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Revisiting the Postulated "Unitary Glutamate Receptor": Electrophysiological and Pharmacological Analysis in Two Heterologous Expression Systems Fails to Detect Evidence for Its Existence

Department of Biochemistry I-Receptor Biochemistry, Ruhr University Bochum, Bochum, Germany

Received July 15, 2005; accepted October 7, 2005

	Abstract

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Several years ago evidence for a so-called "unitary glutamate receptor" was published. This unique type of glutamate receptor was reported to be activated by the traditional agonists of all three major glutamate receptor subfamilies [i.e., -amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), kainate, and N-methyl-D-aspartate (NMDA)] in a glycine-dependent as well as magnesium-blockable manner and was reported to consist of an NR1 subunit coexpressed with the kainate binding protein (KBP) from Xenopus laevis, XenU1. To re-examine the existence of such a receptor, we cloned two splice variants of the X. laevis NMDA receptor subunit NR1, XenNR1-4a and XenNR1-4b, and expressed them in X. laevis oocytes as well as in human embryonic kidney (HEK) 293 cells, either alone or with the X. laevis KBP subunit XenU1. In addition, we coexpressed XenU1 separately with all eight splice variants of the rat NR1 subunit. In no case did we see evidence of a unitary glutamate receptor pharmacology. In HEK293 cells, we did not get receptor response unless an NR2 subunit was coexpressed. In X. laevis oocytes, we did observe responses to glutamate/glycine as well as small responses to glycine alone, but these were independent of coexpressed XenU1. Neither AMPA nor kainate ever elicited significant responses. Because we verified that XenU1 is expressed and inserted into the plasma membrane of HEK293 cells, we conclude that XenU1 and NR1 do not form the postulated unitary glutamate receptor. Furthermore, successful amplification of a fragment of a X. laevis NR2 subunit indicates that X. laevis uses NR2 subunits and not XenU1 to form heteromeric complexes with NR1.

However, several independent studies questioned the commonly used functional classification of the iGluRs and reported, for example, the existence of receptor complexes that united pharmacological properties of NMDA and non-NMDA receptors (Jahr and Stevens, 1987). These findings, which were largely based on pharmacological experiments, were not confirmed at the molecular level until Soloviev et al. (1996) described a "unitary glutamate receptor" with unusual pharmacological properties. This receptor, a heteromeric complex consisting of the Xenopus laevis NMDA receptor splice variant XenNR1-4b and the X. laevis kainate binding protein XenU1, responded equally to AMPA, KA, and NMDA. Even more unusual, all three agonist responses were dependent on glycine as a coagonist, and all three were blocked in a voltage-dependent manner by extracellular magnesium. Soloviev et al. (1996) extensively investigated this unitary glutamate receptor by electrophysiological analysis in HEK293 cells, coimmunoprecipitation studies, and ligand binding studies. The latter experiments also indicated that XenU1 did not only form a unitary glutamate receptor complex with X. laevis NR1 but also with mammalian NR1 subunits (Soloviev et al., 1996). This finding and the observation that XenU1 is expressed in oocytes endogenously (Soloviev and Barnard, 1997) were used to explain the appearance of functional NMDA-gated ion channels when NR1 subunits are expressed in X. laevis oocytes in the absence of NR2 subunits (Moriyoshi et al., 1991; Hollmann et al., 1993).

Our group had cloned XenU1 several years ago for an ion pore transplantation study in which we showed that the ion pore domains of all KBPs can be transplanted into other iGluRs (e.g., the KA receptor subunit GluR6) without loss of ion channel function (Villmann et al., 1997). At the time, we also coexpressed XenU1 with the rat NR1 subunit but failed to observe unusual pharmacological properties. Because the original observation of the unitary glutamate receptor had been made with the X. laevis version of NR1, XenNR1, this left the formal possibility of a species-specific difference in the formation of the unitary glutamate receptor, a possibility that we could not rule out for lack of the XenNR1 cDNA. After a recent study from Steve Heinemann's group (Green et al., 2002) re-examined the interaction of XenU1 and rat NR1-1a and similarly failed to find indications for functional or structural interactions, we decided to take up the issue again. We therefore cloned two splice variants of XenNR1 from X. laevis cDNA and analyzed their functional properties in two heterologous expression systems, X. laevis oocytes as well as HEK293 cells, in each case with and without coexpressed XenU1. In addition, we used all eight known functional splice variants of the rat NR1 subunit (Hollmann et al., 1993) to investigate a possible splice variant-dependent functional interaction with XenU1.

