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Originally published In Press as doi:10.1074/jbc.M413166200 on April 1, 2005

J. Biol. Chem., Vol. 280, Issue 24, 22968-22976, June 17, 2005
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Differential Trafficking of GluR7 Kainate Receptor Subunit Splice Variants*{boxs}

Frédéric Jaskolski, Elisabeth Normand, Christophe Mulle{ddagger}, and Françoise Coussen

From the Laboratoire Physiologie Cellulaire de la Synapse, CNRS Unité Mixte de Recherche 5091, Institut François Magendie, Université Bordeaux 2, Rue C. Saint-Saëns, 33077 Bordeaux Cedex, France

Received for publication, November 22, 2004 , and in revised form, March 9, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Kainate receptors (KARs) are heteromeric ionotropic glutamate receptors that play a variety of roles in the regulation of synaptic network activity. The function of glutamate receptors (GluRs) is highly dependent on their surface density in specific neuronal domains. Alternative splicing is known to regulate surface expression of GluR5 and GluR6 subunits. The KAR subunit GluR7 exists under different splice variant isoforms in the C-terminal domain (GluR7a and GluR7b). Here we have studied the trafficking of GluR7 splice variants in cultured hippocampal neurons from wild-type and KAR mutant mice. We have found that alternative splicing regulates surface expression of GluR7-containing KARs. GluR7a and GluR7b differentially traffic from the ER to the plasma membrane. GluR7a is highly expressed at the plasma membrane, and its trafficking is dependent on a stretch of positively charged amino acids also found in GluR6a. In contrast, GluR7b is detected at the plasma membrane at a low level and retained mostly in the endoplasmic reticulum (ER). The RXR motif of GluR7b does not act as an ER retention motif, at variance with other receptors and ion channels, but might be involved during the assembly process. Like GluR6a, GluR7a promotes surface expression of ER-retained subunit splice variants when assembled in heteromeric KARs. However, our results also suggest that this positive regulation of KAR trafficking is limited by the ability of different combinations of subunits to form heteromeric receptor assemblies. These data further define the complex rules that govern membrane delivery and subcellular distribution of KARs.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ionotropic glutamatergic synaptic transmission is mediated by the N-methyl-D-aspartate, AMPA,1 and kainate-type glutamate receptors (KARs). KARs play a variety of functions in synaptic transmission and neuronal excitability (1–3). At a presynaptic level, KARs modulate transmitter release, thus inducing and regulating short and long term synaptic plasticity. At a post-synaptic level, KARs contribute to the post-synaptic response at some synapses and regulate neuronal excitability by the inhibition of K+ channels. These distinct roles of KARs are likely mediated by KARs with distinct subcellular localization and possibly different biophysical properties. KARs are hetero-oligomeric receptor channels composed of the subunits GluR5, GluR6, GluR7, KA1, and KA2 (4, 5). A diversity of native KARs with distinct heteromeric combinations thus likely exists, although the subunit composition and stoichiometry of KARs are not known in detail. Hitherto we do not understand how subunit composition relates to the different functional roles that KARs play (1–3).

The biogenesis and intracellular trafficking of heteromeric receptor channels such as KARs are likely to be tightly controlled processes requiring proper folding and assembly, with the ER as the primary checkpoint of these complex events (6). The C-terminal domain of ionotropic glutamate receptors contains critical determinants of receptor trafficking and is subject to alternative splicing in most glutamate receptors. Complex interactions of C-terminal domains with proteins that include PDZ proteins are important for the regulated trafficking of AMPA and N-methyl-D-aspartate receptors, especially during synaptic plasticity (7, 8). The GluR5, GluR6, and GluR7 KAR subunits exist under several splice variants isoforms (GluR5a, GluR5b, GluR5c, GluR6a, GluR6b, GluR7a, and GluR7b) that differ in the sequence of their cytoplasmic C-terminal domains. The mechanisms that lead to the differential membrane delivery of KAR subunits and their splice variants have recently been shown to depend on ER retention motifs and forward trafficking signals (9–13). An RXR motif functions as an ER retention signal in GluR5c and KA2, preventing surface expression of these receptors when expressed as homomers (9, 14). A stretch of positively charged amino acids in GluR5b (QRRTQRK) acts as a novel ER retention motif when coupled to a single transmembrane reporter protein (11), although this ER retention signal might be masked in fully assembled homomeric GluR5b receptors. In contrast, GluR6a is highly expressed at the cell surface and promotes membrane delivery of splice variants normally retained in the ER due to the presence of a forward trafficking signal containing a cysteine residue followed by a cluster of positively charged amino acids (9, 12). Thus, several molecular determinants account for the differential surface expression of GluR5 and GluR6 splice variants.

In the present study we investigated the intracellular trafficking and surface expression of the GluR7 splice variants. Although GluR7 is expressed at high levels in various neuronal populations, little is known about its functional properties and its role in glutamatergic synaptic transmission. GluR6 and GluR7 subunits are 86% identical at the amino acid level (15) but display distinct electrophysiological properties. Recombinant GluR7 receptors expressed in HEK 293 cells form functional ion channels that only respond to very high concentrations of glutamate (16). Domains involved in the functional differences between GluR6 and GluR7 have been identified in the L3 extracellular loop (17). GluR7 subunits can also assemble with GluR5, GluR6, and KA2 to form heteromeric channels (13, 16, 18). The two isoforms GluR7a and GluR7b contain distinct C-terminal ends of 64 and 55 amino acids, respectively, and both make functional homomeric channels (16). The alternatively spliced C-terminal domain of GluR7a contains a motif identical to the export signal in GluR6a, whereas GluR7b displays an RXR ER retention motif. We have explored the surface expression and subcellular distribution of the two GluR7 splice variants expressed in cultured hippocampal neurons and analyzed the molecular determinants for their differential trafficking. We have also examined the effects of the co-assembly of GluR6 splice variants on the trafficking of GluR7 isoforms. Our results demonstrate the functional significance of GluR7 alternative splicing.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
RT-PCR Experiments—RNA was prepared from adult C57-BL/6 mice tissues using the Micro-Fast TrackTM protocol (Invitrogen) with 50–100 mg of starting material. The RT step was performed on purified mRNAs (oligo(dT)-cellulose) with a specific reverse primer for the GluR7 subunit, 5'-GCTGGCTATGGAGATGGTCGGTG-3'. RT products were amplified in a first PCR round using the splice variant specific primer pairs 5'-CGGCTCTGAGGTGGTGGAGAATAAC-3' (GluR7a and GluR7b forward), 5'-GCTGGCTATGGAGATGGTCGGTG-3' (GluR7a reverse), and 5'-CTTCCTTCCCTGTCCAACGC-3' (GluR7b reverse). Next, the first round PCR product was amplified in a second PCR round using a second set of splice variant specific primer pairs, 5'-CCCTCGGCTCTGGTGAAGAACAATG-3' (GluR7a and GluR7b forward) (2), 5'-GCTGTGGCTTGTGCTTGAGACGC-3' (GluR7a reverse) (2), and 5'-GCCTCCTCCAGGGCCTCAC-3' (GluR7b reverse) (2). PCR was performed using 30 cycles of 30 s at 94 °C, 30 s at 56 °C, and 45 s at 72 °C. The positive control condition was a two-round PCR experiment on plasmids encoding GluR7a and GluR7b cDNAs.

