GluR6 Kainate Receptor Research Articles

Generation and Analysis of GluR5(Q636R) Kainate Receptor Mutant Mice

The physiological significance of RNA editing of transcripts that code for kainate-preferring glutamate receptor subunits is unknown, despite the fact that the functional consequences of this molecular modification have been well characterized in cloned receptor subunits. RNA editing of the codon that encodes the glutamine/arginine (Q/R) site in the second membrane domain (MD2) of glutamate receptor 5 (GluR5) and GluR6 kainate receptor subunits produces receptors with reduced calcium permeabilities and single-channel conductances. Approximately 50% of the GluR5 subunit transcripts from adult rat brain are edited at the Q/R site in MD2. To address the role of glutamate receptor mRNA editing in the brain, we have made two strains of mice with mutations at amino acid 636, the Q/R-editing site in GluR5, using embryonic stem cell-mediated transgenesis. GluR5(RloxP/RloxP) mice encode an arginine at the Q/R site of the GluR5 subunit, whereas GluR5(wt(loxP)/wt(loxP)) mice encode a glutamine at this site, similar to wild-type mice. Mutant animals do not exhibit developmental abnormalities, nor do they show deficits in the behavioral paradigms tested in this study. Kainate receptor current densities were reduced by a factor of six in acutely isolated sensory neurons of dorsal root ganglia from GluR5(RloxP/RloxP) mice compared with neurons from wild-type mice. However, the editing mutant mice did not exhibit altered responses to thermal and chemical pain stimuli. Our investigations with the GluR5-editing mutant mice have therefore defined a set of physiological processes in which editing of the GluR5 subunit is unlikely to play an important role.

The role of non- N-methyl- d-aspartate ionotropic glutamate receptors in the spinal transmission of nociception in normal animals and animals with carrageenan inflammation

The role of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and kainate receptors in spinal nociceptive transmission in both normal animals and animals with carrageenan inflammation was investigated using the AMPA/kainate receptor antagonist 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2,3-dione (NBQX) and the selective GluR5 kainate receptor antagonist LY382884 [3 S,4a R,6 S,8a R-6-(4-carboxyphenyl)methyl-1,2,3,4,4a,5,6,7,8,8a-deca-hydroisoquinoline-3-carboxylic acid]. In normal animals, spinal administration of 100 μg of LY382884 produced a significant inhibition of both the C-fibre-evoked response and post-discharge of dorsal horn neurons, with the wind-up of the neurons being reduced by both 50 and 100 μg of LY382884. The spinal actions of LY382884 were enhanced following 3 h of carrageenan inflammation, such that doses of 20 μg and above were able to produce significant inhibitions of the noxious-evoked response of the neurons. Spinal administration of NBQX in normal animals (5–50 μg) inhibited the C-fibre-evoked response of the dorsal horn neurons, but only 50 μg of NBQX was able to inhibit the wind-up and post-discharge of the neurons. Following 3 h of carrageenan inflammation, the ability of NBQX to inhibit the wind-up and post-discharge of the neurons was markedly enhanced. These data suggest that both AMPA and kainate GluR5 receptors play an enhanced role in spinal nociceptive processing following the development of peripheral inflammation, as antagonists at both receptors are more effective against nociceptive responses, including wind-up under these inflammatory conditions.

Pharmacological characterization of a GluR6 kainate receptor in cultured hippocampal neurons

We have examined the pharmacology of kainate receptors in cultured hippocampal neurons (6–8 days in vitro (DIV)) from embryonic rats (E17). Cultured neurons were pre-treated with concanavalin A to remove kainate receptor desensitization and whole-cell voltage clamp electrophysiology employed to record inward currents in response to glutamatergic agonists and antagonists. N-methyl- d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptor responses were blocked using MK801 (3 μM) and the 2,3-benzodiazepine, LY300168 (GYKI53655, 50 μM), respectively. Inward currents were recorded in hippocampal neurons upon application of kainate and the 2 S,4 R isomer of 4-methyl glutamic acid (SYM2081) with EC 50 values of 3.4±0.4 μM and 1.6±0.5 μM, respectively ( n=6 cells). The GluR5 selective agonists, LY339434 (100 μM) and ( RS)-2-amino-3-(3-hydroxy-5- tert-butyl-4-isoxazolyl) propionic acid (ATPA) (100 μM), did not evoke detectable inward currents in any cell responding to kainate. LY293558 and the selective GluR5 antagonist, LY382884, had weak antagonist effects on responses evoked by either kainate or (2 S,4 R)-4-methyl glutamate (IC 50 >300 μM). The quinoxalinedione, 2,3-dihyro-6-nitro-7-sulfamoyl-benzo( f)quinoxaline (NBQX), blocked both kainate and (2 S,4 R)-4-methyl glutamate-activated currents at much lower concentrations (IC 50 approximately 10 μM). These results provide pharmacological evidence that ion channels comprised of GluR6 kainate receptor subunits mediate kainate receptor responses in hippocampal neurons cultured 6–8 DIV.

