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Activation of dermal mast cells through the Mas-related G protein-coupled receptor B2 receptor (MrgprB2 in mice and MrgprX2 in humans) is a key component of numerous inflammatory skin diseases, including dermatitis and rosacea. Sensory neurons actively suppress mast cell activation through the regulated release of glutamate, resulting in reduced expression of Mrgprb2 as well as genes associated with proteins found in mast cell granules. To determine whether exogenous glutamate receptor agonism could suppress mast cell function, we determined that mast cells have relatively selective expression of the glutamate receptor ionotropic, kainate 2 (GluK2). A GluK2-specific agonist, SYM2081, effectively inhibited mast cell degranulation in response to MrgprB2 agonism in both murine mast cells and human skin explants in vitro as well as in vivo after both intradermal and topical administration of SYM2081 to mice. Analyses of transcriptomic datasets from SYM2081-treated mast cells using standard differential expression approaches and an interpretable machine learning technique revealed a previously unrecognized cellular program coordinately regulated by GluK2 agonism. GluK2 agonism suppressed the expression of Mrgprb2 and genes associated with mast cell proliferation. Suppression of mast cell proliferation by SYM2081 exposure was confirmed on the basis of reduced Ki-67 expression and BrdU incorporation in vitro and in vivo. Last, pretreatment with SYM2081 reduced skin inflammation in murine models of dermatitis and rosacea. Thus, agonism of GluK2 represents a promising approach to suppress mast cell activation and may prove beneficial as therapy for inflammatory diseases in which mast cell activation is pathogenic.

Kainate receptors (KARs) are a subtype of ionotropic glutamate receptor (iGluR) channels, a superfamily of ligand-gated ion channels which mediate the majority of excitatory neurotransmission in the central nervous system. KARs modulate neuronal circuits and plasticity during development and are implicated in neurological disorders, including epilepsy, depression, schizophrenia, anxiety, and autism. Calcium-permeable KARs undergo ion channel block, but the therapeutic potential of channel blockers remains underdeveloped, mainly due to limited structural knowledge. Here, we present closed-state structures of GluK2 KAR homotetramers in complex with ion channel blockers NpTx-8, PhTx-74, Kukoamine A, and spermine. We find that blockers reside inside the GluK2 ion channel pore, intracellular to the closed M3 helix bundle-crossing gate, with their hydrophobic heads filling the central cavity and positively charged polyamine tails spanning the selectivity filter. Molecular dynamics (MD) simulations of our structures illuminate interactions responsible for different affinity and binding poses of the blockers. Our structures elucidate the trapping mechanism of KAR channel block and provide a template for designing new blockers that can selectively target calcium-permeable KARs in neuropathologies., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Kainate receptors are a subtype of ionotropic glutamate receptors that form transmembrane channels upon binding glutamate. Here, we have investigated the mechanism of partial agonism in heteromeric GluK2/K5 receptors, where the GluK2 and GluK5 subunits have distinct agonist binding profiles. Using single-molecule Förster resonance energy transfer, we found that at the bi-lobed agonist-binding domain, the partial agonist AMPA-bound receptor occupied intermediate cleft closure conformational states at the GluK2 cleft, compared to the more open cleft conformations in apo form and more closed cleft conformations in the full agonist glutamate-bound form. In contrast, there is no significant difference in cleft closure states at the GluK5 agonist-binding domain between the partial agonist AMPA- and full agonist glutamate-bound states. Additionally, unlike the glutamate-bound state, the dimer interface at the agonist-binding domain is not decoupled in the AMPA-bound state. Our findings suggest that partial agonism observed with AMPA binding is mediated primarily due to differences in the GluK2 subunit, highlighting the distinct contributions of the subunits towards activation., (© 2023 Wiley Periodicals LLC.)

Kainate receptors containing the GluK1 subunit have an impact on excitatory and inhibitory neurotransmission in brain regions, such as the amygdala and hippocampus, which are relevant to seizures and epilepsy. Here we used 2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA), a potent and selective agonist of kainate receptors that include the GluK1 subunit, in conjunction with mice deficient in GluK1 and GluK2 kainate receptor subunits to assess the role of GluK1 kainate receptors in provoking seizures and in kindling epileptogenesis. We found that systemic ATPA, acting specifically via GluK1 kainate receptors, causes locomotor arrest and forelimb extension (a unique behavioral characteristic of GluK1 activation) and induces myoclonic behavioral seizures and electrographic seizure discharges in the BLA and hippocampus. In contrast, the proconvulsant activity of systemic AMPA, kainate, and pentylenetetrazol is not mediated by GluK1 kainate receptors, and deletion of these receptors does not elevate the threshold for seizures in the 6 Hz model. ATPA also specifically activates epileptiform discharges in BLA slices in vitro via GluK1 kainate receptors. Olfactory bulb kindling developed similarly in wild-type, GluK1, and GluK2 knock-out mice, demonstrating that GluK1 kainate receptors are not required for epileptogenesis or seizure expression in this model. We conclude that selective activation of kainate receptors containing the GluK1 subunit can trigger seizures, but these receptors are not necessary for seizure generation in models commonly used to identify therapeutic agents for the treatment of epilepsy.

Synapse formation depends on the coordinated expression and regulation of scaffold proteins. The JNK family kinases play a role in scaffold protein regulation, but the nature of this functional interaction in dendritic spines requires further investigation. Here, using a combination of biochemical methods and live-cell imaging strategies, we show that the dynamics of the synaptic scaffold molecule SAP102 are negatively regulated by JNK inhibition, that SAP102 is a direct phosphorylation target of JNK3, and that SAP102 regulation by JNK is restricted to neurons that harbor mature synapses. We further demonstrate that SAP102 and JNK3 cooperate in the regulated trafficking of kainate receptors to the cell membrane. Specifically, we observe that SAP102, JNK3, and the kainate receptor subunit GluK2 exhibit overlapping expression at synaptic sites and that modulating JNK activity influences the surface expression of the kainate receptor subunit GluK2 in a neuronal context. We also show that SAP102 participates in this process in a JNK-dependent fashion. In summary, our data support a model in which JNK-mediated regulation of SAP102 influences the dynamic trafficking of kainate receptors to postsynaptic sites, and thus shed light on common pathophysiological mechanisms underlying the cognitive developmental defects associated with diverse mutations., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Aim: Conservative treatment approaches for thyroid carcinoma (TC) patients with wild-type B-type Raf kinase ( BRAF ) pose risks of long-term recurrence. The association of DNA methylation with TC metastasis is unclear. Patients & methods: Here we analyzed data from 179 BRAF wild-type TC patients in the The Cancer Genome Atlas database, identifying significant metastasis-associated CpGs. A logistic regression model was developed and validated for discriminating lymphatic metastasis in BRAF wild-type TC. Results: The model showed high accuracy (AUC: 0.924 training set; 0.812 and 0.773 external cohorts). TAGLN , MRPL4 , CLDN10 and GRIK2 emerged as diagnostic markers. GRIK2 , downregulated due to promoter hypermethylation, acted as a TC suppressor. Conclusion: Our 5-CpG epigenetic signature effectively discriminates lymphatic metastasis in BRAF wild-type TC, highlighting GRIK2 's tumor-suppressive role influenced by promoter hypermethylation.