DNA Constructs—Six consecutive c-Myc epitopes were introduced after the predicted signal peptide of rat GluR7a(Q) cDNA (Swiss-Prot accession number P42264 [GenBank] -1) and subcloned in pcDNA3 vector. The BstXI site, located before the fourth membrane domain of the protein, was mutated in NheI restriction site by PCR, and an AflII restriction site was introduced after the stop codon. Site-directed mutagenesis was performed using QuikChange XL kit (Stratagene).

Hippocampal Cultures—Primary cultures of hippocampal neurons were obtained from 1-day-old pups of C57-BL/6 and GluR5–/–xGluR6–/– mutant mice (44). Hippocampi were dissociated with papain followed by mechanical trituration and plated at 50,000 cells/cm2 in minimum Eagle's medium supplemented with 0.5% D-glucose, 0.1 mg/ml transferrin, 25 µg/ml insulin, 2 mM GlutaMAX (Invitrogen), and 5 µg/ml gentamycin. 2% B-27 (Sigma) and 1 µM cytosine arabinoside were added 3 days after plating.

Transfection—10–14-day-old hippocampal neurons were transfected using the Lipofectamine 2000 transfection reagent (Invitrogen). 3 µgof DNA in 500 µl of minimum Eagle's medium was mixed with 10 µl of Lipofectamine 2000 in 500 µl of minimum Eagle's medium and incubated at room temperature for 15 min. Cultured medium was then replaced by 1 ml of minimum Eagle's medium complemented with the transfection mix. After 1 h of incubation, the transfection medium was replaced with 3 ml of growth medium. Experiments were performed 24 h after transfection.

Glycosylation Assays—Glycosylation assays were performed using transfected COS-7 cells as described (9). Endoglycosidase H (EndoH) and peptide-N-glycosidase F (PNG-F) were added to protein samples for 12 h at 37 °C.

Immunostaining—To perform double staining for surface expression experiments, 10 µg/ml polyclonal anti-Myc antibody (Ab) (catalog number 06-549, Upstate Biotechnology, Lake Placid, NY) was incubated with the cells for 20 min at 15 °C to limit antibody endocytosis. Cells were washed, fixed in paraformaldehyde (4%), and kept in phosphate-buffered saline with 0.3% bovine serum albumin and 0.05% saponin during the steps that require membrane permeabilization. Intracellular epitopes were detected using 0.5 µg/ml monoclonal anti-Myc Ab (clone 9E10, Roche Applied Science) incubated for 1 h at 20 °C. ER staining was performed with polyclonal anti-calreticulin Ab (1:500; catalog number PA3-900, Upstate Biotechnology) for 1 h at 20 °C. Secondary Abs, which were incubated for 1 h at 20 °C, included Alexa 568- and 488-conjugated anti-rabbit antibody IgGs (catalog numbers A11011 and A11001, Molecular Probes) and Alexa 568- and 488-conjugated anti-mouse IgGs (10 µg/ml; catalog numbers A11008 [GenBank] and A11004 [GenBank] , Molecular Probes). For subcellular distribution of KAR subunits, neurons were incubated with monoclonal anti-Myc Ab or polyclonal anti-Myc Ab for 1 h at 20 °C. Neurons were then permeabilized with saponin and incubated with the monoclonal anti-MAP2 Ab (1:500; clone HM-2, Sigma), the polyclonal anti-vesicular glutamate transporter 1 (anti-VGluT1) Ab (1:500) (26), or the monoclonal anti-TAU1 Ab (1:500; MAB3420, Chemicon) for 1 h at 20 °C. Secondary antibodies were incubated as described above.