	Materials and Methods

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Isolation of the XenNR1-4a cDNA and Construction of the XenNR1-4b and XenNR1-4b(E166G) cDNAs. For the cloning of X. laevis glutamate receptor subunits, three degenerate oligonucleotides were designed as PCR primers based on a sequence alignment of GluR1 (GenBank accession no. X17184 [GenBank] ), KA1 (U08257 [GenBank] ), GluR6 (Z11548 [GenBank] ), NR1-1a (U08261 [GenBank] ), and XenNR1 (X94081 [GenBank] ). These primers, 5'-GGCTWYTGYRTSGACCTG-3' (alternatively, 5'-TGGAAYGGMATGRTKGGMG-3') and 5'-GAARGCWGCCARGTTRGC-3' (with K = G/T, M = A/C, R= A/G, S = G/C, Y = C/T, and W = A/T), were used in an RT-PCR of RNA extracted from adult female X. laevis brain as template. A 594-bp fragment of XenNR1 (Soloviev et al., 1996) beginning at position +1425 was amplified. The PCR product was radiolabeled with [-³²P]dCTP using HexaLabel DNA labeling kit (MBI Fermentas, St. Leon-Rot, Germany) and used as a probe to screen 5 x 10⁵ plaques of a X. laevis embryo cDNA library (Stratagene, La Jolla, CA) at high-stringency conditions (125 mM NaCl, 7.5 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 5x Denhardt's solution, 0.5% SDS, and 100 µg/ml salmon sperm DNA at 65°C). The nylon filters (Roche Diagnostics, Mannheim, Germany) were washed in washing buffer (50 mM NaCl, 3 mM Tris-HCl, pH 7.4, 0.2 mM EDTA, and 0.1% SDS) at 65°C. One clone was selected for further study and was plaque-purified, rescued as pBluescript plasmid, and analyzed by restriction endonuclease digestion and sequencing. We identified the isolated clone as a full-length NMDA receptor subunit from X. laevis lacking a 63-bp sequence (=exon 5 in rat) in the region encoding for the N-terminal domain compared with the published XenNR1 sequence. Further differences to XenNR1 are six single base deviations (positions 120, 557, 756, 2130, 2202, and 2223 of the XenNR1 coding region) of which five do not alter the encoded amino acid. Only the deviation at position 557 alters the amino acid sequence of the encoded protein by replacing glycine 166 of the mature protein by glutamate. Because of the homology of the cloned X. laevis glutamate receptor subunit compared with the NR1-4a splice variant from rat, we called the cloned subunit XenNR1-4a. The XenNR1-4a cDNA sequence has been deposited in GenBank (accession no. DQ066918 [GenBank] ).

The 63-bp sequence that XenNR1-4a is missing compared with the published XenNR1 sequence (which is the XenNR1-4b splice variant) was confirmed to be expressed in frog brain by performing an RT-PCR on adult female X. laevis brain RNA using primers flanking the 63-bp sequence (5'-ATGCCATCCAGATGGCTCTATCTGT-3' and 5'-GAGGAGTATAACTCTGGCTTCCAGT-3'). Fragments of interest were isolated, subcloned into the EcoRV site of pSGEM, and analyzed by sequencing.

For the introduction of the 63-bp sequence into XenNR1-4a, two overlapping tail primers were synthesized: 5'-CCTCGACCAACTTTCCTATGACAACAAGCGTGGACCCAAGGCAGACAAAGTCCTGCAGTT-3' and 5'-AGGAAAGTTGGTCGAGGTTCTCATAGTTCCTTTTTTTACCCTTGGACTCTTTCTCCTCTA-3'. These tail primers were used together with the oligonucleotides 5'-GCTTGGAGCTGAGAGCACCCA-3' and 5'-CATATACAAAAGGTTCTTGGTGGA-3' as primers in an overlap extension PCR. The resulting 1354-bp PCR product and XenNR1-4a were digested with BglII and Bpu10I and ligated to produce XenNR1-4b.

Because our XenNR1-4b contained a glutamate at position 166 instead of a glycine reported for the cDNA clone of Soloviev et al. (1996), we introduced a glycine by PCR-mediated mutagenesis with the mutagenesis primers 5'-TTACTCTTGGACTCTTTCCCCTCTAACAGGGTCTC-3' and 5'-TGGAGACCCTGTTAGAGGGGAAAGAGTCCAAGAGT-3'. The resulting 1354-bp PCR product, which contained the E166G mutation, and XenNR1-4b were digested with BglII and Bpu10I and ligated to produce XenNR1-4b(E166G).

To screen for C-terminally alternatively spliced forms of X. laevis NR1, we performed several RT-PCRs. First, we used oligonucleotides flanking the putative alternatively spliced regions (5'-GAACTCAGCACTATGTACAGACACA-3' and 5'-CCAGTCATTAGCCGTCAGTACA-3'). Second, we used a sense primer lying upstream of the putative splice sites (5'-GAACTCAGCACTATGTACAGACACA-3') and antisense primers located in the exons 21 (5'-TCTCTTGAAGCTGGAGGCCA-3') and 22 (5'-TAACGGGCCGCCTTGTCTGT-3') of Rattus norvegicus.

For electrophysiological investigation of the isolated XenNR1-4 subunits in X. laevis oocytes and HEK293 cells, the cDNAs were subcloned into the expression vectors pSGEM-KS and pcDNA3 (Invitrogen, Karlsruhe, Germany), respectively.

Tagging of Receptor Subunits. For Western blot analysis, XenU1 (GenBank accession no. DQ073428 [GenBank] , isolated in our laboratory previously; Villmann et al., 1997) was C-terminally tagged with a myc epitope-encoding sequence and a polyhistidine tag. To create XenU1-myc-His, the complete XenU1 coding region was amplified by PCR using the primers 5'-CCCAGCTTGCTTGTTCTTT-3' and 5'-AGAATCCTCGAGTGATTTCACACTGTCCACTGCT-3', thus replacing the native stop codon by an XhoI restriction site. The PCR product was digested with EcoRV and XhoI and ligated into the EcoRV/XhoI-cut pcDNA4/TO/myc-His vector (Invitrogen).