Important roles in the initiation and maintenance of postoperative pain are played by the functional control of kainate (KA) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors in the rat dorsal horn (DH). However, the mechanisms underpinning the cross-talk between spinal KA and AMPA receptors in postoperative pain are poorly understood. We hypothesized that after the rat's plantar incision, the synaptic incorporation of AMPA receptor GluR1 subunits in the DH ipsilateral to the incision would increase due to the interaction between GluK2 and neuropilin tolloid-like 2 (NETO2). Our findings showed that incision stimuli caused severe pain responses, as measured by cumulative pain scores. GluK2-NETO2 but not GluK2-NETO1interaction was upregulated in ipsilateral dorsal horn neurons (DHNs) at 6 h post-incision. At 6 h post-incision, NETO2 small interfering ribonucleic acid (siRNA) intrathecal pretreatment increased mechanical withdrawal thresholds to von Freys and decreased ipsilateral paw cumulative pain scores. Further, PKCγactivation and synaptic abundance of GluK2 and GluR1 subunits in the ipsilateral DH were decreased by intrathecal pretreatment with NETO2 siRNA at 6 h post-incision. In conclusion, our findings imply that GluK2-NETO2 interaction could trigger PKCγactivation and the synaptic incorporation of AMPA receptor GluR1 subunits in rat DHs, which in turn led to the enhanced pain hypersensitivity after surgery. It sheds light on the interplay between KA and AMPA receptors in DHNs, which is thought to contribute to postoperative pain., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Kainate receptors (KARs) form a family of ionotropic glutamate receptors that regulate the activity of neuronal networks by both presynaptic and postsynaptic mechanisms. Their implication in pathologies is well documented for epilepsy. The higher prevalence of epileptic symptoms in Alzheimer's disease (AD) patients questions the role of KARs in AD. Here we investigated whether the synaptic expression and function of KARs was impaired in mouse models of AD. We addressed this question by immunostaining and electrophysiology at synapses between mossy fibers and CA3 pyramidal cells, in which KARs are abundant and play a prominent physiological role. We observed a decrease of the immunostaining for GluK2 in the stratum lucidum in CA3, and of the amplitude and decay time of synaptic currents mediated by GluK2-containing KARs in an amyloid mouse model (APP/PS1) of AD. Interestingly, a similar phenotype was observed in CA3 pyramidal cells in male and female mice with a genetic deletion of either presenilin or APP/APLP2 as well as in organotypic cultures treated with γ-secretase inhibitors. Finally, the GluK2 protein interacts with full-length and C-terminal fragments of APP. Overall, our data suggest that APP stabilizes KARs at synapses, possibly through a transsynaptic mechanism, and this interaction is under the control the γ-secretase proteolytic activity of presenilin. SIGNIFICANCE STATEMENT Synaptic impairment correlates strongly with cognitive deficits in Alzheimer's disease (AD). In this context, many studies have addressed the dysregulation of AMPA and NMDA ionotropic glutamate receptors. Kainate receptors (KARs), which form the third family of iGluRs, represent an underestimated actor in the regulation of neuronal circuits and have not yet been examined in the context of AD. Here we provide evidence that synaptic KARs are markedly impaired in a mouse model of AD. Additional experiments indicate that the γ-secretase activity of presenilin acting on the amyloid precursor protein controls synaptic expression of KAR. This study clearly indicates that KARs should be taken into consideration whenever addressing synaptic dysfunction and related cognitive deficits in the context of AD., (Copyright © 2022 the authors.)

Kainate receptors (KARs) are key regulators of synaptic circuits by acting at pre- and postsynaptic sites through either ionotropic or metabotropic actions. KARs can be activated by kainate, a potent neurotoxin, which induces acute convulsions. Here, we report that the acute convulsive effect of kainate mostly depends on GluK2/GluK5 containing KARs. By contrast, the acute convulsive activity of pilocarpine and pentylenetetrazol is not alleviated in the absence of KARs. Unexpectedly, the genetic inactivation of GluK2 rather confers increased susceptibility to acute pilocarpine-induced seizures. The mechanism involves an enhanced excitability of GluK2-/- CA3 pyramidal cells compared with controls upon pilocarpine application. Finally, we uncover that the absence of GluK2 increases pilocarpine modulation of Kv7/M currents. Taken together, our findings reveal that GluK2-containing KARs can control the excitability of hippocampal circuits through interaction with the neuromodulatory cholinergic system., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Abstract: It is well known that GluK2-containing kainate receptors play essential roles in seizure and cerebral ischemia-induced neuronal death, while GluK1-containing kainate receptors could increase tonic inhibition of post-synaptic pyramidal neurons. This research investigated whether GluK1 could inhibit activation of c-Jun N-terminal kinase 3 (JNK3) signaling pathway mediated by the GluK2 in cerebral ischemia–reperfusion. The results show that GluK1 activation by (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA) at 1nmol per rat could inhibit the assembly of GluK2·Postsynaptic density 95·mixed lineage kinase 3 signaling module, activation of JNK3 and its downstream signal molecules. However, the inhibition of ATPA could be prevented by GluK1 antagonist NS3763, GluK1 antisense, and GABAA receptor antagonist bicuculline. In addition, ATPA played a neuroprotective role against cerebral ischemia. In sum, the findings indicate that activation of GluK1 by ATPA at specific dosages may promote GABA release, which then suppresses post-synaptic GluK2–JNK3 signaling-mediated cerebral ischemic injury via GABAAR. [Copyright &y& Elsevier]

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 1-7 . In the brain, the trafficking, gating kinetics and pharmacology of kainate receptors are tightly regulated by neuropilin and tolloid-like (NETO) proteins 8-11 . 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 A/C , the upper lobe of LBD B/D and the lower lobe of LBD A/C , 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., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Most autosomal genes are thought to be expressed from both alleles, with some notable exceptions, including imprinted genes and genes showing random monoallelic expression (RME). The extent and nature of RME has been the subject of debate. Here we investigate the expression of several candidate RME genes in F1 hybrid mouse cells before and after differentiation, to define how they become persistently, monoallelically expressed. Clonal monoallelic expression is not present in embryonic stem cells, but we observe high frequencies of monoallelism in neuronal progenitor cells by assessing expression status in more than 200 clones. We uncover unforeseen modes of allelic expression that appear to be gene-specific and epigenetically regulated. This non-canonical allelic regulation has important implications for development and disease, including autosomal dominant disorders and opens up therapeutic perspectives., (© 2021. The Author(s).)

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.)

Alzheimer's disease (AD) is the most common neurodegenerative disorder. The earliest neuropathology of AD appears in entorhinal cortex (EC) regions. Therapeutic strategies and preventive measures to protect against entorhinal degeneration would be of substantial value in the early stages of AD. In this study, transcriptome based on the Illumina RNA-seq and proteome based on TMT-labelling were performed for RNA and protein profiling on AD EC samples and non-AD control EC samples. Immunohistochemistry was used to validate proteins expressions. After integrated analysis, 57 genes were detected both in transcriptome and proteome data, including 51 in similar altering trends (7 upregulated, 44 downregulated) and 6 in inverse trends when compared AD vs. control. The top 6 genes (GABRG2, CACNG3, CACNB4, GABRB2, GRIK2, and SLC17A6) within the 51 genes were selected and related to "ion transport". Correlation analysis demonstrated negative relationship of protein expression level with the neuropathologic changes. In conclusion, the integrate transcriptome and proteome analysis provided evidence for dysregulation of ion transport across brain regions in AD, which might be a critical signaling pathway that initiates pathology. This study might provide new insight into the earliest changes occurring in the EC of AD and novel targets for AD prevention and treatment., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: The objective of this study was to report the identification of antibodies against the glutamate kainate receptor subunit 2 (GluK2-abs) in patients with autoimmune encephalitis, and describe the clinical-immunological features and antibody effects., Methods: Two sera from 8 patients with similar rat brain immunostaining were used to precipitate the antigen from neuronal cultures. A cell-based assay (CBA) with GluK2-expressing HEK293 cells was used to assess 596 patients with different neurological disorders, and 23 healthy controls. GluK2-ab effects were determined by confocal microscopy in cultured neurons and electrophysiology in GluK2-expressing HEK293 cells., Results: Patients' antibodies precipitated GluK2. GluK2 antibody-specificity was confirmed by CBA, immunoprecipitation, GluK2-immunoabsorption, and GluK2 knockout brain immunohistochemistry. In 2 of 8 samples, antibodies reacted with additional GluK2 epitopes present in GluK1 or GluK3; in both, the reactivity was abrogated after GluK2 immuno-absorption. Six of 8 patients developed acute encephalitis and clinical or magnetic resonance imaging (MRI) features of predominant cerebellar involvement (4 presenting as cerebellitis, which in 2 patients caused obstructive hydrocephalus), and 2 patients had other syndromes (1 with cerebellar symptoms). One of the samples showed mild reactivity with non-kainate receptors (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors [AMPAR] and N-methyl-D-aspartate receptors [NMDAR]) leading to identify 6 additional cases with GluK2-abs among patients with anti-AMPAR (5/71) or anti-NMDAR encephalitis (1/73). GluK2-abs internalized GluK2 in HEK293 cells and neurons; these antibody-effects were reversible in neurons. A significant reduction of GluK2-mediated currents was observed in cells treated with patients' GluK2 serum following the time frame of antibody-mediated GluK2 internalization., Interpretation: GluK2-abs associate with an encephalitis with prominent clinicoradiological cerebellar involvement. The antibody effects are predominantly mediated by internalization of GluK2. ANN NEUROL 2021;90:107-123., (© 2021 American Neurological Association.)