Q/R editing of the rat GluR5 and GluR6 kainate receptors in vivo and in vitro: evidence for independent developmental, pathological and cellular regulation.

Kainate (KA) is a potent neuroexcitatory agent in several areas of the adult brain, with convulsant and excitotoxic properties that increase as ontogeny proceeds. Besides its depolarizing actions, KA may enhance intracellular accumulation of Ca2+ to promote selective neuronal damage. The effects of KA are mediated by specific receptors recently considered to be involved in fast neurotransmission and that can be activated synaptically. KA receptors, e.g. GluR5 and GluR6 have been characterized by molecular cloning. Structure-function relationships indicate that in the MII domain of these KA receptors, a glutamine (Q) or arginine (R) residue determines ion selectivity. The arginine stems from post-transcriptional editing of the GluR5 and GluR6 pre-RNAs, and the unedited and edited versions of GluR6 elicit distinct Ca2+ permeability. Using a PCR-based approach, we show that in vivo, Q/R editing in the GluR5 and GluR6 mRNAs is modulated during ontogeny and differs substantially in a variety of nervous tissues. GluR5 editing is highest in peripheral nervous tissue, e.g. the dorsal root ganglia, where GluR6 expression is barely detectable. In contrast, GluR6 editing is maximal in forebrain and cerebellar structures where GluR5 editing is lower. Intra-amygdaloid injections of KA provide a model of temporal lobe epilepsy, and we show that following seizures, the extent of GluR5 and GluR6 editing is altered in the hippocampus. However, in vitro, high levels of glutamate and potassium-induced depolarizations have no effect on GluR5 and GluR6 Q/R editing. GluR6 editing is rapidly enhanced to maximal levels in primary cultures of cerebellar granule neurons but not in cultured hippocampal pyramidal neurons. Finally, we show that cultured glial cells express partially edited GluR6 mRNAs. Our results indicate that Q/R editing of GluR5 and GluR6 mRNAs is structure-, cell type- and time-dependent, and suggest that editing of these mRNAs is not co-regulated.

Lectin-Induced Inhibition of Desensitization of the Kainate Receptor GluR6 Depends on the Activation State and Can Be Mediated by a Single Native or Ectopic N-Linked Carbohydrate Side Chain

The ionotropic glutamate receptor GluR6 exhibits strongly and rapidly desensitizing current responses. Treatment of heterologically expressed GluR6 with the lectin concanavalin A (ConA) in Xenopus oocytes as well as in human embryonic kidney-293 cells results in a considerable increase of the steady-state current, presumably by inhibiting receptor desensitization. In the present study, we investigated the molecular basis of this effect using a systematic mutagenesis approach. We found that although N-glycosylation is an absolute prerequisite for the lectin-mediated inhibition of desensitization, no single one of the nine extracellular consensus sites for N-glycosylation of GluR6 is required. Rather, each of the nine N-linked carbohydrate side chains is independently capable of modulatory interaction with the lectin. Moreover, even artificially introduced N-glycosylation sites can substitute for native sites. Thus, the specific site of the lectin binding does not appear to be important for its desensitization-inhibiting action. Furthermore, we show that the extent of the receptor's ConA sensitivity depends on its state of activation, because the desensitized GluR6 exhibits significantly lower lectin sensitivity than the nondesensitized receptor. We conclude that binding of ConA "locks" the receptor in the activatable state, thereby inhibiting conformational changes required to shift the receptor to the desensitized state.

Heterogeneity of homomeric GluR5 kainate receptor desensitization expressed in HEK293 cells.