For the analysis of the subcellular localization of X. laevis NR1 receptor subunits and XenU1 in HEK293 cells by confocal microscopy, the subunits were C-terminally tagged with EGFP and DsRed2, respectively. The cDNAs of the XenNR1 subunits were subcloned into the XhoI and SacII restriction sites of pEGFP-N1 (BD Biosciences Clontech, Palo Alto, CA). The stop codon was deleted by performing a PCR using the primers 5'-GAACTCAGCACTATGTACAGACACA-3' and 5'-TGATGGACCGGTGCGACCACAGTGCTAACAGAAG-3', after restriction digest of the PCR product (578 bp) with AgeI and ligation of the fragment into the AgeI-cut XenNR1-4(a/b)/pEGFP-N1, producing XenNR1-4a-EGFP and XenNR1-4b-EGFP. To create XenU1-DsRed2, the XenU1 cDNA was isolated by a restriction digest with XbaI and XhoI and ligated into the NheI/XhoI-cut pDsRed2-N1 vector (BD Biosciences Clontech). The XenU1 C terminus was amplified by PCR using the primers 5'-GATGAACTTCTTGTGAAATCA-3' and 5'-CAGAATGGGCCCATGATTTCACACTGTCCACTG-3'. The PCR product (523 bp) was digested with ApaI and ligated into ApaI-cut XenU1/pDsRed2-N1.

cRNA Synthesis. cRNA synthesis was done as described previously (Hollmann et al., 1994). In brief, template DNA was linearized with NheI. cRNA was synthesized from 1 µg of linearized DNA using an in vitro transcription kit (MBI Fermentas) with a modified protocol that uses 800 µM GpppG (MBI Fermentas) for capping and an extended reaction time of 3 h with T7 polymerase. Trace labeling was performed with [-³²P]UTP to allow calculation of yields and evaluation of transcript quality by agarose gel electrophoresis.

Electrophysiological Studies in X. laevis Oocytes. Frog oocytes of stages V or VI were surgically removed from the ovaries of X. laevis (Nasco, Fort Atkinson, WI) anesthetized with 3-amino-benzoic acid ethylester (1.5 g/l; Sigma, Taufkirchen, Germany). Lumps of 20 oocytes were incubated with 784 U/ml (4 mg/ml) collagenase type I (Worthington Biochemicals, Freehold, NJ) for 1.5 h in Ca²⁺-free Barth's solution (88 mM NaCl, 1.1 mM KCl, 2.4 mM NaHCO₃, 0.8 mM MgSO₄, and 15 mM HEPES, pH adjusted to 7.6 with NaOH) with slow agitation to remove the follicular cell layer and then washed extensively with Barth's solution [88 mM NaCl, 1.1 mM KCl, 2.4 mM NaHCO₃, 0.3 mM Ca(NO)₃, 0.4 mM CaCl₂, 0.8 mM MgSO₄, and 15 mM HEPES, pH adjusted to 7.6 with NaOH)]. Oocytes were maintained in Barth's solution with 100 µg/ml gentamycin, 40 µg/ml streptomycin, and 63 µg/ml penicillin added. Twenty-four hours later, oocytes were injected with a total of 10 ng of cRNA for single expressions and with 20 ng for coexpressions (10 ng each) using a nanoliter injector (WPI, Sarasota, FL). Four to 5 days after injection, oocytes were recorded in normal frog Ringer's solution (NFR; 115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl₂, and 10 mM HEPES, pH adjusted to 7.2 with NaOH) under voltage clamp at –70-mV holding potential [except for current-voltage (I/V) curves], with a TurboTec 10CX amplifier (NPI Electronic GmbH, Tamm, Germany) controlled by Pulse software (HEKA, Lambrecht/Pfalz, Germany). Recording pipettes were pulled from borosilicate glass (Hilgenberg, Malsfeld, Germany) using a PIP5 pipette vertical puller (HEKA). Voltage electrodes had a resistance of 1 to 4 M and were filled with 3 M KCl; current electrodes had a resistance of 0.5 to 1.5 M and were filled with 3 M CsCl. Drugs were applied for 20 s by superfusion at a flow rate of 5 ml/min. Coapplication of agonists is indicated by a slash between the agonists throughout this article (e.g., glutamate/glycine). For I/V curves, agonist was applied by constant superfusion, and a 2-s voltage ramp from –150 to +50 mV was recorded at the peak current, disregarding the initial fast spike that has been shown to represent a Ca²⁺-activated Cl^– conductance endogenous to X. laevis oocytes (Leonard and Kelso, 1990). Voltage ramps recorded before and after agonist application were averaged and subtracted from I/V curves for leakage current correction. To determine EC₅₀ values for glutamate and NMDA, 10 to 12 different agonist concentrations were applied to the same oocyte, and steady-state values of the evoked currents were measured. Data from each oocyte were fitted separately, and EC₅₀ values obtained this way from four oocytes were averaged. For investigation of NMDA receptor-specific Mg²⁺ block, Mg²⁺-Ringer's solution was used (115 mM NaCl, 2.5 mM KCl, 1.8 mM MgCl₂, and 10 mM HEPES, pH adjusted to 7.2 with NaOH).

