Heterogeneity of homomeric GluR5 kainate receptor desensitization expressed in HEK293 cells

Swanson, Geoffrey T.; Heinemann, Stephen F.

doi:10.1111/j.1469-7793.1998.639ba.x

Cited by 52 publications

(49 citation statements)

References 24 publications

(39 reference statements)

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“…8B). Desensitization in the wild type receptor was highly variable (mean des ϭ 5.2 Ϯ 1.0 ms, n ϭ 8), as was seen previously for GluR5-2a receptors expressed in HEK cells (20). In contrast, currents from the mutants were rapidly desensitizing, with the triple mutant GluR5-2b(R896AϩRK/AA) having the fastest des (2.05 Ϯ 0.26, n ϭ 11, p Ͻ 0.05 compared with FIG.…”

Section: Fig 5 Mapping Additional Sequence Determinants For Er Retementioning

confidence: 58%

Cell Surface Expression of GluR5 Kainate Receptors Is Regulated by an Endoplasmic Reticulum Retention Signal

Ren¹,

Riley²,

Needleman

et al. 2003

Journal of Biological Chemistry

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Kainate receptors (KARs) are mediators of excitatory neurotransmission in the mammalian central nervous system, and their efficient targeting and trafficking is critical for normal synaptic function. A key step in the delivery of KARs to the neuronal plasma membrane is the exit of newly assembled receptors from the endoplasmic reticulum (ER). Here we report the identification of a novel ER retention signal in the alternatively spliced C-terminal domain of the GluR5-2b subunit, which controls receptor trafficking in both heterologous cells and neurons. The ER retention motif consists of a critical arginine (Arg-896) and surrounding amino acids, disruption of which promotes ER exit and surface expression of the receptors, as well as altering their physiological properties. The Arg-896-mediated ER retention of GluR5 is regulated by a mutation that mimics phosphorylation of Thr-898, but not by PDZ interactions. Furthermore, two positively charged residues (Arg-900 and Lys-901) in the C terminus were also found to regulate ER export of the receptors. Taken together, our results identify novel trafficking signals in the Cterminal domain of GluR5-2b and demonstrate that alternative splicing is an important mechanism regulating KAR function.Kainate, NMDA, 1 and AMPA receptors mediate glutamatergic excitatory neurotransmission in the mammalian central nervous system (1, 2). The intracellular trafficking and surface delivery of multimeric transmembrane proteins, such as kainate receptors, are likely to be tightly controlled processes requiring proper folding and assembly of constituent subunits, so that only fully assembled, functional receptors can be expressed on the plasma membrane. The endoplasmic reticulum (ER) is the primary checkpoint for these complex events (3). Recently, we demonstrated that an arginine-rich ER retention signal controls the ER egress and surface expression of KA2 kainate receptor subunits (4).Alternative splicing and RNA editing of ionotropic glutamate receptors have been shown to play important roles in receptor assembly and trafficking (5-8). In particular, an RXR (where R is arginine and X a large neutral or positively charged residue) motif was found in the alternatively spliced C1 cassette in the C terminus of NMDA receptor NR1 subunit, mediating the intracellular retention of most C1-containing NR1 isoforms (6, 7). Furthermore, this RXR-mediated ER retention was regulated by protein kinase C (PKC) phosphorylation (6) and a type I PDZ (postsynaptic density-95/Disc large/zona occludens-1)-binding motif in the alternate C2Ј splice cassette (6, 7). In addition, arginine 607 (Arg-607) at a Q/R (glutamine/arginine) mRNA editing site has been reported to determine the ER retention of GluR2 AMPA receptor subunits in cultured neurons (8).The GluR5 kainate receptor subunit also undergoes alternative splicing and RNA editing. Alternative splicing of the Nterminal region of the rat GluR5 mRNA yields two different isoforms, GluR5-1 and GluR5-2, where GluR5-1 contains an extra 15 amino acids compared wi...

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Section: Fig 5 Mapping Additional Sequence Determinants For Er Retementioning

confidence: 58%

Cell Surface Expression of GluR5 Kainate Receptors Is Regulated by an Endoplasmic Reticulum Retention Signal

Ren¹,

Riley²,

Needleman

et al. 2003

Journal of Biological Chemistry

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“…The significance of these changes in desensitization rates is unclear but generally suggests that interactions with other proteins or modification of residues in the C terminal influence kinetic behavior of kainate receptors, as has been shown previously (Traynelis and Wahl, 1997;Garcia et al, 1998;Swanson and Heinemann, 1998).…”