1. Kainate receptors with pharmacological properties similar to those of the GluR5 subunit have been shown to modulate inhibitory synaptic transmission in the CA1 region of the hippocampus. The kinetic properties of currents gated by GluR5 receptors have not been examined in detail. Here we describe several biophysical features of recombinant GluR5 receptors expressed in HEK293 cells. 2. We found that homomeric GluR5 receptors can exhibit striking inter-cell variability in channel kinetics in response to the agonists kainate and glutamate. Desensitization rates in response to kainate varied between individual cells by nearly 1000-fold (range, 1.5 ms to 1.5 s), while glutamate desensitization rates differed by 9-fold (range, 1.0 to 9.0 ms). 3. The time course of recovery from desensitization in response to glutamate also showed inter-cell variation. The majority of glutamate currents in GluR5-expressing cells recovered from desensitization with two widely separated exponential components: 50 +/- 10 ms and 5.1 +/- 1.0 s (contributing 37.6 % and 62.4 % of the sum of the exponential fits, respectively). In contrast, currents with the fastest desensitization kinetics had a recovery time course of 4.8 +/- 0.3 s. 4. Kainate receptors in murine dorsal root ganglion neurons are likely to be composed of homomeric GluR5 subunits. These receptor currents recovered from glutamate desensitization with a biexponential time course of 36 +/- 4 ms and 4.7 +/- 0.7 s. 5. These results suggest that aspects of GluR5 kainate receptor function are modulated by intracellular mechanism(s). At synapses such mechanisms could regulate the frequency- response relationship of synaptic kainate receptors by altering their rate of entry into and recovery from desensitization.

LY339434, a GluR5 kainate receptor agonist

The activity of a γ-substituted glutamate analogue, (2 S, 4 R, 6E)-2-amino-4-carboxy-7-(2-naphthyl)hept-6-enoic acid (LY339434) and (2 S,4 R)-4-methylglutamic acid at ionotropic glutamate receptors has been examined. Ligand binding studies were performed using [ 3H] AMPA binding to membranes expressing either homomeric recombinant GluR1, GluR2, GluR4 receptors, and [ 3H] kainate binding to GluR5 and GluR6 kainate receptors. LY339434 and (2 S,4 R)-4-methylglutamic acid showed selectivity in ligand binding studies for kainate receptors over AMPA receptors. Within the kainate class of glutamate receptors, LY339434 showed selectivity for GluR5 over GluR6 whereas (2 S,4 R)-4-methylglutamic acid showed high affinity for both GluR5 and GluR6 kainate receptors. Examination of the functional activity of LY339434 and (2 S,4 R)-4-methylglutamic acid showed that both compounds evoked inward currents in dorsal root ganglion neurons (DRG) with estimated EC 50 values of 0.8±0.2 μM and 0.17±0.04 μM, respectively. In GluR5 expressing HEK 293 cells, LY339434 evoked inward currents with an estimated EC 50 value of 2.5±0.9 μM but had little effect on GluR6 expressing cells at concentrations less than 100 μM. LY339434 was a weak AMPA receptor agonist ( EC 50 values>300 μM) as determined by activity in acutely isolated cerebellar Purkinje neurons. LY339434 and (2 S,4 R)-4-methylglutamic acid had agonist activity at NMDA receptors studied in cultured hippocampal neurons with EC 50s of 2.5 μM and 11.7 μM, respectively. These results indicate that both LY339434 and (2 S,4 R)-4-methyl glutamic acid may be useful pharmacological tools for the examination of kainate receptors.

GluR5 kainate receptor activation in interneurons increases tonic inhibition of pyramidal cells.

We studied the modulation of GABAergic inhibition by glutamate and kainate acting on GluR5-containing kainate receptors in the CA1 hippocampal region. Glutamate, kainate or ATPA, a selective agonist of GluR5-containing receptors, generates an inward current in inhibitory interneurons and cause repetitive action potential firing. This results in a massive increase of tonic GABAergic inhibition in the somata and apical dendrites of pyramidal neurons. These effects are prevented by the GluR5 antagonist LY 293558. Electrical stimulation of excitatory afferents generates kainate receptor-mediated excitatory postsynaptic currents (EPSCs) and action potentials in identified interneurons that project to the dendrites and somata of pyramidal neurons. Therefore glutamate acting on kainate receptors containing the GluR5 subunit may provide a protective mechanism against hyperexcitability.