HEK293 Cell Transfection. HEK293 cells were transfected with recombinant vector DNA using a modified calcium phosphate precipitation technique (Chen and Okayama, 1987). Exponentially growing cells in polyornithine-coated 35-mm dishes were transfected with 2 to 5 µg of DNA. For precipitation, a mixture of 2 µg of each DNA, 10 µl of CaCl₂ (2.5 M), and water (ad 100 µl) was incubated for 20 min at room temperature. Next, 100 µl of 2x HEPES-buffered saline [280 mM NaCl, 1.5 mM Na₂HPO₄, and 40 mM HEPES, pH adjusted to 7.1 with NaOH] was added, and the mixture was incubated for 20 min at room temperature. Then, 500 µl of cell culture medium (Dulbecco's modified Eagle's medium and 10% fetal bovine serum) was added in droplets. The whole mixture was transferred to a 35-mm dish, and the cells were incubated for 8 h at 37°C with 3% CO₂. After incubation, the cells were washed twice with 2 ml of phosphate-buffered saline before growth medium was added (for patch clamp and Western blots, minimal essential medium; for confocal microscopy, Earle's minimal essential medium). After transfection, HEK293 cells were allowed to express receptor for 48 to 96 h at 37°C with 5% CO₂.

If the transfected cells were analyzed by the patch-clamp technique, 1 µg of EGFP/pcDNA3 was added to the precipitation mixture. Thus, transfected cells could be identified by their green fluorescence when excited at 488 nm.

For Western blot analysis, 85-mm dishes were used. Therefore, the amount of all ingredients of the precipitation mixture was increased 6-fold.

Electrophysiological Studies in HEK293 Cells. Whole cell recordings were performed using a HEKA EPC-9 amplifier (HEKA) controlled by Pulse software (HEKA). Recording pipettes were pulled from borosilicate glass (GC150TL-10; Clarke Electromedical Instruments, Pangbourne, UK) using a PIP5 pipette vertical puller (HEKA). Ligand was applied using a theta glass capillary (Hilgenberg) that bathed the suspended cell in a laminar flow of solution, giving a time resolution for equilibration of 10 to 30 ms (Udgaonkar and Hess, 1987). The external buffer consisted of 140 mM NaCl, 4 mM KCl, 2 mM CaCl₂, and 10 mM HEPES, pH adjusted to 7.3 with NaOH; the internal buffer was 110 mM Cs gluconate, 20 mM CsCl, 4 mM NaCl, 1 mM MgCl₂, 0.5 mM CaCl₂, 5 mM EGTA, and 10 mM HEPES, pH adjusted to 7.4 with KOH. The current responses were measured at room temperature at a holding potential of –60 mV.

Subunit Expression Analysis by Western Blotting. Western blot analysis was performed on HEK293 cells transfected with XenU1-myc-His DNA 40 h after transfection. Cells were washed in phosphate-buffered saline and swelling buffer [10 mM HEPES (pH adjusted to 7.9 with KOH), 1.5 mM MgCl₂, 10 mM KCl, and 0.5 mM dithiothreitol] and incubated on ice in swelling buffer afterward. Next, cells were homogenized using a douncer, and then 1/10 volume of stabilization buffer [300 mM HEPES (pH adjusted to 7.9 with KOH), 30 mM MgCl₂, and 1.4 M KCl] was added. Nuclei and crude cell fragments were removed by low-speed centrifugation (2500g; 1 min; 4°C). The supernatant was used in an ultracentrifugation (100,000g; 1 h; 4°C), and to the resulting membrane pellet urea buffer (8 M urea, 375 mM Tris-HCl, pH 6.8, and 0.1% SDS) and SDS-polyacrylamide gel electrophoresis loading buffer (25 mM Tris-HCl, pH 6.8, 6% SDS, 800 mM -mercaptoethanol, 20% glycerol, 0.1% bromphenol blue, and 8 M urea) were added. Then, the sample was incubated in boiling water (10 min). Proteins were separated by SDS-polyacrylamide gel electrophoresis on an 8% gel and electroblotted on nitrocellulose membranes (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The nitrocellulose membranes were blocked with 4% nonfat dry milk (Gluecksklee, Muenchen, Germany) in Tris-buffered saline/Tween 20 (140 mM NaCl, 20 mM Tris-HCl, pH 7.6, and 0.1% Tween 20), and detection of proteins was carried out using mouse anti-myc (gift from B. J. Benecke, Ruhr University, Bochum, Germany) and donkey anti-mouse (Dianova, Hamburg, Germany) antibodies. Blots were developed using enhanced chemiluminescence solutions (Pierce Chemical, Rockford, IL).

