rectification is regulated by NETO2.
Compared to the other glutamate receptors, progress in the understanding of the functions of kainate receptors (KARs) has lagged behind, due mainly to the relative lack of specific pharmacological ...tools. Over the last decade subunit selective agonists (e.g. ATPA and 5-iodowillardiine) and orthosteric (e.g. LY382884 and ACET) and allosteric antagonists for KARs that contain GluK1 (GluR5) subunits have been developed. However, no selective ligands for the other KAR subunits have been identified. The use of GluK1 antagonists has enabled several functions of KARs, that contain this subunit, to be identified. Thus, KARs have been shown to regulate excitatory and inhibitory synaptic transmission. In the case of the regulation of l-glutamate release, they can function as facilitatory autoreceptors or inhibitory autoreceptors during repetitive synaptic activation and can respond to ambient levels of l-glutamate to provide a tonic regulation of l-glutamate release. KARs also contribute a component of excitatory synaptic transmission at certain synapses. They can also act as triggers for both long-term potentiation (LTP) and long-term depression (LTD) and rapid alterations in their trafficking can result in altered synaptic transmission during both synaptic plasticity and neuronal development. KARs also contribute to synchronised rhythmic activity in the brain and are involved in forms of learning and memory. With respect to therapeutic indications, antagonists for GluK1 have shown positive activity in animal models of pain, migraine, epilepsy, stroke and anxiety. This potential has now been confirmed in dental pain and migraine in initial studies in man.
Kainic acid (KA) is an analogue of the excitatory neurotransmitter glutamate that, when injected systemically into adult rats, can trigger seizures and progressive neuronal loss in a manner that ...mirrors the neuropathology of human mesial temporal lobe epilepsy. However, biomolecular mechanisms responsible for the neuronal loss that occurs as a consequence of this treatment remains elusive. We have recently reported that toxicity induced by KA can partly be mediated by astrocyte-derived amyloid β (Aβ) peptides, which are critical in the development of Alzheimer’s disease (AD). Nonetheless, little is known how KA can influence amyloid precursor protein (APP) levels and processing in astrocytes. Thus, in the present study using human U-373 astrocytoma and rat primary astrocytes, we evaluated the role of KA on APP metabolism. Our results revealed that KA treatment increased the levels of APP and its cleaved products (α-/β-CTFs) in cultured U-373 astrocytoma and primary astrocytes, without altering the cell viability. The cellular and secretory levels of Aβ
1–40
/Aβ
1–42
were markedly increased in KA-treated astrocytes. We also demonstrated that the steady-state levels of APP-secretases were not altered but the activity of γ-secretase is enhanced in KA-treated U-373 astrocytoma. Furthermore, using selective receptor antagonists, we showed that the effects of KA is mediated by activation of kainate receptors and not NMDA or AMPA receptors. These results suggest that KA can enhance amyloidogenic processing of APP by activating its own receptor leading to increased production/secretion of Aβ-related peptides from activated astrocytes which may contribute to the pathogenesis of temporal lobe epilepsy.
Oceanic harmful algal blooms of
diatoms produce the potent mammalian neurotoxin domoic acid (DA). Despite decades of research, the molecular basis for its biosynthesis is not known. By using growth ...conditions known to induce DA production in
, we implemented transcriptome sequencing in order to identify DA biosynthesis genes that colocalize in a genomic four-gene cluster. We biochemically investigated the recombinant DA biosynthetic enzymes and linked their mechanisms to the construction of DA's diagnostic pyrrolidine skeleton, establishing a model for DA biosynthesis. Knowledge of the genetic basis for toxin production provides an orthogonal approach to bloom monitoring and enables study of environmental factors that drive oceanic DA production.
Both inhibitory and excitatory neurotransmitter receptors can influence maturation and survival of adult-born neurons in the dentate gyrus; nevertheless, how these two neurotransmitter systems affect ...integration of new neurons into the existing circuitry is still not fully characterized. Here, we demonstrate that glutamate receptors of the kainate receptor (KAR) subfamily are expressed in adult-born dentate granule cells (abDGCs) and that, through their interaction with GABAergic signaling mechanisms, they alter the functional properties of adult-born cells during a critical period of their development. Both the intrinsic properties and synaptic connectivity of young abDGCs were affected. Timed KAR loss in a cohort of young adult-born neurons in mice disrupted their performance in a spatial discrimination task but not in a hippocampal-dependent fear conditioning task. Together, these results demonstrate the importance of KARs in the proper functional development of young abDGCs.
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•Kainate receptors are expressed on developing adult-born dentate granule cells•KARs in adult-born neurons affect their synaptic and intrinsic properties•Timed ablation of KARs during a critical period disrupts spatial discrimination in mice
Zhu et al. record from birth-dated, adult-born granule cells in the hippocampus to determine how their properties are affected by KARs. Both the synaptic and intrinsic membrane properties are disrupted during a short developmental window, and mice with a loss of KARs in young neurons have impairments in spatial discrimination.