GluR5 kainate receptor mediated synaptic transmission in rat basolateral amygdala in vitro

A non-NMDA and non-AMPA receptor mediated excitatory synaptic response was identified in intracellularly recorded basolateral amygdala (BLA) neurons in an in vitro slice preparation. Synaptic potentials were evoked by stimulation of either the external capsule (EC) or basal amygdala (BA). NMDA and GABA A receptors were blocked by inclusion of 100 μM (±)-2-amino-5-phosphonopentanoic acid and 10 μM bicuculline in the perfusion solution. The AMPA receptor-selective allosteric antagonists GYKI 52466 (50 μM) and GYKI 53655 (50 μM) partially suppressed depolarizing synaptic responses evoked by single shock EC stimulation, but fully blocked synaptic responses evoked by BA stimulation. In recordings carried out in the presence of the AMPA receptor antagonists, EC stimulation with pulse trains (5–8 pulses at 50–100 Hz) evoked a large increase in the amplitude of synaptic responses. The AMPA receptor-independent component of the train-induced synaptic response had a null potential near 0 mV. Such AMPA receptor-independent, train-evoked synaptic responses were largely blocked by the AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (20 μM; 85±4%). In addition, the responses were blocked by the GluR5-selective kainate receptor antagonist LY293558 (10 μM; 95±2%). These results indicate that a component of the EC (but not the BA) synaptic response is mediated by kainate receptors containing the GluR5 subunit.

Antagonism of neuronal kainate receptors by lanthanum and gadolinium

The effects of lanthanum and gadolinium on currents evoked by excitatory amino acids were studied in cultured rat hippocampal and cortical neurons, in freshly dissociated dorsal root ganglion neurons, and in human embryonic kidney 293 cells expressing the GluR6 kainate receptor subunit. In all of these cells, currents mediated by kainate-preferring receptors were antagonized by low micromolar concentrations of the trivalent ions. At negative holding potentials, the IC 50 values for inhibition in DRG cells were 2.8 μM for La and 2.3 μM for Gd. Kainate receptor-mediated currents in hippocampal neurons and in 293 cells expressing GluR6 were blocked by La with IC 50 values of 2.1 and 4.4 μM, respectively. Steady-state inhibition by the lanthanides showed very slight dependence on membrane potential, however, we were not able to resolve any systematic variation with membrane potential in the kinetics of block onset or recovery. Inhibition was not use-dependent and was not overcome by increasing the concentration of agonist. These results indicate that lanthanides probably do not bind deep within the ion pore or directly compete for the agonist binding site. In contrast to neuronal AMPA receptors, which require more than 100 μM lanthanides for half-maximal blockade, the inhibition of neuronal and recombinant kainate receptors by these ions displays significantly higher potency.

Acute Effects of Ethanol on Recombinant Kainate Receptors: Lack of Role of Protein Phosphorylation

This study examined the acute actions of ethanol on recombinant rat GluR6 kainate receptors expressed in Xenopus oocytes and HEK 293 cells. Electrophysiological recordings showed that co-application of ethanol with submaximal kainate concentrations resulted in similar inhibition of kainate-gated currents in both expression systems. Manipulation of intracellular phosphorylation pathways by intracellular dialysis with a solution without ATP and GTP did not modify the inhibitory effects of ethanol. Moreover, co-transfection of GluR6 receptor subunits with PKA-alpha catalytic subunit or the calcium/calmodulin-dependent protein kinase II (CamKII) catalytic fragment did not change the sensitivity of the receptor to ethanol. Treatment of Xenopus oocytes with specific inhibitors of PKC, PKA, CamKII, tyrosine kinases, and serine-threonine protein phosphatases did not affect the 100 mM ethanol-induced inhibition of GluR6 receptor-mediated currents. Biochemical experiments with transiently transfected HEK 293 cells confirmed published reports that GluR6 receptors are minimally phosphorylated under basal conditions in these cells and also revealed that acute ethanol did not increase GluR6 phosphorylation. These results suggest that, under our experimental conditions, ethanol inhibits recombinant GluR6 receptor function by a direct effect on the receptor rather than an indirect action via protein phosphorylation.

Identification of Amino Acid Residues that Control Functional Behavior in GluR5 and GluR6 Kainate Receptors

GluR5 and GluR6 kainate receptors differ in their responses to a variety of agonists, despite their relatively high primary sequence homology. We carried out a structure–function study to identify amino acids underlying these divergent responses. Patch clamp analysis of chimeric GluR5-GluR6 receptors indicated that several functionally dominant sites were localized to the C-terminal side of M1. All nonconserved amino acids in the region between M3 and M4 of GluR6 were then individually mutated to their GluR5 counterparts. We found that a single amino acid (N721 in GluR6) controls both AMPA sensitivity and domoate deactivation rates. Additionally, mutation of A689 in GluR6 slowed kainate desensitization. These functional effects were accompanied by alterations in binding affinities. These results support a critical role for these residues in receptor binding and gating activity.