Immunostaining Data Analysis—Confocal images were acquired on a Leica TCS SP2 microscope. The exposure settings and gain of laser were kept the same for each compared condition corresponding to undersaturated acquisitions. Images in the displayed figures were overexposed for a better demonstration but do not correspond to the analyzed images. The relative surface labeling value was analyzed as follows. Signal detection was performed using the Sobel edge detection method (approximation of the locale derivate by convolutions). Surface and intracellular labeled areas defined by the edge detection were measured (number of pixels, {cong}10 nm2 per pixel). Relative surface labeling was calculated as the surface labeled area divided by the sum of surface labeled area and intracellular labeled area (percentage). 10–20 cells were measured in each condition to calculate the mean relative surface labeling. Colocalization was quantified after signal detection (Sobel method) by correlation calculation in each pixel of paired images as in (24). Correlation images (normalized mean deviation product (nMDP) of the images) displays the pixel map of nMDP calculated for paired images. The Icorr index corresponds to the number of correlated pixels (nMDP > 0) over total number of pixels in the correlation image. The Icorr varies from 0 to 1, where 0.5 corresponds to randomly distributed signals. 10 paired images were measured in each condition so that we could calculate the corresponding mean Icorr. Image analysis was performed using MATLAB software (MathWorks). Statistical significance was tested using two-way analysis of variance followed by a rank sum nonparametric test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Splice Variant Isoforms of GluR7, mRNA Expression, and Membrane Delivery—GluR7a and GluR7b differ in terms of C-terminal cassettes of 64 and 55 residues, respectively, localized 14 amino acids after the end of the fourth membrane domain (Fig. 1A). Probes to detect GluR7 mRNA in previous experiments have not been designed to differentiate between the expression of the two splice variants (15, 19). We first examined whether both splice variants were expressed in selected mouse brain regions with RT-PCR using specific oligonucleotides (Fig. 1B). Both GluR7 subunit splice variants were detected in the mouse nervous system with no apparent regional specificity of expression. To examine whether alternative splicing regulates subcellular distribution and surface expression of GluR7 splice variants, we tagged each splice variant by inserting six successive c-Myc epitopes in the extracellular N-terminal domain, just after the signal peptide. Myc-tagged GluR7a and GluR7b were transfected in cultured hippocampal neurons. Twenty-four hours after transfection, neurons were stained for surface expressed receptors (on living cells) and intracellular receptors (Fig. 1D; neurons from wild type mice). GluR7a and GluR7b were both expressed at the cell surface and distributed in clusters in the cell membrane (Fig. 1D, left column). Qualitatively, the surface staining was weaker for GluR7b than for GluR7a, suggesting differential membrane delivery. To quantify the surface expression of these isoforms, we used an image analysis method adapted from our previous work (9) (see "Materials and Methods"). In neurons from wild type mice, GluR7a was detected at higher levels than GluR7b at the cell surface (Fig. 1E; relative surface labeling (percentage) was 48 ± 2% for GluR7a and 19 ± 3% for GluR7b, p < 0.005, n = 20). We validated our image analysis method by performing biotinylation assays in COS-7 cells transfected with GluR7a and GluR7b that yielded comparative results (see supplemental data available in the on-line version of this article). Cultured neurons derived from wild-type mice express endogenous KAR subunits (20, 21) that may co-assemble with GluR7 and potentially regulate trafficking of the transfected subunits. Cultured hippocampal neurons from GluR5–/–xGluR6–/– mice (22) have allowed the study of transfected GluR5 and GluR6 splice variants in the absence of these endogenous subunits (9). In fact, cultured hippocampal neurons derived from wild-type mice do not express GluR7 mRNA (20, 21). We further verified the absence of the GluR7 protein in cultured hippocampal neurons from GluR5–/–xGluR6–/– mice in Western blots probed with a GluR6/7 antibody that should only detect GluR7a (see supplemental data). We thus used cultured neurons derived from GluR5–/–xGluR6–/–, which are devoid of GluR5, GluR6, and GluR7 subunits, to explore surface expression of GluR7 splice variants. In these cultured neurons, the transfected GluR7a splice variant was still expressed at a high level on the cell surface (Fig. 1E; in GluR5–/–xGluR6–/– neurons the relative surface labeling (percentage) for GluR7a was 45 ± 5%, not significantly different from that of WT). Thus, GluR7a readily traffics to the plasma membrane even in the absence of endogenous KAR subunits such as GluR6a. In contrast, the level of surface-expressed GluR7b was significantly lower than in neurons from WT mice (in GluR5–/–xGluR6–/– neurons the relative surface labeling (percentage) for GluR7b was 9 ± 1%; p < 0.01 for WT versus GluR5–/–xGluR6–/–). This result suggests that GluR7b is poorly expressed at the plasma membrane as a homomer and likely co-assembles with endogenous GluR5 or the GluR6 subunit when expressed in WT neurons.



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  		FIG. 1.
C-terminal isoforms of GluR7 subunit, mRNA expression, and surface expression in neurons. A, schematic representation of GluR7 C-terminal (C-ter) isoforms generated by alternative splicing. N-Ter, N-terminal end. B, RT-PCR products of GluR7 isoforms in ethidium bromide-agarose gels. GluR7a and GluR7b are both detected in the mouse nervous system (the lane marked Control represents a PCR product with corresponding plasmids as positive controls; the lane labeled Blank corresponds to RT-PCR without mRNA). C, ClustalW alignment of GluR7 C-terminal splice variants (the number after the last transmembrane domain, at the end of sequence, is the size in amino acids). The gray box corresponds to homologous regions between isoforms. D, sample images of surface staining (left column) and intracellular staining (right column) of transfected neurons (from WT mice) with epitope-tagged GluR7a and GluR7b. Scale bar, 10 µm. E, relative surface labeling of GluR7a and GluR7b in cultured neurons from WT (black) or GluR5–/–xGluR6–/– mice (gray) (n = 20; **, p < 0.01 for GluR5–/–xGluR6–/– versus WT).

 
GluR7 Isoforms Are Differentially Retained in the ER—The low expression of GluR7b at the plasma membrane of neurons could be caused by retention in the ER, which serves as a quality control checkpoint for multimeric receptors and channels before export to the Golgi and to the plasma membrane (6). The GluR7b splice variant strongly colocalized with calreticulin, an ER resident chaperone protein (23) (Fig. 2A). This colocalization appears in yellow in the merge image and in hot colors in the calculated correlation (nMDP) images (Fig. 2A; see "Materials and Methods") (24). In contrast, GluR7a staining was only partly colocalized with calreticulin in bright puncta, which may correspond to ER exit sites (Fig. 2A, left column, white arrowhead) (25). Using our correlation image analysis, we quantified the level of colocalization between the two markers by calculating for each paired image a correlation index, Icorr (see "Materials and Methods"). The level of colocalization between the ER marker and the two splice variants differed markedly (Fig. 2B) (mean Icorr was 0.58 ± 0.02 for GluR7a and 0.76 ± 0.02 for GluR7b; n = 10, p < 0.005), indicating preferential retention of GluR7b in the ER. To verify that overexpression of recombinant proteins does not saturate cell sorting capacity and lead to retention within the ER, we have transfected DNA mixes comprising various proportions of coding and empty vectors (coding/empty ratios of 2:1, 1:1, 1:2, and 1:4). Under all of these conditions GluR7b strongly colocalized with the ER marker, whereas GluR7a was expressed on the cell surface and partly colocalized with the ER in discrete dots. We also measured colocalization of GluR7 splice variants with the ER marker 4 and 10 days after transfection, because longer expression time leads to degradation of overloaded proteins. In this experiment, although the overall level of recombinant proteins decreases, differences between GluR7a and GluR7b in terms of colocalization with the ER marker are maintained (data not shown).