Analysis of Subcellular Localization by Confocal Microscopy of Fluorescently Labeled Subunits. Confocal microscopy was performed on a Leica TCS SP2 (Leica, Mannheim, Germany) laser scanning confocal microscope using a Leica 63x, 1.3 numerical aperture, water immersion lens. HEK293 cells expressing X. laevis glutamate receptor subunits were plated on 35-mm glass-bottomed culture dishes and were kept in minimal essential medium with Earl's salts and 10% fetal calf serum but without phenol red (Sigma). During measurement, the dishes were preserved in a heated microscope chamber (H. Saur, Reutlingen, Germany), which adjusted the temperature to 37°C and the CO₂ percentage to 7% permanently. Colocalization studies were performed using dual excitation and emission filter sets. Specificity of labeling was established by examination of single labeled samples, and signal detection was optimized to ensure absence of signal crossover. For the analysis of the laser scanning confocal micrographs (including identification of colocalization), the Leica software was used.

	Results

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We initially set out to test whether coexpression of the X. laevis KBP XenU1 with any of the eight known rat NR1 splice variants (Hollmann et al., 1993) can generate the reported unitary glutamate receptor. For electrophysiological investigation, the eight rat NR1 splice variants were expressed alone and together with XenU1 in X. laevis oocytes. To test whether functional glutamate receptors were present, we applied glutamate (100 µM) together with the coagonist glycine (10 µM). Furthermore, we tested for the reported unique unitary glutamate receptor pharmacology by application of kainate (100 µM) or AMPA (100 µM), each with and without glycine. Because it is known that glycine applied alone can generate currents from NMDA receptors (Kleckner and Dingledine, 1988; Moriyoshi et al., 1991; Laube et al., 1993), we additionally applied glycine alone in all experiments. In all oocytes injected with NR1 with or without XenU1, we found significant current responses upon application of glutamate/glycine. Some oocytes showed small currents also upon application of kainate/glycine, AMPA/glycine, or glycine alone, which were all equal in size. By contrast, we never obtained a current response upon application of kainate or AMPA alone (Table 1). We therefore conclude that these small currents were induced by glycine alone without any effect exerted by AMPA or kainate. In addition, we found no significant difference in current amplitudes between oocytes expressing NR1 alone or together with XenU1. This finding was independent of the NR1 splice variant and included the NR1-4b splice variant (Fig. 1), which originally was reported to form ion channels of the unitary glutamate receptor type, at least for the X. laevis homolog of NR1-4b (Soloviev et al., 1996).

View larger version (21K): [in this window] [in a new window]   Fig. 1. Comparison of agonist-induced current responses recorded from voltage-clamped X. laevis oocytes injected with cRNAs of the rat NR1 splice variants NR1-4a and NR1-4b alone and together with XenU1. The experimental details were as described under Materials and Methods. The applied agonist concentrations were 100 µM glutamate, 100 µM KA, 100 µM AMPA, and 10 µM glycine. The application of agonists is indicated by black bars.

Although we found no evidence of a unitary glutamate receptor when we combined rat NR1 subunits with XenU1, we could not rule out its existence because Soloviev et al. (1996) had used the X. laevis homolog of NR1-4b, XenNR1-4b, in combination with XenU1 when they observed the unitary glutamate receptor properties. To be able to analyze the exact same subunit combination as Soloviev et al. (1996) in their original description of the unitary glutamate receptor, we screened a cDNA library from X. laevis embryo for XenNR1 cDNAs using as a probe a 594-bp fragment of a X. laevis NR1 subunit amplified by PCR from X. laevis brain reverse-transcribed total RNA. One clone was identified corresponding to a full-length X. laevis NMDA receptor cDNA. The cDNA lacked a 63-nucleotide sequence in the N-terminal domain compared with the published XenNR1; additionally, six nucleotides differed. Five of these differences did not alter the encoded amino acids (see Materials and Methods), whereas one alternate nucleotide replaced glycine 166 of the mature protein reported by Soloviev et al. (1996) by glutamate. Sequence comparison with the rat NR1 splice variants showed our clone to be the X. laevis homolog of the rat NR1-4a subunit, XenNR1-4a. To obtain the X. laevis NR1 subunit XenNR1-4b, we introduced the missing 63-bp sequence (=exon 5 in rat), which is characteristic for b splice variants (Hollmann et al., 1993), by overlap extension PCR. In addition, we amplified the sequence around the X. laevis exon 5 homolog by RT-PCR from X. laevis brain mRNA and obtained two specific bands (455 and 518 bp), which prove that a and b splice variants were expressed in adult female X. laevis brain. The fact that the 455-bp band was much stronger than the 518-bp band indicates that NR1 a splice variants are more common in X. laevis brain than b splice variants.

To check the X. laevis exon 5-homologous sequence, we subcloned the 518-bp fragment and analyzed it by sequencing. We found our exon 5-homologous sequence to be identical to that reported by Soloviev et al. (1996). However, we confirmed that the G166E amino acid deviation mentioned above was genuine. To screen for further XenNR1 splice variants, we performed several PCRs with oligonucleotides recognizing the sequences upstream and downstream of the C-terminally spliced exons 21 and 22 known from rat NR1 (Hollmann et al., 1993), but we did not obtain evidence for the existence of more alternatively spliced forms of XenNR1. By contrast, if we used a sense primer located upstream of the putative alternatively spliced exons and an antisense primer located in the sequence homologous to rat exon 21, we amplified one specific band (588 bp). Determination of the sequence of the fragment proved that a sequence homologous to rat exon 21 exists in X. laevis NR1 subunits. Using the same strategy with an exon 22-specific antisense primer, we obtained no specific bands. These results are evidence that in addition to NR1-4, at least the NR1–3 splice variants exist in X. laevis.