The mechanisms underlying postoperative pain differ from the inflammatory or neuropathic pain. Previous studies have demonstrated that intrathecal α-amino-3-hydroxy-5-methy-4-isoxazole propionate (AMPA) -kainate (KA) receptor antagonist inhibits the guarding pain behavior and mechanical hyperalgesia, indicating a critical role of spinal KA receptors in postoperative pain hypersensitivity. However, how the functional regulations of spinal KA receptor subunits are involved in the postoperative pain hypersensitivity remains elusive. Therefore, in the current study, we investigated the synaptic delivery of spinal KA receptor subunits and the interaction between KA receptor subunits and glutamate receptor-interacting protein (GRIP) during the postoperative pain. Our data indicated that plantar incision induced the synaptic delivery of GluK2, but not GluK1 or GluK3 in ipsilateral spinal cord dorsal horns. The co-immunoprecipitation showed an increased GluK2 -GRIP interaction in ipsilateral dorsal horn neurons at 6 h post-incision. Interestingly, Intrathecal pretreatment of GRIP siRNA increased the paw withdrawal thresholds to mechanical stimuli and decreased the cumulative pain scores in the paws ipsilateral to the incision at 6 h post-incision. Additionally, Intrathecal pretreatment of GRIP siRNA reduced the synaptic abundance of GluK2 in ipsilateral spinal dorsal horn at 6 h after plantar incision. In general, our data have demonstrated that the GluK2- GRIP interaction-mediated synaptic abundance of GluK2 in dorsal horn neurons plays an important role in the postoperative pain hypersensitivity. Disrupting the GluK2- GRIP interaction may provide a new approach for relieving postoperative pain.

Synapse and synapse-associated proteins (SAPs) play critical roles in various neurodegeneration diseases and brain tumors. However, in lower-grade gliomas (LGG), SAPs have not been explored systematically. Herein, we are going to explore SAPs expression profile and its clinicopathological significance in LGG which can offer new insights to glioma therapy. In the present study, we integrate a list of SAPs that covered 231 proteins with synaptogenesis activity and post synapse formation. The LGG RNA-seq data were downloaded from GEO, TCGA and CGGA database. The prognosis associated SAPs in key modules of PPI (protein-protein interaction networks) was regarded as hub SAPs. Western blot, quantitative reverse transcription PCR (qRT-PCR) and immunochemistry results from HPA database were used to verify the expression of hub SAPs. There were 68 up-regulated SAPs and 44 down-regulated SAPs in LGG tissue compared with normal brain tissue. Data from function enrichment analysis revealed functions of differentially expressed SAPs in synapse organization and glutamatergic receptor pathway in LGGs. Survival analysis revealed that four SAPs, GRIK2, GABRD, GRID2 and ARC were correlate with the prognosis of LGG patients. Interestingly, we found that GABRD were up-regulated in LGG patients with seizures, indicating that SAPs may link to the pathogenesis of seizures in glioma patients. The four-SAPs signature was revealed as an independent prognostic factor in gliomas. Our study presented a novel strategy to assess the prognostic risks of LGGs, based on the expression of SAPs., (© 2021 The Author(s).)

Kainate receptors (KARs) are members of the glutamate receptor family that regulate synaptic function in the brain. Although they are known to be associated with psychiatric disorders, how they are involved in these disorders remains unclear. KARs are tetrameric channels assembled from a combination of GluK1-5 subunits. Among these, GluK2 and GluK5 subunits are the major heteromeric subunits in the brain. To determine the functional similarities and differences between GluK2 and GluK5 subunits, we generated GluK2 KO and GluK5 KO mice on a C57BL/6N background, a well-characterized inbred strain, and compared their behavioral phenotypes. We found that GluK2 KO and GluK5 KO mice exhibited the same phenotypes in many tests, such as reduced locomotor activity, impaired motor function, and enhanced depressive-like behavior. No change was observed in motor learning, anxiety-like behavior, or sociability. Additionally, we identified subunit-specific phenotypes, such as reduced motivation toward their environment in GluK2 KO mice and an enhancement in the contextual memory in GluK5 KO mice. These results revealed that GluK2 and GluK5 subunits not only function in a coordinated manner but also have a subunit-specific role in regulating behavior. To summarize, we demonstrated subunit-specific and common behavioral effects of GluK2 and GluK5 subunits for the first time. Moreover, to the best of our knowledge, this is the first evidence of the involvement of the GluK5 subunit in the expression of depressive-like behavior and contextual memory, which strongly indicates its role in psychiatric disorders., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Ozone is widely used to relieve chronic pain clinically, but the precise mechanisms governing its action have yet to be elucidated. The present study aimed to investigate the mechanisms underlying the pain‑alleviating effect of ozone in the chronic constriction injury (CCI) model of sciatic nerve in rats. Pain behaviours of rats were assessed by mechanical allodynia and thermal hyperalgesia. The expression of spinal glutamate receptor 6 (GluR6) and NF‑κB/p65 was detected by western blotting and reverse transcription‑quantitative PCR. Meanwhile, the expression of spinal IL‑1β, IL‑6 and TNF‑α was detected by ELISA. GluR6 short interfering (si)RNAs were used intrathecally immediately following CCI once per day. Ozone (10, 20 or 30  µ g/ml) or oxygen was injected intrathecally on day 7 after CCI. The expression level of spinal GluR6 increased on day 3 and reached a peak on day 7 after CCI. The expression level of spinal IL‑1β, IL‑6, TNF‑α and NF‑κB/p65 also increased on day 7 after CCI. In addition, pre‑intrathecal injection of GluR6 siRNAs inhibited pain behaviours and suppressed the expression of spinal GluR6, IL‑1β, IL‑6, TNF‑α and NF‑κB/p65 in CCI rats on day 7. Intrathecal injection of ozone was also observed to inhibit pain behaviours and suppress the expression of spinal GluR6, IL‑1β, IL‑6, TNF‑α and NF‑κB/p65 in CCI rats on day 7. The present study suggested that GluR6 served a pivotal role in neuropathic pain and that intrathecal injection of ozone may alleviate neuropathic pain via the GluR6‑NF‑κB/p65 signalling pathway.