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  		FIG. 2.
Colocalization of GluR7 isoforms with the ER marker calreticulin. A, transfected cultured hippocampal neurons from GluR5–/–xGluR6–/– mice. Cells were co-stained for total c-Myc epitopes (top row; red in row labeled merge) and for the ER resident protein calreticulin (second row from top; green in the row labeled merge). Bottom row is the correlation image (nMDP; see "Materials and Methods") quantifying local colocalization in hot colors (nMDP > 0, red to white) and non-colocalization in cold colors (nMDP < 0, blue variations). GluR7a partly colocalizes with the ER marker (white arrowheads), whereas GluR7b localization is strongly restricted to calreticulin-positive compartments (hot colored in nMDP images). Scale bar, 2 µm. B, quantification of image correlation (mean Icorr) between GluR7 isoforms and calreticulin staining (n = 10; ***, p < 0.001 for GluR7a versus GluR7b). C, Western blot analysis of protein extracts from transfected COS-7 cells blotted with an anti-Myc antibody. Lane labeled Input is the total protein sample; EndoH and PNG-F correspond to digestion products of the corresponding enzymes, respectively. GluR7a is partly resistant to EndoH treatment, whereas GluR7b is completely sensitive (black arrowheads indicates the predicted molecular mass of the un-glycosylated tagged isoforms).

 
To confirm the differential trafficking of GluR7 isoforms from the ER to the plasma membrane, we next examined the pattern of glycosylation of these subunits. In Western blots from total protein extracts of transfected COS-7 cells, GluR7a and GluR7b were detected at a higher molecular masses than predicted from the amino acid sequence of the tagged isoforms (Fig. 2C, lanes marked Input; black arrowheads), 110 kDa for GluR7a and 109 kDa for GluR7b. These bands likely correspond to glycosylated subunits, because GluR7 carries eight predicted N-glycosylation sites (Swiss-Prot accession number P42264 [GenBank] ). We treated protein extracts with PNG-F, which removes N-glycans. GluR7 proteins treated with PNG-F migrated to the molecular mass predicted for non-glycosylated proteins. Resistance to EndoH was conferred by mannosidase 2 processing of the protein that occurs in the Golgi apparatus. GluR7a was partially resistant to the treatment by EndoH, whereas no EndoH-resistant fraction could be detected for GluR7b (Fig. 2C), reflecting the low amount of proteins that have undergone the last phases of glycosylation in the Golgi apparatus. Native GluR7a was also partially resistant to deglycosylation by EndoH in protein extracts from mouse brain (see supplemental data in the on-line version of this article). The low surface expression of GluR7b can be explained by restricted export of the subunits from the ER, whereas GluR7a readily traffics from the ER to the plasma membrane.



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  		FIG. 3.
Subcellular distribution of GluR7 subunits in cultured hippocampal neurons. A, co-staining of surface GluR7 isoforms with the somatodendritic marker MAP2 and the marker for glutamatergic synapses, VGluT1, in neurons from GluR5–/–xGluR6–/– mice. The images in the top row show the distribution of each isoform (green, Myc) in somatic compartments (red, MAP2). The middle row shows the same labeling at high magnification in dendrites. The bottom row shows the dendritic localization of GluR7 isoforms in reference to glutamatergic synapses (green, VGluT1). None of these subunits colocalize with the glutamatergic synaptic marker. Scale bars, 5 µm for images in the top row and 2 µm for images in the bottom two rows. B, co-staining of surface GluR7 isoforms with the axonal marker TAU1 and the marker for glutamatergic synapses, VGluT1, in neurons from GluR5–/–xGluR6–/– mice. Images in the top row display GluR7 isoforms (green, Myc) in axons (blue, TAU1). Images in the bottom rows depict co-staining of GluR7 isoforms and VGluT1 (red). GluR7a is concentrated on pre-synaptic glutamatergic sites (white arrowheads), whereas GluR7b is not. Scale bars, 2 µm for the top row and 1 µm for the bottom two rows.

 
Subcellular Distribution of GluR7 Isoforms—The various roles of KARs in synaptic transmission depend critically on their subcellular localization in specific membrane domains in the axonal or somatodendritic compartments. It is not yet clear whether subunit composition and splice variants might play a role in the polarized trafficking of KARs in these neuronal domains. We examined the subcellular localization of tagged GluR7a and GluR7b transfected in cultured hippocampal neurons derived from GluR5–/–xGluR6–/– mice. To explore the distribution of tagged subunits in regards to axonal or somatodendritic compartments, we performed double labeling studies using antibodies either against the microtubule-associated proteins Tau1 (a marker of axons) or MAP2 (a marker of somatodendritic compartments) (Fig. 3A). We found that both GluR7 isoforms were present at the surface of MAP2-positive compartments (Fig. 3A, sections marked Soma and Dendrites). Similarly, both GluR7 isoforms were detected at the surface of axonal processes (Fig. 3B, row marked Axons). To examine the potential localization of GluR7 isoforms at glutamatergic synapses, cultured neurons were labeled with an antibody against VGluT1, a presynaptic marker (26). No colocalization was found between VGluT1 staining and somatodendritic GluR7 isoforms, indicating that GluR7 is not targeted to post-synaptic sites in cultured hippocampal neurons (Fig. 3A, row marked Post-Synaptic) (colocalization index (Icorr) at post-synaptic sites is 0.51 ± 0.04 for GluR7a and 0.48 ± 0.01 for GluR7b, n = 10). We next explored the distribution of GluR7a and GluR7b at presynaptic sites on glutamatergic inputs. Both GluR7a and GluR7b concentrated at VGlut1 positive clusters on axonal processes (i.e. thin processes extending by >100 µm from the soma) of the transfected neuron (Fig. 3B, row marked Pre-Synaptic; white arrowheads) (colocalization index (Icorr) at presynaptic sites was 0.65 ± 0.01 for GluR7a and 0.62 ± 0.03 for GluR7b, n = 10 neurons; for both isoforms the difference between pre- and post-synaptic colocalization is significant, with p < 0.05). These results suggest that alternative splicing of GluR7 subunits might not be critical for the polarized targeting of KARs in axons or dendrites. In cultured hippocampal neurons, both GluR7 isoforms are targeted to glutamatergic synaptic terminals but not to post-synaptic sites.