A hippocampal GluR5 kainate receptor regulating inhibitory synaptic transmission.

The principal excitatory neurotransmitter in the vertebrate central nervous system, L-glutamate, acts on three classes of ionotripic glutamate receptors, named after the agonists AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxalole-4-propionic acid), NMDA (N-methyl-D-aspartate) and kainate. The development of selective pharmacological agents has led to a detailed understanding of the physiological and pathological roles of AMPA and NMDA receptors. In contrast, the lack of selective kainate receptor ligands has greatly hindered progress in understanding the roles of kainate receptors. Here we describe the effects of a potent and selective agonist, ATPA ((RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid) and a selective antagonist, LY294486 ((3SR, 4aRS, 6SR, 8aRS)-6-((((1H-tetrazol-5-yl) methyl)oxy)methyl)-1, 2, 3, 4, 4a, 5, 6, 7, 8, 8a-decahydroisoquinoline-3-carboxylic acid), of the GluR5 subtype of kainate receptor. We have used these agents to show that kainate receptors, comprised of or containing GluR5 subunits, regulate synaptic inhibition in the hippocampus, an action that could contribute to the epileptogenic effects of kainate.

Identification of kainate receptor-mediated intracellular calcium increases in cultured rat cerebellar granule cells.

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

Allelic association of juvenile absence epilepsy with a GluR5 kainate receptor gene (GRIK1) polymorphism

Allelic association of juvenile absence epilepsy with a GluR5 kainate receptor gene (GRIK1) polymorphism

Allelic association of juvenile absence epilepsy with a GluR5 kainate receptor gene (GRIK1) polymorphism

Juvenile absence epilepsy (JAE) is a common subtype of idiopathic generalized epilepsy (IGE). Hereditary factors play a major role in its etiology. The important function of glutamate receptors (GluRs) in excitatory neurotransmission, synaptic plasticity, and neurodevelopment suggests their involvement in epileptogenesis. A tetranucleotide repeat polymorphism in the non-coding region of the kainate-selective GluR5 receptor gene (GRIK1) on chromosome 21q22.1 provides the tool to investigate this candidate gene. The present association and linkage study tested the hypothesis that allelic variants of GRIK1 confer genetic susceptibility to the pathogenesis of JAE. Our family-based association analysis using the haplotype-based haplotype relative risk statistic revealed an association of JAE with the nine-repeat containing allele of the GRIK1 tetranucleotide polymorphism (chi2 = 8.31, df = 1, P = 0.004). Supportive evidence for linkage to a JAE related IGE spectrum (Zmax = 1.67 at GRIK1) under an autosomal dominant mode of inheritance and significant allele sharing (P < 0.05) among the affected family members suggest that allelic variants of GRIK1 contribute a major genetic determinant to the pathogenesis of JAE-related phenotypes.

Desensitization of Kainate Receptors by Kainate, Glutamate and Diastereomers of 4-Methylglutamate

The potencies of kainate, glutamate and diastereomers of 4-methylglutamate were determined for activation and steady-state desensitization of GluR6 and dorsal root ganglion-type kainate receptors using whole-cell voltage clamp. In HEK293 cells expressing GluR6, all four diastereomers induced desensitizing inward currents at relatively high concentrations (>50 μM), however, the 2S,4R diastereomer (2S,4R-4MG; SYM 2081) was approximately 100-fold more potent than the other three. The ec 50 for receptor activation by 2S,4R-4MG (1.0 μM) was similar to that for kainic acid (1.8 μM), but 2S,4R-4MG was significantly more potent than kainate, glutamate or the other diastereomers of 4-methylglutamate at producing steady-state desensitization of GluR6 receptors. ic 50 s for desensitization quantified using a fixed concentration of kainate as a test agonist were 7.6, 31 and 667 nM for 2S,4R-4MG, kainate and glutamate, respectively. In addition, 2S,4R-4MG fully desensitized native kainate receptors (of the GluR5 subtype) in dorsal root ganglion neurons with an ic 50 of 11 nM, compared to 3.4 μM for glutamate. For GluR6, recovery from desensitization displayed a similar time course for kainate and glutamate (τ = 3–4 s) but was roughly 20-fold slower for 2S,4R-4MG, which suggests that the rate of recovery is not entirely dependent on the affinity of ligand for the desensitized receptor. Following exposure to concanavalin A, application of kainate, glutamate and 2S,4R-4MG evoked very similar maximal currents that showed little or no desensitization. Lectin pretreatment produced a leftward shift in the concentration-response relationship for 2S,4R-4MG with an 11-fold reduction in the ec 50; however, no significant change in the ec 50 for kainate was observed. The characteristic of 2S,4R-4MG to potently and completely desensitize both recombinant GluR6 receptors and native receptors on dorsal root ganglion neurons suggests that this compound will be useful to study selective blockade of these receptors in the nervous system. © 1997 Elsevier Science Ltd.