Section: Resultsmentioning

confidence: 99%

A C-Terminal Determinant of GluR6 Kainate Receptor Trafficking

Yan¹,

Sanders²,

Xu³

et al. 2004

J. Neurosci.

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Intracellular trafficking of ionotropic glutamate receptors is regulated predominantly by determinants in the cytoplasmic C-terminal domain of the subunit proteins. Although AMPA receptors are found at the vast majority of excitatory synapses, synaptic kainate receptors exhibit a much more restricted distribution, suggesting that specific mechanisms exist for selective trafficking of these receptor proteins. In this report, we define a critical forward trafficking motif that is necessary for surface expression of the glutamate receptor 6 (GluR6) kainate receptor as well as chimeric proteins containing only the GluR6 C-terminal domain. The trafficking determinant was identified by tracking surface expression of green fluorescent protein-tagged GluR6 receptors with confocal immunofluorescence in COS-7 cells and cultured neurons and patch-clamp electrophysiology in human embryonic kidney 293 cells. Serial truncation and alanine site mutagenesis of the GluR6 subunit C terminus localized the critical motif to a seven amino acid stretch of predominantly basic residues. Alanine mutation of the trafficking motif reduced kainate receptor current amplitudes by Ͼ90% and resulted in retention of the mutated receptors in the endoplasmic reticulum. This forward trafficking domain is the first such identified for kainate receptors.

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“…Previous studies also showed that CTZ enhanced AMPAinduced [Ca 2ϩ ] i responses with great variability (Hoyt et al, 1995;Sekiguchi et al, 1998;Ambrósio et al, 2000). Moreover, it is well established that the desensitization kinetics of kainate receptors depends of the subunit composition (Swanson et al, 1998b), and that brief exposure to the lectin Con A eliminates desensitization of current gated by kainate and domoate. Using the more selective agonist to kainate receptors (MGA) (Jones et al, 1997;Donevan et al, 1998), we observed that, in the presence of Con A, to eliminate receptor desensitization, MGA did mediate the [Ca 2ϩ ] i increase.…”

Section: Discussionmentioning

confidence: 99%

Role of kainate receptor activation and desensitization on the [Ca²⁺]_i changes in cultured rat hippocampal neurons

Silva¹,

Malva²,

Ambrósio³

et al. 2001

J of Neuroscience Research

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We investigated the role of kainate (KA) receptor activation and desensitization in inducing the increase in the intracellular free Ca 2ϩ concentration ([Ca 2ϩ ] i ) in individual cultured rat hippocampal neurons. The rat hippocampal neurons in the cultures were shown to express kainate receptor subunits, KA2 and GluR6/7, either by immunocytochemistry or by immunoblot analysis. The effect of LY303070, an ␣-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptor antagonist, on the alterations in the [Ca 2ϩ ] i caused by kainate showed cell-tocell variability. The [Ca 2ϩ ] i increase caused by kainate was mostly mediated by the activation of AMPA receptors because LY303070 inhibited the response to kainate in a high percentage of neurons. The response to kainate was potentiated by concanavalin A (Con A), which inhibits kainate receptor desensitization, in 82.1% of the neurons, and this potentiation was not reversed by LY303070 in about 38% of the neurons. Also, upon stimulation of the cells with 4-methylglutamate (MGA), a selective kainate receptor agonist, in the presence of Con A, it was possible to observe [Ca 2ϩ ] i changes induced by kainate receptor activation, because LY303070 did not inhibit the response in all neurons analyzed. In toxicity studies, cultured rat hippocampal neurons were exposed to the drugs for 30 min, and the cell viability was evaluated at 24 hr using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The selective activation of kainate receptors with MGA, in the presence of Con A, induced a toxic effect, which was not prevented by LY303070, revealing a contribution of a small subpopulation of neurons expressing kainate receptors that independently mediate cytotoxicity. Taken together, these results indicate that cultured hippocampal neurons express not only AMPA receptors, but also kainate receptors, which can modulate the [Ca 2ϩ ] i and toxicity. J. Neurosci. Res. 65:378 -386, 2001.

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Heterogeneity of homomeric GluR5 kainate receptor desensitization expressed in HEK293 cells

Cited by 52 publications

References 24 publications

Cell Surface Expression of GluR5 Kainate Receptors Is Regulated by an Endoplasmic Reticulum Retention Signal

Cell Surface Expression of GluR5 Kainate Receptors Is Regulated by an Endoplasmic Reticulum Retention Signal

A C-Terminal Determinant of GluR6 Kainate Receptor Trafficking

Role of kainate receptor activation and desensitization on the [Ca²⁺]_i changes in cultured rat hippocampal neurons

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Heterogeneity of homomeric GluR5 kainate receptor desensitization expressed in HEK293 cells

Cited by 52 publications

References 24 publications

Cell Surface Expression of GluR5 Kainate Receptors Is Regulated by an Endoplasmic Reticulum Retention Signal

Cell Surface Expression of GluR5 Kainate Receptors Is Regulated by an Endoplasmic Reticulum Retention Signal

A C-Terminal Determinant of GluR6 Kainate Receptor Trafficking

Role of kainate receptor activation and desensitization on the [Ca2+]i changes in cultured rat hippocampal neurons

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Role of kainate receptor activation and desensitization on the [Ca²⁺]_i changes in cultured rat hippocampal neurons