Molecular Determinants for the Surface Expression of GluR7 Isoforms—In its C-terminal domain, the GluR7a subunit shares with GluR6a a stretch of amino acids that acts as an ER export motif (9, 12) (Fig. 4A; see supplemental data for BLAST search and ClustalW alignment in the on-line version of this article). This motif is composed of a cysteine residue followed by a cluster of positively charged (underlined) amino acids, CQRRLKHK. We tested the hypothesis that this motif was also acting as an ER export signal for GluR7a. We examined the effects of mutating the four charged amino acid residues into alanines (Fig. 4A, GluR7a-4A). Whereas GluR7a-4A was detectable at a high level on the cell surface of neurons from WT mice, surface staining for GluR7a-4A was weak in neurons from GluR5–/–xGluR6–/– mice (Fig. 4, B and C) (relative surface labeling for GluR7a-4A was 39 ± 2% for WT and 22 ± 2% for GluR5–/–xGluR6–/–, p < 0.01). Cotransfection of GluR7a-4A with untagged GluR7a increased surface expression of the mutated subunit (Fig. 4C) (relative surface labeling for GluR7a-4A cotransfected with GluR7a in GluR5–/–xGluR6–/– was 43 ± 7%, p < 0.05). Mutation of the forward trafficking motif of GluR7a was compensated by assembly with a non-mutated GluR7a subunit. To confirm that the CQRRLKHK motif of GluR7a acted as an ER exit signal, we co-stained Myc-GluR7a with the ER marker calreticulin (Fig. 4D). GluR7a-4A colocalized with calreticulin in neurons from GluR5–/–xGluR6–/– mice (Fig. 4D; yellow in the merged image (Merge) and hot colors in the row marked Image nMDP). Quantification of this colocalization revealed that GluR7a-4A was more colocalized with the ER marker than GluR7a was (Fig. 4D; mean Icorr was 0.58 ± 0.02 for GluR7a and 0.71 ± 0.02 for GluR7a-4A, p < 0.01). These results indicate that the stretch of charged (underlined) amino acid residues (RRXKXK) shared by GluR6a and GluR7a acts in both proteins as an essential determinant for their high surface expression in the plasma membrane.



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  		FIG. 4.
GluR7a carries an ER export motif. A, aligned amino acids (870–980) of GluR7a and its mutated form GluR7a-4A. B, double staining (left column, surface; right column, intracellular) of GluR7b-ALA in transfected neurons from WT mice (top row) and GluR5–/–xGluR6–/– mice (bottom row). GluR7b-ALA is detected less on the cell surface of GluR5–/–xGluR6–/–-type neurons than WT neurons. Scale bar, 10 µm. C, relative surface labeling of GluR7a-4A. Black bar is WT condition, gray bar is GluR5–/–xGluR6–/–, and green bar is co-expression with untagged GluR7a (n = 20; **, p < 0.01 for GluR7a-4A WT versus GluR7a-4A GluR5–/–xGluR6–/–; *, p < 0.05 for GluR7a-4A GluR5–/–xGluR6–/– versus GluR7a-4A GluR5–/–xGluR6–/–+GluR7a). Mutation of the four basic amino acids (873RRXKXK878) critically decreases the surface expression of GluR7a. D, co-staining of GluR7a-4A (red in the merged image) with calreticulin (green in the merged image) in neurons from GluR5–/–xGluR6–/–-type mice; GluR7a-4A localizes in the ER as shown by the hot colors in the correlation nMDP image (yellow in merged image). Scale bar, 2 µm. Scatter plot is the quantification of colocalization (mean Icorr ± S.E., n = 10; **, p < 0.01 for GluR7a versus GluR7a-4A).

 
GluR7b contains an RXR motif (Fig. 5A) that was identified as an ER retention signal that limits surface expression of potassium channels, G-protein coupled receptors, and some ionotropic glutamate receptor subunits (6, 27, 28). The subunits KA2 and GluR5c expressed as homomers fail to reach the plasma membrane and are confined to the ER because of the presence of an RXR sequence (9, 10). Disruption of these motifs in GluR5c and KA2 results in ER exit and surface expression. At variance with GluR5c and KA2, which were undetectable in the plasma membrane (9, 10), GluR7b was detected, although at a low level, at the cell surface of neurons from GluR5–/–xGluR6–/– mice (Fig. 1E). We thus examined the role of this motif in the surface trafficking of GluR7b in neurons from GluR5–/–xGluR6–/– mice. We replaced the two arginines by alanine residues in Myc-tagged GluR7b (GluR7b-ALA). GluR7b-ALA was detectable as small clusters along neuronal processes of neurons from WT mice as well as from GluR5–/–xGluR6–/– mice (Fig. 5B). Unexpectedly, mutating the RXR motif in GluR7b slightly decreased its surface expression in neurons from WT mice (relative surface labeling was 19 ± 3% for GluR7b and 10 ± 1% for GluR7b-ALA, p < 0.05) (Fig. 5C). In addition, the level of surface expression of GluR7b-ALA did not significantly increase when expressed in neurons derived from GluR5–/–xGluR6–/– mice, even when cotransfected with untagged GluR7a (relative surface labeling of GluR7b-ALA was 14 ± 2% in GluR5–/–xGluR6–/– mice and 12 ± 4% in GluR5–/–xGluR6–/– with GluR7a) (Fig. 5C). Similar results were obtained when GluR7b-ALA was cotransfected with GluR6a (relative surface labeling of GluR7b-ALA was 15 ± 4% in GluR5–/–xGluR6–/– mice with GluR6a). These results indicate that the RLR motif in GluR7b might not act as an ER retention signal in contrast with GluR5c and KA2.



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  		FIG. 5.
Molecular determinants of GluR7b assembly. A, aligned amino acids of GluR7b containing the RXR site (858–868) and its mutated form, GluR7b-ALA. B, double staining (left column, surface; right column, intracellular) of GluR7b-ALA in transfected neurons from WT (images in top row) and GluR5–/–xGluR6–/– mice (images in bottom row). GluR7b-ALA is detected on the cell surface of both types of neurons. Scale bar, 10 µm. C, relative surface labeling of GluR7b and GluR7b-ALA. The left bar is WT condition, the middle bar is GluR5–/–xGluR6–/– condition, and the right bar is co-expression with untagged GluR7a (n = 20; *, p < 0.05 for GluR7b WT versus GluR7b-ALA WT). Mutating the RXR motif in GluR7b does not increase surface expression of the subunit but inhibits the effect of GluR7a on promoting GluR7b surface expression.