It had been reported that the sequence encoding the N-terminal domain of XenNR1 showed significant differences compared with rat NR1, and it had been suggested that these differences alter the receptor properties (Soloviev et al., 1996). Because an extensive characterization of the X. laevis NR1 subunit had never been reported, we tested whether the two isolated XenNR1-4 splice variants are functional subunits and behave similar to mammalian NR1 subunits. We initially expressed XenNR1-4a and XenNR1-4b alone or with the rat NR2B subunit in X. laevis oocytes as well as in HEK293 cells. In oocytes, both homomerically expressed X. laevis NR1 subunits showed small currents upon application of 100 µM glutamate with 10 µM glycine (XenNR1-4a, 3.5 ± 0.5 nA, n = 8; XenNR1-4b, 12.0 ± 1.6 nA, n = 5) and 100 µM NMDA with 10 µM glycine (XenNR1-4a, 1.3 ± 0.4 nA, n = 8; XenNR1-4b, 2.6 ± 1.3 nA, n = 5; Fig. 2A). When we expressed the X. laevis NR1 splice variants together with rat NR2B, the current amplitudes were greatly increased as is found with rat NR1 subunits when coexpressed with NR2B (Ikeda et al., 1992; Kutsuwada et al., 1992; Meguro et al., 1992; Ishii et al., 1993). We obtained steady-state currents of 1100 ± 218 nA (n = 16) and 5917 ± 166 nA (n = 7) upon application of glutamate/glycine for XenNR1-4a/NR2B and XenNR1-4b/NR2B, respectively, and 411 ± 77 nA (n = 16) and 826 ± 65 nA (n = 7) upon application of NMDA/glycine (Fig. 2B). We then recorded dose-response curves for glutamate and NMDA, of XenNR1-4a/NR2B and XenNR1-4b/NR2B (Fig. 2D). We calculated EC₅₀ values of 2.0 ± 0.3 µM (glutamate) and 60 ± 13 µM (NMDA) for XenNR1-4a/NR2B and 2.8 ± 0.5 µM (glutamate) and 240 ± 25 µM (NMDA) for XenNR1-4a/NR2B. Next, we investigated the NMDA receptor-specific glycine dependence of glutamate-induced currents known from mammalian NMDA receptors (Johnson and Ascher, 1987; Kleckner and Dingledine, 1988). We found that XenNR1-4a/NR2B as well as XenNR1-4b/NR2B were activated by glutamate applied alone, but the steady-state currents obtained were relatively small, 10 ± 3nA(n = 6) and 94 ± 20 nA (n = 9), respectively. Likewise, upon application of glycine alone, small currents were obtained for XenNR1-4a/NR2B (17 ± 5 nA; n = 6) and XenNR1-4b/NR2B (211 ± 74 nA; n = 9). Coapplication of glutamate and glycine increased the current amplitudes for XenNR1-4a/NR2B by approximately 136-fold (1361 ± 446 nA; n = 6) compared with glutamate-induced currents, and for XenNR1-4b/NR2B by approximately 77-fold (7283 ± 658 nA; n = 9; Fig. 2C). Another NMDA receptor-specific property of the X. laevis NR1 splice variants could be shown as we analyzed the glutamate/glycine-induced current flow in the presence and absence of extracellular Mg²⁺. We found that I/V curves recorded in Mg²⁺-free NFR were linear for both XenNR1 splice variants investigated, whereas in the presence of 1.8 mM extracellular Mg²⁺ ions the X. laevis NMDA receptor complexes showed rectifying I/V with no significant current flow at negative membrane potentials, but linearly increasing currents at positive membrane potentials (Fig. 2E). Further experiments showed that the receptors are not blocked by divalent cations in general but that the receptor complexes containing X. laevis NR1 subunits are highly permeable for Ca²⁺ ions (data not shown). In addition to the Mg²⁺ block, X. laevis NMDA receptors can be blocked by MK-801. A concentration of 1 µM MK-801 was sufficient to reduce the glutamate/glycine-induced current response of XenNR1-4a/NR2B and XenNR1-4b/NR2B to 7 ± 2% (n = 9) and 5 ± 3% (n = 6), of the maximal currents, respectively. All these experiments demonstrate that X. laevis NR1 subunits are functionally equivalent to the homologous subunits from R. norvegicus.