Kainate receptors (KARs) are L-glutamate-gated ion channels that regulate synaptic transmission and modulate neuronal circuits. KARs have strict assembly rules and primarily function as heteromeric receptors in the brain. A longstanding question is how KAR heteromer subunits organize and coordinate together to fulfill their signature physiological roles. Here we report structures of the GluK2/GluK5 heteromer in apo, antagonist-bound, and desensitized states. The receptor assembles with two copies of each subunit, ligand binding domains arranged as two heterodimers and GluK5 subunits proximal to the channel. Strikingly, during desensitization, GluK2, but not GluK5, subunits undergo major structural rearrangements to facilitate channel closure. We show how the large conformational differences between antagonist-bound and desensitized states are mediated by the linkers connecting the pore helices to the ligand binding domains. This work presents the first KAR heteromer structure, reveals how its subunits are organized, and resolves how the heteromer can accommodate functionally distinct closed channel structures., Competing Interests: NK, PB, AP, DB, JM No competing interests declared, (© 2021, Khanra et al.)

Trigeminal neuralgia is a debilitating condition, and the pain easily spreads to other parts of the face. Here, we established a mouse model of partial transection of the infraorbital nerve (pT-ION) and found that the Connexin 36 (Cx36) inhibitor mefloquine caused greater alleviation of pT-ION-induced cold allodynia compared to the reduction of mechanical allodynia. Mefloquine reversed the pT-ION-induced upregulation of Cx36, glutamate receptor ionotropic kainate 2 (GluK2), transient receptor potential ankyrin 1 (TRPA1), and phosphorylated extracellular signal regulated kinase (p-ERK) in the trigeminal ganglion. Cold allodynia but not mechanical allodynia induced by pT-ION or by virus-mediated overexpression of Cx36 in the trigeminal ganglion was reversed by the GluK2 antagonist NS102, and knocking down Cx36 expression in Nav1.8-expressing nociceptors by injecting virus into the orofacial skin area of Nav1.8-Cre mice attenuated cold allodynia but not mechanical allodynia. In conclusion, we show that Cx36 contributes greatly to the development of orofacial pain hypersensitivity through GluK2, TRPA1, and p-ERK signaling.

Mutations in the PARK2 gene encoding the protein parkin cause autosomal recessive juvenile Parkinsonism (ARJP), a neurodegenerative disease characterized by dysfunction and death of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). Since a neuroprotective therapy for ARJP does not exist, research efforts aimed at discovering targets for neuroprotection are critically needed. A previous study demonstrated that loss of parkin function or expression of parkin mutants associated with ARJP causes an accumulation of glutamate kainate receptors (KARs) in human brain tissues and an increase of KAR-mediated currents in neurons in vitro. Based on the hypothesis that such KAR hyperactivation may contribute to the death of nigral DA neurons, we investigated the effect of KAR antagonism on the DA neuron dysfunction and death that occur in the parkinQ311X mouse, a model of human parkin-induced toxicity. We found that early accumulation of KARs occurs in the DA neurons of the parkinQ311X mouse, and that chronic administration of the KAR antagonist UBP310 prevents DA neuron loss. This neuroprotective effect is associated with the rescue of the abnormal firing rate of nigral DA neurons and downregulation of GluK2, the key KAR subunit. This study provides novel evidence of a causal role of glutamate KARs in the DA neuron dysfunction and loss occurring in a mouse model of human parkin-induced toxicity. Our results support KAR as a potential target in the development of neuroprotective therapy for ARJP.

In this study, we split 2156 individuals from the Chinese Longitudinal Healthy Longevity Survey (CLHLS) data into two groups, establishing a phenotype of exceptional longevity & normal cognition versus cognitive impairment. We conducted a genome-wide association study (GWAS) to identify significant genetic variants and biological pathways that are associated with cognitive impairment and used these results to construct polygenic risk scores. We elucidated the important and robust factors, both genetic and non-genetic, in predicting the phenotype, using several machine learning models. The GWAS identified 28 significant SNPs at p-value [Formula: see text] significance level and we pinpointed four genes, ESR1, PHB, RYR3, GRIK2, that are associated with the phenotype though immunological systems, brain function, metabolic pathways, inflammation and diet in the CLHLS cohort. Using both genetic and non-genetic factors, four machine learning models have close prediction results for the phenotype measured in Area Under the Curve: random forest (0.782), XGBoost (0.781), support vector machine with linear kernel (0.780), and [Formula: see text] penalized logistic regression (0.780). The top four important and congruent features in predicting the phenotype identified by these four models are: polygenic risk score, sex, age, and education.

Background/aim: Glutamate receptor GRIK2, previously designated as GluR6, is best described in neuronal cells. However, its biological relevance in non-neuronal cells is not well understood. We have investigated the expression of this important protein in normal human fibroblasts as a function of cell proliferation., Materials and Methods: We introduced expression constructs of all five isoforms (A-E) of GRIK2 in normal human fibroblasts and investigated the cells for the presence and localization of GRIK2, as well as for cell proliferation and senescence over a period of 24 days., Results: The expression of GRIK2-A isoform led to immediate cessation of cell proliferation. However, the cell numbers increased by 1.5- to 9.0-fold in 24 days upon transfection with B, C, D and E isoforms, after which they entered a state of senescence. The decreased proliferation was reflected by incorporation of BrdU in only 2-8% of transfected cells even after culturing them for 16 days., Conclusion: Our results are indicative of an association between GRIK2 and aging of fibroblasts., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

We first explore the features of GluK2 endocytosis during kainate excitotoxicity and then explore the role of Ca 2+ in the regulation of GluK2 endocytosis. The roles of Ca 2+ were examined by treating cells with Ca 2+ inhibitors or chelators. Surface biotinylation was used to examine the surface localization of GluK2. Immunoprecipitation followed by immunoblotting was used to identify the interaction of GluK2 with the endocytosis regulator protein-interacting with C kinase 1 and dynamin. Dynamin phosphorylation was examined by immunoblotting with the corresponding antibodies. Our results show that GluK2 internalization is blocked by inhibitors of clathrin-independent endocytosis and relies on intracellular Ca 2+ /calcineurin signaling. Protein-interacting with C kinase 1-GluK2 interaction is regulated by Ca 2+ /calcineurin signaling. Dynamin participates in the regulation of GluK2 surface localization. Also, calcineurin activation is related to dynamin function during kainate excitotoxicity. In conclusion, GluK2 receptor endocytosis is probably a clathrin-independent and dynamin-dependent process regulated by the peak Ca 2+ transient. This work indicates the roles of the Ca 2+ network in the regulation of GluK2 endocytosis during kainate excitotoxicity., Competing Interests: All authors declare no conflicts of interest., (© 2020 Du et al. Published by IMR press.)

Seizure protein 6 (SEZ6) is required for the development and maintenance of the nervous system, is a major substrate of the protease BACE1 and is linked to Alzheimer's disease (AD) and psychiatric disorders, but its molecular functions are not well understood. Here, we demonstrate that SEZ6 controls glycosylation and cell surface localization of kainate receptors composed of GluK2/3 subunits. Loss of SEZ6 reduced surface levels of GluK2/3 in primary neurons and reduced kainate-evoked currents in CA1 pyramidal neurons in acute hippocampal slices. Mechanistically, loss of SEZ6 in vitro and in vivo prevented modification of GluK2/3 with the human natural killer-1 (HNK-1) glycan, a modulator of GluK2/3 function. SEZ6 interacted with GluK2 through its ectodomain and promoted post-endoplasmic reticulum transport of GluK2 in the secretory pathway in heterologous cells and primary neurons. Taken together, SEZ6 acts as a new trafficking factor for GluK2/3. This novel function may help to better understand the role of SEZ6 in neurologic and psychiatric diseases., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Kainate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are two major, closely related receptor subtypes in the glutamate ion channel family. Excessive activities of these receptors have been implicated in a number of central nervous system diseases. Designing potent and selective antagonists of these receptors, especially of kainate receptors, is useful for developing potential treatment strategies for these neurological diseases. Here, we report on two RNA aptamers designed to individually inhibit kainate and AMPA receptors. To improve the biostability of these aptamers, we also chemically modified these aptamers by substituting their 2'-OH group with 2'-fluorine. These 2'-fluoro aptamers, FB9s-b and FB9s-r, were markedly resistant to RNase-catalyzed degradation, with a half-life of ∼5 days in rat cerebrospinal fluid or serum-containing medium. Furthermore, FB9s-r blocked AMPA receptor activity. Aptamer FB9s-b selectively inhibited GluK1 and GluK2 kainate receptor subunits, and also GluK1/GluK5 and GluK2/GluK5 heteromeric kainate receptors with equal potency. This inhibitory profile makes FB9s-b a powerful template for developing tool molecules and drug candidates for treatment of neurological diseases involving excessive activities of the GluK1 and GluK2 subunits., (© 2020 Jaremko et al.)