 



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  		FIG. 6.
Co-immunoprecipitation of GluR6 and GluR7 isoforms. A, immunoprecipitation (IP) of Myc-GluR6b or Myc-GluR7b with GluR6a or GluR7a (untagged). Protein extracts of transfected COS-7 cells were prepared, immunoprecipitated, and Western blotted as described (45). Immunoprecipitation was done with the anti-GluR6/7 Ab (directed against "a" isoforms) (catalog number 06-309, Upstate Biotechnology) and Western blotting with a polyclonal anti-Myc Ab (see "Materials and Methods"). GluR6b and GluR7b assemble with a homologous subunit rather than a heterologous subunit. FT, flow through. B, immunoprecipitation (IP) of GluR6a and GluR7a in transfected COS-7 cells. Cells were transfected with GFP-GluR6a and Myc-GluR7a, and protein extracts were precipitated with and anti-GFP Ab (A11122 [GenBank] , Molecular Probes) or an anti-Myc Ab (see "Materials and Methods") and Western blotted with a corresponding alternate Ab. Assembly of GluR6 and GluR7 is increased when both subunits carries a forward trafficking motifs. FT, flow through. C, quantification of immunoprecipitated (IP) fraction (mean immunoprecipitated fraction as percentage of input, n = 4) for all the combinations tested (numbered 1–6). Loaded sample volumes were adapted for quantification (see 1x and 10x in panels A and B). Mean immunoprecipitated fraction as percentage of input (±S.E.) is 22.3 ± 4.3% for GluR6b/GluR6a, 5.2 ± 1.3% for GluR6b/GluR7a, 25.2 ± 3.8% for GluR7b/GluR7a, 6.2 ± 2.1% for GluR7b/GluR6a (p < 0.05), 69.2 ± 3.3% for immunoprecipitated GFP/Western blotted Myc, and 65.2 ± 1.9% for immunoprecipitated Myc/Western blotted GFP. Quantification was done using National Institutes of Health Image software.

 
Assembly and Trafficking of GluR6/GluR7 Heteromers—Although various heteromeric combinations of KAR subunits have been shown to form functional ion channels, the composition of native KARs is not precisely known. In the brain, GluR7 mRNA is found in neuronal populations that also express GluR6, such as dentate granule cells of the hippocampus or pyramidal cells in the neocortical layer VI (19). Because of its very low affinity for glutamate, the GluR7 subunit is expected to co-assemble with other ion-forming KAR subunits such as GluR6 (29). Here, we examined whether GluR6 and GluR7 splice variants co-assembled and how these different isoforms interacted to regulate membrane delivery of heteromeric KARs. We checked for co-assembly between different GluR6 and GluR7 isoforms transfected in COS-7 cells, immunoprecipitated with an anti GluR6a/7a antibody, and probed with an anti-Myc antibody (Fig. 6A). Although heteromerization between the two subunit isoforms was observed for all paired combinations tested, co-transfection of isoforms of the same subunit (GluR6a/GluR6b and GluR7a/GluR7b) yielded more efficient co-assembly than cross-combinations between GluR6 and GluR7 isoforms (GluR6b/GluR7a and GluR6a/GluR7b) (mean immunoprecipitated fraction as the percentage of input (±S.E.) was 22 ± 4% for GluR6a/GluR6b, 25 ± 4% for GluR7a/GluR7b, 5 ± 1% for GluR6b/GluR7a, and 6 ± 2% for GluR6a/GluR7b, n = 4 for all conditions) (Fig. 6C). To test for the co-assembly of GluR6a and GluR7a, we co-transfected a GFP-GluR6a construct with Myc-GluR7a (Fig. 6B). The combination of these two isoforms sharing the same forward trafficking motif yielded a high degree of co-assembly (mean immunoprecipitated fraction as the percentage of input ± S.E. was 69 ± 3% for immunoprecipitated anti-GFP/Western blot anti-Myc and 65 ± 2% for immunoprecipitated anti-Myc/Western blot anti-GFP). Thus, GluR6 and GluR7 subunit splice variants can co-assemble. Apart from the GluR6a/GluR7a pair, there is a preferential assembly of subunits of the same type (GluR6 or GluR7). The degree of co-assembly also depends largely on the nature of the C-terminal domain.



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  		FIG. 7.
Surface expression of GluR6 and GluR7 combinations in neurons. Relative surface labeling of GluR6b and GluR7b transfected in several combinations in cultured hippocampal neurons. Black bar is single transfection in neurons from wild type mice, gray bar is single transfection in neurons from GluR5–/–xGluR6–/– mice, red bar is co-transfection with untagged GluR6a in neurons from GluR5–/–xGluR6–/– mice, and green bar is co-transfection with untagged GluR7a in neurons from GluR5–/–xRGlu6–/– mice (n = 20; **, p < 0.01 for GluR5–/–xGluR6–/– versus WT; *, p < 0.05 for GluR5–/–xGluR6–/– with GluR6a versus GluR5–/–xGluR6–/– with GluR7a).

 
We then examined how heteromerization between GluR6 and GluR7 isoforms affected the trafficking of KARs to the plasma membrane. GluR6a is a key subunit promoting surface expression of KAR subunits that are retained in the ER when expressed as homomers, such as KA2, GluR5, or GluR6b (9, 12). Because GluR7a is expressed at a high level in the plasma membrane and shares with GluR6a the same export motif, we examined whether GluR7a can also promote surface expression of GluR6b, a GluR6 splice variant that is largely retained in the ER. As described previously (9), the surface expression level of GluR6b was significantly lower in neurons from GluR5–/–xGluR6–/– mice than in neurons from WT mice (the surface expression level of GluR6b was 48 ± 3% in WT and 12 ± 3% in GluR5–/–xGluR6–/–, p < 0.01) (Fig. 7). When co-transfected with GluR7a in neurons from GluR5–/–xGluR6–/– mice, the surface expression level of GluR6b increased markedly (surface expression level was 32 ± 4%). However, GluR6a was more efficient than GluR7a in promoting surface expression of GluR6b (relative surface labeling for GluR6b was 47 ± 5% in GluR5–/–xGluR6–/– mice with GluR6a, p < 0.05) (Fig. 7). The surface expression level of GluR6b transfected with GluR6a in neurons from GluR5–/–xGluR6–/– mice was similar to that of GluR6b in neurons from wild-type mice (Fig. 6) (9). GluR7a also promoted surface expression of GluR5c, a subunit retained strictly in the ER when expressed as a homomer (relative surface labeling for GluR5c was 2 ± 1% in GluR5–/–xGluR6–/– mice and 10 ± 4% in GluR5–/–xGluR6–/– with GluR7a; p < 0.05 for GluR5c alone versus cotransfection). Interestingly, GluR6a promoted the surface expression of GluR5c to the same extent as GluR7a (relative surface expression of GluR5c was 9 ± 3% in GluR5–/–xGluR6–/– with GluR6a).