View larger version (32K): [in this window] [in a new window]   Fig. 2. Properties of currents recorded from X. laevis oocytes injected with combinations of receptor subunit cRNAs. Experimental details are described under Materials and Methods. Glu, 100 µM glutamate; NMDA, 100 µM NMDA; Gly, 10 µM glycine, unless stated differently. Coapplication of agonists is indicated by black bars. A, representative recordings from oocytes injected with XenNR1-4a (top trace) or XenNR1-4b (bottom trace) cRNAs. B, representative recordings from voltage-clamped oocytes injected with rat NR2B and XenNR1-4a (top trace) or XenNR1-4b (bottom trace) cRNAs. C, representative recordings from oocytes injected with XenNR1-4a plus NR2B (top trace) or XenNR1-4b plus NR2B (bottom trace) cRNAs showing the glycine dependence of glutamate-induced currents. D, glutamate and NMDA dose-response curves determined from oocytes injected with NR2B and XenNR1-4a (top graph) or XenNR1-4b cRNAs (bottom graph). Glutamate () and NMDA () concentrations were varied whereas the coagonist concentration was maintained at 10 µM glycine. Values are the means ± S.E.M. of four experiments, normalized to the responses to 100 µM glutamate/10 µM glycine or 1 mM NMDA/10 µM glycine. The EC50 values are given in the text. E, I/V curves recorded from oocytes injected with rat NR2B and XenNR1-4a (top graph) or XenNR1-4b (bottom graph) cRNAs. Recordings of all cells were done either in Mg2+-free NFR (black curves) or in Mg2+-Ringer's solution (gray curves). Note absence of outward rectification in Mg2+-free NFR.

View larger version (19K): [in this window] [in a new window]   Fig. 3. Representative current traces from patch-clamped HEK293 cells transfected with X. laevis NR1 splice variants alone and together with rat NR2B. The application of agonists (100 µM glutamate with 10 µM glycine) is indicated by black bars.

We then analyzed the electrophysiological properties of XenNR1-4 splice variants coexpressed with XenU1, the putative unitary glutamate receptor, in X. laevis oocytes as well as in HEK293 cells. As a control, we also expressed the XenNR1 subunits alone. In oocytes, we performed applications of glutamate (100 µM), KA (100 µM), and AMPA (10 µM), each alone and together with 10 µM glycine. In addition, glycine was applied alone (Fig. 4A). We found that XenNR1-4a never showed current responses to KA (with or without glycine), AMPA (with or without glycine), or glycine, neither when expressed alone nor when coexpressed with XenU1. Only application of glutamate/glycine induced small currents in oocytes expressing XenNR1-4a or XenNR1-4a plus XenU1. However, comparing the current amplitudes recorded from XenNR1-4a with those obtained from XenNR1-4a plus XenU1, no significant differences were obtained (Table 2). When we investigated XenNR1-4b, we similarly found no significant current responses upon application of KA (with or without glycine), AMPA (with or without glycine), or glycine. Exclusively glutamate was able to induce currents in the presence of glycine. When we coexpressed XenNR1-4b and XenU1, which is the reported subunit combination of the unitary glutamate receptor (Soloviev et al., 1996, 1998), we found in one of five oocytes tiny currents upon application of KA/glycine (1.4 nA) and AMPA/glycine (1.5 nA). However, glycine alone induced currents that were equal in size (1.0 nA), whereas application of KA or AMPA alone never induced current responses. Therefore, we conclude these currents to be induced by glycine alone, as was also shown above for rat NMDA receptor subunits with and without XenU1. Glutamate/glycine induced larger currents (19.2 ± 4.6 nA; n = 9) that however, showed no significant difference in their amplitudes compared with the currents recorded in oocytes expressing XenNR1-4b alone (17.9 ± 3.1 nA; n = 7; Table 2).

View larger version (26K): [in this window] [in a new window]   Fig. 4. Pharmacology of the putative unitary glutamate receptor in X. laevis oocytes and HEK293 cells. Applied agonist concentrations: Glu, 100 µM glutamate; KA, 100 µM kainate; AMPA, 100 µM AMPA; and Gly, 10 µM glycine. The application of agonists is indicated by black bars. A, comparison of agonist-induced current responses recorded from voltage-clamped oocytes injected with cRNAs of X. laevis NR1 splice variants XenNR1-4a and XenNR1-4b alone and together with XenU1. B, representative current responses from patch-clamped HEK293 cells transfected with either XenU1 alone or together with one of the two X. laevis NR1-4 splice variants.

However, as mentioned above, our XenNR1-4b showed a one-amino acid difference in the N-terminal domain compared with the XenNR1-4b of Soloviev et al. (1996). Because we cannot rule out that this minor difference has an effect on the receptor properties, we constructed Soloviev's XenNR1-4b(E166G) cDNA by PCR-mediated mutagenesis (for details, see Materials and Methods). This subunit was expressed alone and together with XenU1 in X. laevis oocytes to test for pharmacological properties that might indicate the existence of a unitary glutamate receptor. Whereas XenNR1-4b(E166G) forms perfectly functional glutamate/glycine-activated ion channels in oocytes, we could not find any of the unique unitary glutamate receptor properties. As observed for our original XenNR1-4b subunit, kainate or AMPA applied alone never elicited current response. Coapplication of any of those agonists together with glycine in some oocytes yielded tiny currents (1.0–1.8 nA), which in every case were equal in size to currents induced by glycine applied alone (Table 2). Therefore, we interpret these small responses to represent pure glycine currents, as has been shown for all other NMDA receptor subunits investigated. Thus, the single amino acid deviation at position 166 of the mature XenNR1-4b protein does not seem to alter the ion channel properties.