Introduction: The male-predominant sex difference through the spectrum of erosive esophagitis to Barrett's esophagus is widely known. We conducted a genome-wide association study (GWAS) stratified by sex for identifying factors that can predict the endoscopically diagnosed erosive esophagitis., Methods: Erosive esophagitis was diagnosed by endoscopy and assessed for severity. We identified genetic factors associated with erosive esophagitis that accounted for the sex differences in a cohort of 4,242 participants via a GWAS. After quality control and imputation, genetic associations with erosive esophagitis were investigated by multivariate linear regression in 3,620 subjects. Single-nucleotide polymorphisms (SNPs) with P < 5.0 × 10 were considered significant genome wide, and a genetic risk score was constructed for the prediction of erosive esophagitis risk., Results: Six genome-wide significant SNPs near the GRIK2 gene on chromosome 6 were found to be associated with erosive esophagitis only in male subjects. These were predictive of severity through a genetic risk score (P < 0.05), and the findings were validated in a cohort of 622 subjects (P < 0.05)., Discussion: This is the first GWAS of erosive esophagitis, and we identified 6 genome-wide significant SNPs in male subjects. These SNPs could help explain the pathogenesis of erosive esophagitis and contribute to the understanding of sex differences. Further genetic investigation could allow for the prediction of high risk for erosive esophagitis and development of new treatment options.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases. In most cases, the development of the disease is sporadic and is not associated with any currently known mutations associated with PD. It is believed that changes associated with the epigenetic regulation of gene expression may play an important role in the pathogenesis of this disease. The study of individuals with an almost identical genetic background, such as monozygotic twins, is one of the best approaches to the analysis of such changes. A whole-transcriptome analysis of dermal fibroblasts obtained from three pairs of monozygotic twins discordant for PD was carried out in this work. Twenty-nine differentially expressed genes were identified in the three pairs of twins. These genes were included in seven processes within two clusters, according to the results of an enrichment analysis. The cluster with the greatest statistical significance included processes associated with the regulation of the differentiation of fat cells, the action potential, and the regulation of glutamatergic synaptic transmission. The most significant genes, which occupied a central position in this cluster, were PTGS2, SCN9A, and GRIK2. These genes can be considered as potential candidate genes for PD.

Fusions of the Runt-related transcription factor 1 (RUNX1) with different partner genes have been associated with various hematological disorders. Interestingly, the C-terminally truncated form of RUNX1 and RUNX1 fusion proteins are similarly considered important contributors to leukemogenesis. Here, we describe a 59-year-old male patient who was initially diagnosed with acute myeloid leukemia, inv(16)(p13;q22)/CBFB-MYH11 (FAB classification M4Eo). He achieved complete remission and negative CBFB-MYH11 status with daunorubicin/cytarabine combination chemotherapy but relapsed 3 years later. Cytogenetic analysis of relapsed leukemia cells revealed CBFB-MYH11 negativity and complex chromosomal abnormalities without inv(16)(p13;q22). RNA-seq identified the glutamate receptor, ionotropic, kinase 2 (GRIK2) gene on 6q16 as a novel fusion partner for RUNX1 in this case. Specifically, the fusion of RUNX1 to the GRIK2 antisense strand (RUNX1-GRIK2as) generated multiple missplicing transcripts. Because extremely low levels of wild-type GRIK2 were detected in leukemia cells, RUNX1-GRIK2as was thought to drive the pathogenesis associated with the RUNX1-GRIK2 fusion. The truncated RUNX1 generated from RUNX1-GRIK2as induced the expression of the granulocyte colony-stimulating factor (G-CSF) receptor on 32D myeloid leukemia cells and enhanced proliferation in response to G-CSF. In summary, the RUNX1-GRIK2as fusion emphasizes the importance of aberrantly truncated RUNX1 in leukemogenesis., (© 2020 S. Karger AG, Basel.)

Kainate receptors, which are glutamate activated excitatory neurotransmitter receptors, predominantly exist as heteromers of GluK2 and GluK5 subunits in the mammalian central nervous system. There are currently no structures of the full-length heteromeric kainate receptors. Here, we have used single molecule FRET to determine the specific arrangement of the GluK2 and GluK5 subunits within the dimer of dimers configuration in a full-length receptor. Additionally, we have also studied the dynamics and conformational heterogeneity of the amino-terminal and agonist-binding domain interfaces associated with the resting and desensitized states of the full-length heteromeric kainate receptor using FRET-based methods. The smFRET data are compared to similar experiments performed on the homomeric kainate receptor to provide insight into the differences in conformational dynamics that distinguish the two functionally. This article is part of a Special Issue entitled: Molecular biophysics of membranes and membrane proteins., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The excitatory neurotransmitter glutamate is essential in basal ganglia motor circuits and has long been thought to contribute to cell death and degeneration in Parkinson's disease (PD). While previous research has shown a significant role of NMDA and AMPA receptors in both excitotoxicity and PD, the third class of ionotropic glutamate receptors, kainate receptors, have been less well studied. Given the expression of kainate receptor subunits GluK1-GluK3 in key PD-related brain regions, it has been suggested that GluK1-GluK3 may contribute to excitotoxic cell loss. Therefore the neuroprotective potential of the kainate receptor antagonist UBP310 in animal models of PD was investigated in this study. Stereological quantification revealed administration of UBP310 significantly increased survival of dopaminergic and total neuron populations in the substantia nigra pars compacta in the acute MPTP mouse model of PD. In contrast, UBP310 was unable to rescue MPTP-induced loss of dopamine levels or dopamine transporter expression in the striatum. Furthermore, deletion of GluK1, GluK2 or GluK3 had no effect on MPTP or UBP310-mediated effects across all measures. Interestingly, UBP310 did not attenuate cell loss in the midbrain induced by intrastriatal 6-OHDA toxicity. These results indicate UBP310 provides neuroprotection in the midbrain against MPTP neurotoxicity that is not dependent on specific kainate receptor subunits., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Sex differences in opioid dependence (OD) are genetically influenced. We conducted genomewide gene-by-sex interaction scans for the DSM-IV diagnosis of OD in 8,387 African-American (AA) or European-American subjects (43.6% women; 4,715 OD subjects). Among AAs, 9 SNPs were genome-wide significant at ADGRV1 (adhesion G-protein-coupled receptor V1, lead-SNP rs2366929*(C/T), p = 1.5 × 10 -9 ) for sex-different risk of OD, with the rs2366929*C-allele increasing OD risk only for men. The top co-expressions in brain were between ADGRV1 and GRIK2 in substantia nigra and medullary inferior olivary nucleus, and between ADGRV1 and EFHC2 in frontal cortex and putamen. Significant sex-differential ADGRV1 expression from GTEx was detected in breast (Bonferroni-corrected-p < 0.002) and in heart (p < 0.0125), with nominal significance identified in brain, thyroid, lung, and stomach (p < 0.05). ADGRV1 co-expression and disease-enrichment analysis identifying the top 10 diseases showed strikingly sexually dimorphic risks. The enrichment and transcriptome analyses provided convergent support that ADGRV1 exerts a sex-different effect on OD risk. This is the first study to identify genetic variants contributing to sex differences in OD. It shows that ADGRV1 contributes to OD risk only in AA men, a finding that warrants further study.