We finally examined whether surface expression of GluR7b was facilitated to a similar extent by GluR6a and by GluR7a. Co-transfection of GluR7b with GluR6a in neurons from GluR5–/–xGluR6–/– mice promoted surface expression of GluR7b to levels observed when GluR7b was transfected alone in WT neurons (relative surface labeling of GluR7b was 25 ± 3% in GluR5–/–xGluR6–/– with GluR6a). These data further support the notion that GluR6a is endogenously expressed in neuronal cultures from WT mice and can promote forward trafficking of ER-retained KAR subunits. Interestingly, GluR7a was more efficient than GluR6a in promoting surface expression of GluR7b in neurons from GluR5–/–xGluR6–/– mice (relative surface labeling of GluR7b was 46 ± 5% in GluR5–/–xGluR6–/– with GluR7a, p < 0.05). These data indicate that GluR7a is another key subunit positively regulating the trafficking of KARs to the plasma membrane. These data also suggest that the efficacy of GluR6a and GluR7a in promoting surface expression of ER-retained subunits is limited by the variable ability of distinct KAR subunits to co-assemble in heteromers.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Regulation of the trafficking of glutamate receptors to the plasma membrane contributes to the control of synaptic strength (30, 31). Recent progress in the study of KAR trafficking has highlighted a role for subunit composition and alternative splicing in ER sorting of GluR5, GluR6, and KA2 (9–11, 13). Nothing is known about the subcellular localization and synaptic function of the GluR7 KAR subunit. This lack of information is due first to lack of specific agonists and antagonists for this receptor subunit, which shows only a very low affinity for glutamate when expressed as a recombinant homomer in heterologous cells (16). In addition, a specific GluR7 antibody for determining the regional and subcellular localization of the GluR7 protein is still lacking, although in situ hybridization studies have revealed high expression of GluR7 in the cerebellum, the cortex, or the hippocampus (19, 32). The present study represents a first step in understanding the subcellular localization and the role of the GluR7 subunit. We have compared the subcellular distribution of two GluR7 splice variants that differ in their C-terminal domains. In the absence of a specific anti-GluR7 antibody, we have tagged both GluR7 splice variants and expressed them in hippocampal neuronal cultures derived from KAR subunit mutant mice that are devoid of endogenous GluR5, GluR6, and GluR7. We report that the two splice variants traffic differentially from the ER to the plasma membrane. The GluR7a splice variant displays a high surface expression level, whereas GluR7b is in large part retained in the ER. Furthermore, GluR7a promotes surface expression of KAR subunits that tend to be retained in the ER. We show that all paired combinations of the GluR6/GluR7 subunit yield subunit co-assembly. However, the co-assembly of subunits and splice variants largely depends on subunit types and C-terminal domains, in addition to the previously characterized N-terminal determinants (33).

Forward Trafficking Signal of GluR7a—In this study we defined a C-terminal motif necessary for the high surface expression of GluR7a that is not present in GluR7b. This motif (CQRRLKHK) contains four positively charged amino acids and is identical to the forward trafficking motif described for GluR6a (9, 12). A BLAST search and ClustalW alignment (see supplemental data in the on-line version of this article) only detected these motifs in GluR6a and GluR7a. Mutation of the four positively charged amino acids induced low surface expression and retention of homomeric GluR7a receptors in the ER when the subunit was expressed in neurons derived from cultured neurons devoid of GluR5, GluR6, and GluR7. The mechanism by which the CQRRLKHK motif operates as a forward trafficking signal is unknown. In the case of GluR6a, Yan et al. proposed that, in addition to the four positively charged amino acids, the cysteine residue also played a role in forward trafficking (12). This cysteine residue, which is also present in GluR7a, is known to be palmitoylated in GluR6a (34), suggesting a role in anchoring the C-terminal domain of these proteins in the membrane. But it is not yet known how membrane association or palmitoylation of the cytoplasmic tail of GluR6a (or possibly GluR7a) promotes surface expression of KARs. In the closely related AMPA receptor family, surface expression of GluR1 and GluR4 depends on basic residues that reside in the juxtamembrane segment of their C-terminal domains. These residues are important for interactions with the spectrin-binding protein 4.1N (35, 36). Point mutations of these three basic residues decrease surface expression of GluR4 and prevent interaction with 4.1 proteins (36). Apart from the presence of basic residues, this sequence bears little similarity with the forward trafficking motif in GluR6a and GluR7a. An anterograde trafficking motif has also been found in the N-terminal extracellular domain of AMPA receptors, with the possibility that this motif might act during the assembly process (37). It seems unlikely that KAR subunits possess an important forward trafficking motif in their N-terminal domain, because the different isoforms for GluR6 and GluR7 display very different levels of cell surface expression with identical N-terminal domains. Finally, it should be noted that GluR6a and GluR7a are highly expressed at the surface of COS-7 cells and the surface neurons in culture, suggesting that efficient trafficking of KARs containing these isoforms does not require the presence of neuronal specific machinery.