Genotypes at the GluR6 kainate receptor locus are associated with variation in the age of onset of Huntington disease.

Huntington disease (HD) is associated with abnormal expansions of a CAG repeat close to the 5' end of the IT15 gene. We have assembled a set of 293 HD subjects whose ages of onset were known and sized their HD CAG repeats. These repeats accounted for 69% of the variance of age of onset when we used the most parsimonious model, which relates the logarithm of age of onset to a function of CAG repeat number. Since other familial factors have been proposed to influence the age of onset of HD, we have examined a number of candidate loci. The CAG repeat number on normal chromosomes, the delta2642 polymorphism in the HD gene, and apolipoprotein E genotypes did not affect the age of onset of HD. Although mitochondrial energy production defects in HD have led to suggestions that variants in the mitochondrial genome may be associated with clinical variability in HD, this suggestion was not supported by our preliminary experiments that examined the DdeI mitochondrial restriction fragment length polymorphism at position 10,394. Excitotoxicity has been a favored mechanism to explain the cell death in HD, particularly since intrastriatal injection of excitatory amino acids in animals creates HD-like pathology. Accordingly, we investigated the GluR6 kainate receptor. Of the variance in the age of onset of HD that was not accounted for by the CAG repeats, 13% could be attributed to GluR6 genotype variation. These data implicate GluR6-mediated excitotoxicity in the pathogenesis of HD and highlight the potential importance of this process in other polyglutamine repeat expansion diseases.

P-10-28 - Anticonvulsant and anxiolytic effects of LY300164, an orally effective AMPA antagonist

The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) has been implicated in the processing of nociceptive information from the orofacial region. Kainate receptors (KARs) play an important role in sensory transmission. Five different KAR subunits have been cloned and the expression of the KAR subunits showed developmental changes. In this study, RT-PCR, western blotting, immunohistochemistry and a patch clamp technique were used examine the functional expression of the GluR5 subunit in the SG of the Vc in juvenile, peripubertal and/or adult mice. The levels of mRNA and protein expression of the GluR5 subunit in the SG of the Vc were higher in the juvenile mice than in the peripubertal or adult mice. In addition, the KA and ATPA, a GluR5 KAR agonist, induced membrane depolarization on the SG neurons in both juvenile and adult mice in a concentration-dependent manner. However, the juvenile SG neurons showed a stronger response to KA and ATPA than those of adults. The membrane depolarization by KA was suppressed slightly in the presence of the AMPA receptor antagonist, GYKI 52466. These results show that the GluR5 KAR subunits are expressed functionally on the SG neurons of the Vc in mice, and the expression levels of the GluR5 subunits decrease with postnatal development. These postnatal changes in the GluR5 KAR subunit may be a possible mechanism for age-dependent pain processing.

Refinement of map position of the human GluR6 kainate receptor gene (GRIK2) and lack of association and linkage with idiopathic generalized epilepsies

Hereditary factors play a major role in the etiology of idiopathic generalized epilepsies (IGEs). The pivotal function of glutamate receptors (GluRs) in excitatory neurotransmission implicates their involvement in epileptogenesis and genetic susceptibility to IGEs. A trinucleotide repeat polymorphism detected in the 3' untranslated region of the kainate-selective GluR6 receptor gene (GRIK2) on chromosome 6 makes it possible to perform linkage and association studies with this high-ranking candidate gene. The present study tested the hypothesis that allelic variants of GRIK2 contribute to the genetic susceptibility to the common IGEs. Linkage and association analyses were conducted in 63 families ascertained through IGE patients with juvenile myoclonic epilepsy, juvenile absence epilepsy, or childhood absence epilepsy. Our linkage and association results suggest that allelic variants of GRIK2 are not involved in the expression of the common familial IGEs, and radiation hybrid mapping assigns GRIK2 to the chromosomal region 6q16.3-q21. This localization excludes GRIK2 as a candidate for the putative IGE susceptibility locus "EJM1" on the short arm of chromosome 6.