Objective: Islets secrete neurotransmitters including glutamate which participate in fine regulation of islet function. The excitatory ionotropic glutamate receptor GluK2 of the kainate receptor family is widely expressed in brain and also found in islets, mainly in α and γ cells. α cells co-release glucagon and glutamate and the latter increases glucagon release via ionotropic glutamate receptors. However, neither the precise nature of the ionotropic glutamate receptor involved nor its role in glucose homeostasis is known. As isoform specific pharmacology is not available, we investigated this question in constitutive GluK2 knock-out mice (GluK2 -/- ) using adult and middle-aged animals to also gain insight in a potential role during aging., Methods: We compared wild-type GluK2 +/+ and knock-out GluK2 -/- mice using adult (14-20 weeks) and middle-aged animals (40-52 weeks). Glucose (oral OGTT and intraperitoneal IPGTT) and insulin tolerance as well as pyruvate challenge tests were performed according to standard procedures. Parasympathetic activity, which stimulates hormones secretion, was measured by electrophysiology in vivo. Isolated islets were used in vitro to determine islet β-cell electrical activity on multi-electrode arrays and dynamic secretion of insulin as well as glucagon was determined by ELISA., Results: Adult GluK2 -/- mice exhibit an improved glucose tolerance (OGTT and IPGTT), and this was also apparent in middle-aged mice, whereas the outcome of pyruvate challenge was slightly improved only in middle-aged GluK2 -/- mice. Similarly, insulin sensitivity was markedly enhanced in middle-aged GluK2 -/- animals. Basal and glucose-induced insulin secretion in vivo was slightly lower in GluK2 -/- mice, whereas fasting glucagonemia was strongly reduced. In vivo recordings of parasympathetic activity showed an increase in basal activity in GluK2 -/- mice which represents most likely an adaptive mechanism to counteract hypoglucagonemia rather than altered neuronal mechanism. In vitro recording demonstrated an improvement of glucose-induced electrical activity of β-cells in islets obtained from GluK2 -/- mice at both ages. Finally, glucose-induced insulin secretion in vitro was increased in GluK2 -/- islets, whereas glucagon secretion at 2 mmol/l of glucose was considerably reduced., Conclusions: These observations indicate a general role for kainate receptors in glucose homeostasis and specifically suggest a negative effect of GluK2 on glucose homeostasis and preservation of islet function during aging. Our observations raise the possibility that blockade of GluK2 may provide benefits in glucose homeostasis especially during aging., (Copyright © 2019 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The neuropilin and tolloid-like (Neto) proteins Neto1 and Neto2 are auxiliary subunits of kainate-type glutamate receptors (KARs) that regulate KAR trafficking and gating. However, how Netos bind and regulate the biophysical functions of KARs remains unclear. Here, we found that the N-terminal domain (NTD) of glutamate receptor ionotropic kainate 2 (GluK2) binds the first complement C1r/C1s-Uegf-BMP (CUB) domain of Neto proteins ( i.e. NTD-CUB1 interaction) and that the core of GluK2 (GluK2ΔNTD) binds Netos through domains other than CUB1s (core-Neto interaction). Using electrophysiological analysis in HEK293T cells, we examined the effects of these interactions on GluK2 gating, including deactivation, desensitization, and recovery from desensitization. We found that NTD deletion does not affect GluK2 fast gating kinetics, the desensitization, and the deactivation. We also observed that Neto1 and Neto2 differentially regulate GluK2 fast gating kinetics, which largely rely on the NTD-CUB1 interactions. NTD removal facilitated GluK2 recovery from desensitization, indicating that the NTD stabilizes the GluK2 desensitization state. Co-expression with Neto1 or Neto2 also accelerated GluK2 recovery from desensitization, which fully relied on the NTD-CUB1 interactions. Moreover, we demonstrate that the NTD-CUB1 interaction involves electric attraction between positively charged residues in the GluK2&#95;NTD and negatively charged ones in the CUB1 domains. Neutralization of these charges eliminated the regulatory effects of the NTD-CUB1 interaction on GluK2 gating. We conclude that KARs bind Netos through at least two sites and that the NTD-CUB1 interaction critically regulates Neto-mediated GluK2 gating., (© 2019 Li et al.)

Objectives: Animal studies have shown that glutamate receptor ionotropic kainate 2 (GRIK2) gene knockout mice are more impulsive and aggressive. This study aims to verify whether the rs6922753 and rs2227283 polymorphisms of the GRIK2 gene are associated with both aggressive behavior and bipolar mania in the Chinese Han population., Methods: Polymerase chain reaction (PCR) was applied in the genotype rs6922753 and rs2227283 polymorphisms of the GRIK2 gene in 201 bipolar manic patients with aggressive behaviors, 198 bipolar manic patients without aggressive behaviors, and 132 healthy controls. The Modified Overt Aggression Scale (MOAS) was used to evaluate aggressive behavior in patients with bipolar mania., Results: No correlation was found between aggressive behavior and the rs6922753 polymorphism in the three groups. The A/A genotype and A allele of the rs2227283 polymorphism were found significantly more frequently in patients with aggressive behavior than in healthy controls (p = .004 and p = .013, respectively) and in patients with nonaggressive behavior (p = .002 and p = .018, respectively). The A/A genotype and A allele were associated with an increased risk of aggressive behavior., Conclusion: This study suggests that the rs2227283 polymorphism of the GRIK2 gene is related to aggressive behaviors in bipolar manic patients and that the A/A genotype and A allele may increase the risk of the aggressive behavior in bipolar manic patients., (© 2019 The Authors. Brain and Behavior published by Wiley Periodicals, Inc.)

The RISE model is an effective system to study the underlying molecular and cellular mechanisms involved in the initiation and maintenance of epilepsy in vivo. Here we profiled the expression of excitatory and inhibitory neurotransmitter receptor subunits and synaptic scaffolding proteins in the hippocampus and temporal lobe and compared these changes with alterations in network activity at specific timepoints during epileptogenesis. Significant changes occurred in all of the ionotropic glutamate receptor subunits tested during epilepsy induction and progression and the profile of these changes differed between the hippocampus and temporal lobe. Notably, AMPAR subunits were dramatically decreased during the latent phase of epilepsy induction, matched by a profound decrease in the network response to kainate application in the hippocampus. Moreover, decreases in the GABA A β3 subunit are consistent with a loss of inhibitory input contributing to the perturbation of excitatory/inhibitory balance and seizure generation. These data highlight the synaptic reorganisation that mediates the relative hypoexcitability prior to the manifestation of seizures and subsequent hyperexcitability when spontaneous seizures develop. These patterns of changes give new insight into the mechanisms underpinning epilepsy and provide a platform for future investigations targeting particular receptor subunits to reduce or prevent seizures., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

During neuronal development, AMPA receptors (AMPARs) and NMDA receptors (NMDARs) are important for neuronal differentiation. Kainate receptors (KARs) are closely related to AMPARs and involved in the regulation of cortical network activity. However, their role for neurite growth and differentiation of cortical neurons is unclear. Here, we used KAR agonists and overexpression of selected KAR subunits and their auxiliary neuropilin and tolloid-like proteins, NETOs, to investigate their influence on dendritic growth and network activity in organotypic cultures of rat visual cortex. Kainate at 500 nM enhanced network activity and promoted development of dendrites in layer II/III pyramidal cells, but not interneurons. GluK2 overexpression promoted dendritic growth in pyramidal cells and interneurons. GluK2 transfectants were highly active and acted as drivers for network activity. GluK1 and NETO1 specifically promoted dendritic growth of interneurons. Our study provides new insights for the roles of KARs and NETOs in the morphological and physiological development of the visual cortex.