The RXR Motif of GluR7b Does Not Act as an ER Retention Signal—Like GluR5c and KA2, GluR7b carries an RXR motif in its C-terminal domain. A similar motif functions as a strong ER retention signal in KATP channels (27), {gamma}-aminobutyric acid type B receptors (28, 38), and some N-methyl-D-aspartate receptor splice variants (39, 40). In GluR5c and KA2, the RXR signal reliably prevents expression of the homomeric receptors to the plasma membrane (9, 10). Mutation of the two arginine residues to alanine residues greatly enhances surface expression of GluR5c or KA2. In addition, the retention signal of GluR5c or KA2 can be masked during assembly with the GluR6a subunit splice variant. At variance with homomeric GluR5c and KA2 subunits, which are strictly retained in the ER, GluR7b receptors were in part exported to the plasma membrane, although they mainly co-localized with the ER marker calreticulin. We found that introducing the same alanine mutations in the RLR motif did not increase surface expression of GluR7b, indicating that this motif does not act as an ER retention signal. Small changes in the local sequence context could markedly affect the RXR signal strength (41), explaining why the RXR signal does not act as an ER retention motif in GluR7b. In the KATP channel subunit Kir6.2, 14-3-3 proteins interact specifically with a cytoplasmic RXR motif and promote efficient ER exit (42). Similarly, putative binding of a 14-3-3-related protein to the ER retention motif of GluR7b might serve to overcome retention in the ER. Homomeric GluR7b would then be addressed to the plasma membrane through a default biosynthetic pathway when not co-assembled with GluR6a or GluR7a. A similar default pathway is likely used by GluR5a, which is only weakly expressed at the cell surface (9, 11) despite a short C-terminal domain (16 amino acids). Interestingly, introducing the alanine mutations in the RLR motif of GluR7b markedly affected the ability of GluR7a to promote surface expression of GluR7b. A possible explanation is that mutation of the RXR motif in GluR7b, by preventing binding of a scaffold protein such as the 14-3-3 protein, affects cross-assembly with other KAR subunit splice variants. These results indicate differences in the involvement of RXR motifs in the C-terminal domains of KAR subunits and suggest that the RXR motif of GluR7b might be important during the biogenesis and assembly process.

Assembly and Surface Expression of GluR6/GluR7 Heteromeric Receptors—Although GluR6 and GluR7 share 86% amino acid sequence identity, they differ considerably in their ion channel properties. Whereas recombinant GluR6 receptors show large currents in response to kainate and glutamate, GluR7a and GluR7b receptors only respond to unphysiological concentrations of glutamate (16). The relative difference in agonist efficacy has been attributed to residues localized in the third extracellular loop (17). Because of the very low efficacy of glutamate for the activation of GluR7 receptors, it can be hypothesized that, in native KARs, GluR7 co-assembles with other KAR subunits. GluR7 is expressed in neuronal populations with high expression of GluR6, such as pyramidal cells of the inner layer of the neocortex or dentate granule cells (15, 19, 32). GluR7 (GluR7a) co-assembles with GluR6 (GluR6a) in heterologous cell systems, but co-assembly dramatically reduces the amplitude of GluR6 responses to kainate, possibly as a consequence of reduced functional expression of GluR6/GluR7 heteromers (17, 29). The C-terminal domain of GluR7 plus the C-terminal part of the third extracellular loop was reported to be responsible for the decreased functional expression of GluR6/GluR7 heteromers (17). We show that all paired combinations of GluR6 and GluR7 splice variants yield heteromeric cross-assembly when expressed in COS-7 cells and probed by immunoprecipitation. The degree of heteromerization was very variable, depending on two factors. First, GluR6 splice variants assemble more efficiently with one another that with GluR7 splice variants (and vice versa). In addition, the efficacy of co-assembly depends on the nature of the C-terminal domain. These experiments are in agreement with the notion that efficient heteromeric assembly of KAR subunits requires a combination of extracellular and C-terminal cytoplasmic domains (17, 33). However, co-expression of GluR6a and GluR7a, both of which traffic efficiently along the biosynthetic pathway, yields efficient heteromerization in contrast with what was previously suggested to explain the decreased current amplitude of GluR6/GluR7 heteromers (17, 29). The reasons for inefficient co-assembly of various GluR6 and GluR7 splice variants might in fact lie in the differential subcellular localization of the subunits through the biosynthetic pathway, which would lessen the probability for cross-assembly of certain paired combinations. In the absence of a suitable GluR7 antibody, it is not yet known if GluR7 assembles with other subunits in native KARs and what would be the functional consequences of this co-assembly. Our results on recombinant heteromeric receptors indicate that GluR7a, like GluR6a, facilitates membrane delivery of ER-retained KAR subunits such as GluR5c, GluR6b, and GluR7b. In keeping with this finding, electrophysiological and cobalt uptake experiments have previously given functional evidence for the surface expression of KA2 when expressed with GluR7a (13, 16). It would thus be very interesting to test whether GluR7a serves to drive ER-retained subunits to neuronal compartments where these subunits might play their functional roles. Conversely, GluR7b might need to be co-assembled in heteromers with either GluR6a or GluR7a to be functionally expressed at the membrane. Because KA2 is expressed in cultured hippocampal neurons (20) (but see Ref. 21), it is also possible that heteromerization with KA2 influences the trafficking of GluR7. It should be mentioned that KA2 proteins are down-regulated in neurons from mice lacking the GluR5 or GluR6 gene (43). Thus, we cannot exclude the fact that differences observed in GluR7b trafficking between wild type and GluR5–/–xGluR6–/– neurons would be due to the lack of KA2.

Altogether, much remains to be known about the subcellular distribution and functional properties of GluR7-containing KARs in synaptic function. Recent studies on the trafficking of KARs raise the question of how subunit composition and alternative splicing, which give rise to a large number of KAR combinations, may subserve the wide spectrum of roles played by these receptors.


   FOOTNOTES
 
* This work was supported by grants by the Centre National de la Recherche Scientifique, the Ministère de la Recherche of France, and the Conseil Régional d'Aquitaine, as well as by European Commission Contract QLRT-2000-02089. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at www.jbc.org) contains supplemental material in the form of Figs. sup 1 (biotinylation assay), sup 2 (expression of GluR5–7 subunits in cultured hippocampal neurons), sup 3 (glycosylation state of GluR7a in brain extracts), and sup 4 (BLAST search and ClustalW alignment). Back

{ddagger} To whom correspondence should be addressed. Tel.: 33-5-5757-4086; Fax: 33-5-5757-4082; E-mail: mulle{at}u-bordeaux2.fr.

1 The abbreviations used are: AMPA, {alpha}-amino-3-hydroxy-5-methylisoxazole-4-propionic acid; Ab, antibody; ALA, replacement of two arginines by two alanines; EndoH, endoglycosidase H; ER, endoplasmic reticulum; GFP, green fluorescent protein; GluR, glutamate receptor; KA, kainate; KAR, kainate receptor; MAP2, microtubule-associated protein 2; nMDP, normalized mean deviation product; PNG-F, peptide N-glycosidase-F; RT, reverse transcription; VGluT1, vesicular glutamate transporter 1; WT, wild type. Back


   ACKNOWLEDGMENTS
 
We thank Steve Heinemann and the Salk Institute for making mutant mice available.



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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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