GluK3-kainate receptors are atypical members of the iGluR family that reside at both the pre- and postsynapse and play a vital role in the regulation of synaptic transmission. For a better ...understanding of structural changes that underlie receptor functions, GluK3 receptors were trapped in desensitized and resting/closed states and structures analyzed using single particle cryo-electron microscopy. While the desensitized GluK3 has domain organization as seen earlier for another kainate receptor-GluK2, antagonist bound GluK3 trapped a resting state with only two LBD domains in dimeric arrangement necessary for receptor activation. Using structures as a guide, we show that the N-linked glycans at the interface of GluK3 ATD and LBD likely mediate inter-domain interactions and attune receptor-gating properties. The mutational analysis also identified putative N-glycan interacting residues. Our results provide a molecular framework for understanding gating properties unique to GluK3 and exploring the role of N-linked glycosylation in their modulation.
The kainate receptors GluK1‐3 (glutamate receptor ionotropic, kainate receptors 1–3) belong to the family of ionotropic glutamate receptors and are essential for fast excitatory neurotransmission in ...the brain, and are associated with neurological and psychiatric diseases. How these receptors can be modulated by small‐molecule agents is not well understood, especially for GluK3. We show that the positive allosteric modulator BPAM344 can be used to establish robust calcium‐sensitive fluorescence‐based assays to test agonists, antagonists, and positive allosteric modulators of GluK1‐3. The half‐maximal effective concentration (EC50) of BPAM344 for potentiating the response of 100 μm kainate was determined to be 26.3 μm for GluK1, 75.4 μm for GluK2, and 639 μm for GluK3. Domoate was found to be a potent agonist for GluK1 and GluK2, with an EC50 of 0.77 and 1.33 μm, respectively, upon co‐application of 150 μm BPAM344. At GluK3, domoate acts as a very weak agonist or antagonist with a half‐maximal inhibitory concentration (IC50) of 14.5 μm, in presence of 500 μm BPAM344 and 100 μm kainate for competition binding. Using H523A‐mutated GluK3, we determined the first dimeric structure of the ligand‐binding domain by X‐ray crystallography, allowing location of BPAM344, as well as zinc‐, sodium‐, and chloride‐ion binding sites at the dimer interface. Molecular dynamics simulations support the stability of the ion sites as well as the involvement of Asp761, Asp790, and Glu797 in the binding of zinc ions. Using electron microscopy, we show that, in presence of glutamate and BPAM344, full‐length GluK3 adopts a dimer‐of‐dimers arrangement.
How kainate receptors can be modulated by small‐molecule agents is not well understood. Establishment of calcium‐sensitive fluorescence‐based assays at GluK1‐3 allowed for high‐throughput screening for modulators and other ligands. Using X‐ray crystallography and molecular dynamics of an H523A mutant of the GluK3 ligand‐binding domain, here, we unraveled ion binding sites and dimer interface stabilization by the positive allosteric modulator BPAM344. Using electron microscopy, we show that BPAM344 and glutamate introduced an active‐like shape of full‐length GluK3. This figure was prepared with pymol (Version 2.0.3, Schrödinger, LLC).
Ionotropic glutamate receptors are ligand-gated ion channels that mediate excitatory synaptic transmission in the vertebrate brain. To gain a better understanding of how structural changes gate ion ...flux across the membrane, we trapped rat AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) and kainate receptor subtypes in their major functional states and analysed the resulting structures using cryo-electron microscopy. We show that transition to the active state involves a 'corkscrew' motion of the receptor assembly, driven by closure of the ligand-binding domain. Desensitization is accompanied by disruption of the amino-terminal domain tetramer in AMPA, but not kainate, receptors with a two-fold to four-fold symmetry transition in the ligand-binding domains in both subtypes. The 7.6 Å structure of a desensitized kainate receptor shows how these changes accommodate channel closing. These findings integrate previous physiological, biochemical and structural analyses of glutamate receptors and provide a molecular explanation for key steps in receptor gating.
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 heterozygous 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.
Ionotropic glutamate receptors principally mediate fast excitatory transmission in the brain. Among the three classes of ionotropic glutamate receptors, kainate receptors (KARs) have a unique brain ...distribution, which has been historically defined by (3)H-radiolabeled kainate binding. Compared with recombinant KARs expressed in heterologous cells, synaptic KARs exhibit characteristically slow rise-time and decay kinetics. However, the mechanisms responsible for these distinct KAR properties remain unclear. We found that both the high-affinity binding pattern in the mouse brain and the channel properties of native KARs are determined by the KAR auxiliary subunit Neto1. Through modulation of agonist binding affinity and off-kinetics of KARs, but not trafficking of KARs, Neto1 determined both the KAR high-affinity binding pattern and the distinctively slow kinetics of postsynaptic KARs. By regulating KAR excitatory postsynaptic current kinetics, Neto1 can control synaptic temporal summation, spike generation and fidelity.
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