Excitatory synapses onto somatostatin (SOM) interneurons show robust short-term facilitation. This hallmark feature of SOM interneurons arises from a low initial release probability that regulates the recruitment of interneurons in response to trains of action potentials. Previous work has shown that Elfn1 (extracellular leucine rich repeat and fibronectin Type III domain containing 1) is necessary to generate facilitating synapses onto SOM neurons by recruitment of two separate presynaptic components: mGluR7 (metabotropic glutamate receptor 7) and GluK2-KARs (kainate receptors containing glutamate receptor, ionotropic, kainate 2). Here, we identify how a transsynaptic interaction between Elfn1 and mGluR7 constitutively reduces initial release probability onto mouse cortical SOM neurons. Elfn1 produces glutamate-independent activation of mGluR7 via presynaptic clustering, resulting in a divergence from the canonical "autoreceptor" role of Type III mGluRs, and substantially altering synaptic pharmacology. This structurally induced determination of initial release probability is present at both layer 2/3 and layer 5 synapses. In layer 2/3 SOM neurons, synaptic facilitation in response to spike trains is also dependent on presynaptic GluK2-KARs. In contrast, layer 5 SOM neurons do not exhibit presynaptic GluK2-KAR activity at baseline and show reduced facilitation. GluK2-KAR engagement at synapses onto layer 5 SOM neurons can be induced by calmodulin activation, suggesting that synaptic function can be dynamically regulated. Thus, synaptic facilitation onto SOM interneurons is mediated both by constitutive mGluR7 recruitment by Elfn1 and regulated GluK2-KAR recruitment, which determines the extent of interneuron recruitment in different cortical layers. SIGNIFICANCE STATEMENT This study identifies a novel mechanism for generating constitutive GPCR activity through a transsynaptic Elfn1/mGluR7 structural interaction. The resulting tonic suppression of synaptic release probability deviates from canonical autoreceptor function. Constitutive suppression delays the activation of somatostatin interneurons in circuits, necessitating high-frequency activity for somatostatin interneuron recruitment. Furthermore, variations in the synaptic proteome generate layer-specific differences in facilitation at pyr → SOM synapses. The presence of GluK2 kainate receptors in L2/3 enhances synaptic transmission during prolonged activity. Thus, layer-specific synaptic properties onto somatostatin interneurons are mediated by both constitutive mGluR7 recruitment and regulated GluK2 kainate receptor recruitment, revealing a mechanism that generates diversity in physiological responses of interneurons., (Copyright © 2019 the authors.)

Aims: The association of single nucleotide polymorphisms (SNPs) of glutamate receptor 2 (GRIK2) gene, as well as gene-gene interaction with the risk of early-onset epilepsy susceptibility, was studied in Chinese children., Methods: Generalized multi-factor dimension reduction (GMDR) is used to identify the optimal linkage between interaction among four SNPs and early-onset epilepsy susceptibility. Logistic regression was performed to assess association between four SNPs within GRIK2 gene and the risk of epilepsy., Results: The results show that the risk of epilepsy in the rs4840200-T allele carriers was significantly higher than CC (CT/TT vs CC), adjusted OR (95% CI) = 1.74 (1.31-2.20), and the carrier of rs3213607-A allele was also higher than CC (CG/GG vs CC) with adjusted OR (95% CI) = 1.61 (1.23-2.10). We did not detect significant association between rs9390754 and rs2235076 within GRIK2 gene and epilepsy risk. In the GMDR analysis for the gene/gene interaction (2-4 locus models), we found a significant two-locus model (P = 0.001) involving rs4840200 and rs9390754. The cross-validation consistency was 10/10, and the prediction error was 0.632. Participants with rs4840200-CT/TT and rs9390754-GA/AA genotype within GRIK2 gene have the highest epilepsy risk, compared to participants with rs4840200-CC and rs9390754-GG genotype within GRIK2 gene, OR (95% CI) = 2.42 (1.78-3.11), after covariates adjustment for age and gender., Conclusions: Both rs4840200-T and rs3213607-A, and the interactions between rs4840200 and rs9390754 are related to the increased risk of epilepsy risk., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Kainate receptors are glutamate-gated cation-selective channels involved in excitatory synaptic signaling and are known to be modulated by ions. Prior functional and structural studies suggest that the dimer interface at the agonist-binding domain plays a key role in activation, desensitization, and ion modulation in kainate receptors. Here we have used fluorescence-based methods to investigate the changes and conformational heterogeneity at these interfaces associated with the resting, antagonist-bound, active, desensitized, and ion-modulated states of the receptor. These studies show that in the presence of Na + ions the interfaces exist primarily in the coupled state in the apo, antagonist-bound and activated (open channel) states. Under desensitizing conditions, the largely decoupled dimer interface at the agonist-binding domain as seen in the cryo-EM structure is one of the states observed. However, in addition to this state there are several additional states with lower levels of decoupling. Replacing Na + with Cs + does not alter the FRET efficiencies of the states significantly, but shifts the population to the more decoupled states in both resting and desensitized states, which can be correlated with the lower activation seen in the presence of Cs + .

Kainate receptors are ligand-gated ion channels that have two major roles in the central nervous system: they mediate a postsynaptic component of excitatory neurotransmission at some glutamatergic synapses and modulate transmitter release at both excitatory and inhibitory synapses. Accumulating evidence implicates kainate receptors in a variety of neuropathologies, including epilepsy, psychiatric disorders, developmental delay, and cognitive impairment. Here, to gain a deeper understanding of the conformational changes associated with agonist binding and channel opening, we generate a series of Cys substitutions in the GluK2 kainate receptor subunit, focusing on the M3 helices that line the ion pore and form the bundle-crossing gate at the extracellular mouth of the channel. Exposure to 50 µM Cd produces direct activation of homomeric mutant channels bearing Cys substitutions in (A657C), or adjacent to (L659C), the conserved SYTANLAAF motif. Activation by Cd is occluded by modification with 2-aminoethyl MTS (MTSEA), indicating that Cd binds directly and specifically to the substituted cysteines. Cd potency for the A657C mutation (EC 50 = 10 µM) suggests that binding involves at least two coordinating residues, whereas weaker Cd potency for L659C (EC 50 = 2 mM) implies that activation does not require tight coordination by multiple side chains for this substitution. Experiments with heteromeric and chimeric channels indicate that activation by Cd requires Cys substitution at only two of the four subunits within a tetrameric receptor and that activation is similar for substitution within subunits in either the A/C or B/D conformations. We develop simple kinetic models for the A657C substitution that reproduce several features of Cd activation as well as the low-affinity inhibition observed at higher Cd concentrations (5-20 mM). Together, these results demonstrate rapid and reversible channel activation, independent of agonist site occupancy, upon Cd binding to Cys side chains at two specific locations along the GluK2 inner helix., (© 2019 Wilding and Huettner.)

Key Points: Potassium-chloride co-transporter 2 (KCC2) plays a critical role in regulating chloride homeostasis, which is essential for hyperpolarizing inhibition in the mature nervous system. KCC2 interacts with many proteins involved in excitatory neurotransmission, including the GluK2 subunit of the kainate receptor (KAR). We show that activation of KARs hyperpolarizes the reversal potential for GABA (E GABA ) via both ionotropic and metabotropic signalling mechanisms. KCC2 is required for the metabotropic KAR-mediated regulation of E GABA , although ionotropic KAR signalling can hyperpolarize E GABA independent of KCC2 transporter function. The KAR-mediated hyperpolarization of E GABA is absent in the GluK1/2 -/- mouse and is independent of zinc release from mossy fibre terminals. The ability of KARs to regulate KCC2 function may have implications in diseases with disrupted excitation: inhibition balance, such as epilepsy, neuropathic pain, autism spectrum disorders and Down's syndrome., Abstract: Potassium-chloride co-transporter 2 (KCC2) plays a critical role in the regulation of chloride (Cl - ) homeostasis within mature neurons. KCC2 is a secondarily active transporter that extrudes Cl - from the neuron, which maintains a low intracellular Cl - concentration [Cl - ]. This results in a hyperpolarized reversal potential of GABA (E GABA ), which is required for fast synaptic inhibition in the mature central nervous system. KCC2 also plays a structural role in dendritic spines and at excitatory synapses, and interacts with 'excitatory' proteins, including the GluK2 subunit of kainate receptors (KARs). KARs are glutamate receptors that display both ionotropic and metabotropic signalling. We show that activating KARs in the hippocampus hyperpolarizes E GABA , thus strengthening inhibition. This hyperpolarization occurs via both ionotropic and metabotropic KAR signalling in the CA3 region, whereas it is absent in the GluK1/2 -/- mouse, and is independent of zinc release from mossy fibre terminals. The metabotropic signalling mechanism is dependent on KCC2, although the ionotropic signalling mechanism produces a hyperpolarization of E GABA even in the absence of KCC2 transporter function. These results demonstrate a novel functional interaction between a glutamate receptor and KCC2, a transporter critical for maintaining inhibition, suggesting that the KAR:KCC2 complex may play an important role in excitatory:inhibitory balance in the hippocampus. Additionally, the ability of KARs to regulate chloride homeostasis independently of KCC2 suggests that KAR signalling can regulate inhibition via multiple mechanisms. Activation of kainate-type glutamate receptors could serve as an important mechanism for increasing the strength of inhibition during periods of strong glutamatergic activity., (© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.)

The biological mechanisms underlying complex forms of learning requiring the understanding of rules based on previous experience are not yet known. Previous studies have raised the intriguing possibility that improvement in complex learning tasks requires the long-term modulation of intrinsic neuronal excitability, induced by reducing the conductance of the slow calcium-dependent potassium current (sI AHP ) simultaneously in most neurons in the relevant neuronal networks in several key brain areas. Such sI AHP reduction is expressed in attenuation of the postburst afterhyperpolarization (AHP) potential, and thus in enhanced repetitive action potential firing. Using complex olfactory discrimination (OD) learning as a model for complex learning, we show that brief activation of the GluK2 subtype glutamate receptor results in long-lasting enhancement of neuronal excitability in neurons from controls, but not from trained rats. Such an effect can be obtained by a brief tetanic synaptic stimulation or by direct application of kainate, both of which reduce the postburst AHP in pyramidal neurons. Induction of long-lasting enhancement of neuronal excitability is mediated via a metabotropic process that requires PKC and ERK activation. Intrinsic neuronal excitability cannot be modulated by synaptic activation in neurons from GluK2 knock-out mice. Accordingly, these mice are incapable of learning the complex OD task. Moreover, viral-induced overexpression of Gluk2 in piriform cortex pyramidal neurons results in remarkable enhancement of complex OD learning. Thus, signaling via kainate receptors has a central functional role in higher cognitive abilities.

The kainate family of ionotropic glutamate receptors (iGluRs) mediates pre- and postsynaptic neurotransmission. Previously computed conformational potentials of mean force (PMFs) for iGluR ligand-binding domains (LBDs) revealed subtype-dependent conformational differences between α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartic acid (NMDA) iGluR subfamilies. Here we report PMFs for the kainate receptor GluK2 in apo and glutamate-bound states. Apo and glutamate-bound GluK2 LBDs preferentially access closed-cleft conformations. Apo GluK2 exhibits a surprisingly high degree of conformational flexibility, accessing open and closed states. Comparing across iGluR subtypes, these results are similar to glycine-binding GluN1 and GluN3A NMDA subunits and differ from glutamate-binding GluA2 and GluN2A subunits. To test the contribution of cross-lobe interactions on closed-cleft LBD stability, we computed PMFs for two GluK2 mutants, D462A and D656S. D462A, but not D656S, weakens closed-cleft conformations of the glutamate-bound LBD. Theoretical Boltzmann-weighted small-angle X-ray scattering profiles improve agreement with experimental results compared with calculations from the LBD crystal structure alone., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: The aberrant regulation of growth and proliferation is a key feature of carcinoma cells. In order to use molecular strategies to correct these defects toward therapeutic purposes, it is important to characterize the entire spectrum of causative molecules., Materials and Methods: By using gene transfer technique, SKOV3 ovarian carcinoma cells were transduced with an expression construct of glutamate receptor 6 (glutamate ionotropic receptor kainate type subunit 2, GRIK2) in retroviral vector PQCXIP. The senescence of transduced cells was subsequently characterized., Results: Our results demonstrated that retroviral transduction occurs with high frequency and transduced cells continue to proliferate, albeit at a significantly reduced rate, up to 39 days. Some transduced colonies stopped proliferating after 12 days, and none of the clones proliferated beyond 37 days. The doubling time for these transduced cells increased progressively until they reached a complete cell-cycle arrest. The proliferating cells were distinguished by bromodeoxyuridine incorporation and 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide assay. The growth and cell cycle arrest in transduced cells accompanied activation of senescence-associated β-galactosidase. Furthermore, we have demonstrated a decrease in the levels of active protein kinase B and increase in the abundance of inactive cyclin-dependent kinase 1., Conclusion: These results indicate involvement of GRIK2 in senescence and suggests GRIK2 as a potential target for therapeutic intervention of cancer cells., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Kainate receptors (KARs) regulate neuronal excitability and network function. Most KARs contain the subunit GluK2 (also known as GRIK2), and the properties of these receptors are determined in part by ADAR2 (also known as ADARB1)-mediated mRNA editing of GluK2, which changes a genomically encoded glutamine residue into an arginine residue (Q/R editing). Suppression of synaptic activity reduces ADAR2-dependent Q/R editing of GluK2 with a consequential increase in GluK2-containing KAR surface expression. However, the mechanism underlying this reduction in GluK2 editing has not been addressed. Here, we show that induction of KAR upscaling, a phenomenon in which surface expression of receptors is increased in response to a chronic decrease in synaptic activity, results in proteasomal degradation of ADAR2, which reduces GluK2 Q/R editing. Because KARs incorporating unedited GluK2(Q) assemble and exit the ER more efficiently, this leads to an upscaling of KAR surface expression. Consistent with this, we demonstrate that partial ADAR2 knockdown phenocopies and occludes KAR upscaling. Moreover, we show that although the AMPA receptor (AMPAR) subunit GluA2 (also known as GRIA2) also undergoes ADAR2-dependent Q/R editing, this process does not mediate AMPAR upscaling. These data demonstrate that activity-dependent regulation of ADAR2 proteostasis and GluK2 Q/R editing are key determinants of KAR, but not AMPAR, trafficking and upscaling.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Kainate-type glutamate receptors play critical roles in excitatory synaptic transmission and synaptic plasticity in the brain. GluK1 and GluK2 possess fundamentally different capabilities in surface trafficking as well as synaptic targeting in hippocampal CA1 neurons. Here we find that the excitatory postsynaptic currents (EPSCs) are significantly increased by the chimeric GluK1(SP GluK2 ) receptor, in which the signal peptide of GluK1 is replaced with that of GluK2. Coexpression of GluK1 signal peptide completely suppresses the gain in trafficking ability of GluK1(SP GluK2 ), indicating that the signal peptide represses receptor trafficking in a trans manner. Furthermore, we demonstrate that the signal peptide directly interacts with the amino-terminal domain (ATD) to inhibit the synaptic and surface expression of GluK1. Thus, we have uncovered a trafficking mechanism for kainate receptors and propose that the cleaved signal peptide behaves as a ligand of GluK1, through binding with the ATD, to repress forward trafficking of the receptor.