Because we could not detect any of the unique non-NMDA receptor agonist-induced currents that were reported to be characteristic for the unitary glutamate receptor, we tried to find other hints for its existence. However, the lack of AMPA-induced currents minimized our chances to re-examine functional properties described by Soloviev et al. (1996). Two characteristics could still be tested: the reported inhibitory effects of 6,7-dinitroquinoxaline-2,3-dione (DNQX) and AMPA. We found that currents induced by coapplication of glutamate (100 µM) and glycine (10 µM) surprisingly were slightly inhibited by coapplied AMPA (100 µM). However, this effect was entirely independent of coexpressed XenU1. Thus, the glutamate/glycine-induced currents were reduced to 70 ± 3% (n = 5) and 81 ± 6% (n = 5) for XenNR1-4a and XenNR1-4a plus XenU1, respectively. Likewise, we found the currents of XenNR1-4b and XenNR1-4b plus XenU1 to be reduced by coapplication of AMPA to 81 ± 5% (n = 5) and 72 ± 6% (n = 5), respectively. To test whether this inhibitory effect by AMPA is dependent on the coexpressed XenU1, we coexpressed the XenNR1 subunits with rat NR2B. In this experiment, we also found an AMPA inhibition, to 59 ± 4% (n = 5) and 28 ± 7% (n = 4) for XenNR1-4a/NR2B and XenNR1-4b/NR2B, respectively. Likewise, an inhibitory effect of DNQX (50 µM) on currents induced by application of NMDA (500 µM) plus glycine (10 µM) was observed. Currents of oocytes expressing any XenNR1-4 splice variant, with or without XenU1, were significantly decreased when DNQX was coapplied (XenNR1-4a, 49 ± 7%, n = 5; XenNR1-4a + XenU1, 54 ± 4%, n = 5; XenNR1-4b, 63 ± 7%, n = 5; and XenNR1-4b + XenU1, 62 ± 7%, n = 5). As for AMPA, this effect was also observed in oocytes expressing heteromeric NMDA receptor complexes (XenNR1-4a/NR2B, 32 ± 2%, n = 5; and XenNR1-4b/NR2B, 33 ± 8%, n = 4). Therefore, the inhibitory effects of DNQX and AMPA do not seem to be unique unitary glutamate receptor properties as postulated (Soloviev et al., 1996) but rather are a normal feature of X. laevis NMDA receptors.

Because we did not find any of the reported unique unitary glutamate receptor properties in oocytes, we turned to investigating the existence of this unique receptor type in a mammalian cell line that unlike X. laevis oocytes does not express XenU1 endogenously. We expressed XenNR1-4a as well as XenNR1-4b and XenNR1-4b(E166G) together with XenU1 in HEK293 cells. To rule out that XenU1 generates functional homomeric receptor complexes in HEK293 cells, we also expressed XenU1 alone. However, we never obtained significant current on any cell analyzed, regardless whether it expressed XenU1 alone (n = 13; Fig. 4B) or XenNR1-4a/XenU1 (n = 30), XenNR1-4b/XenU1 (n = 34). Because Soloviev et al. (1996) based all their evidence for the existence of a unitary glutamate receptor on patch-clamp experiments in HEK293 cells, we additionally investigated electrophysiologically the XenNR1-4b(E166G)/XenU1 subunit combination in HEK293 cells. In none of the cells tested (n = 12) did we get a current response upon application of glutamate/glycine, although XenNR1-4b(E166G) forms functional ion channels in coexpression with rat NR2B (data not shown). Because the lack of current response may have been due to a lack of expression of XenU1 protein, expression was tested by Western blot analysis. Because there is no commercially available antibody against XenU1, we C-terminally tagged the protein with a myc-/polyhistidine-tag (for details, see Materials and Methods). The cDNA encoding XenU1-myc-His was transfected into HEK293 cells and 40 h after transfection, we performed a membrane preparation. Membrane proteins were solubilized and separated by SDS-polyacrylamide gel electrophoresis, blotted onto a nitrocellulose membrane, and the presence of XenU1-myc-His was checked using a mouse anti-myc antibody. This experiment confirmed that XenU1-myc-His is expressed in HEK293 cells (Fig. 5). Although lack of XenU1 expression was thus ruled out, it was still possible that XenU1 was not located in the plasma membrane and therefore was unable to interact with XenNR1 to form a unitary glutamate receptor. We therefore investigated the intracellular localization of the X. laevis glutamate receptor subunits by means of confocal microscopy. We C-terminally tagged XenU1 with DsRed2 and the two XenNR1-4 splice variants with EGFP. cDNAs encoding these fusion proteins were transfected into HEK293 cells either separately or in combinations: XenNR1-4a-EGFP, XenNR1-4b-EGFP, XenU1-DsRed2, XenNR1-4a-EGFP plus XenU1-DsRed2, and XenNR1-4b-EGFP plus XenU1-DsRed2 (Fig. 6). We found that XenU1-DsRed2 was strongly expressed in the plasma membrane. When we expressed the EGFP-tagged X. laevis NR1-4 splice variants alone, we found green fluorescence in the plasma membrane as well as inside the cells (Fig. 6A). Upon coexpression of XenNR1 and XenU1, we found that each subunit was localized to the same regions as when it was expressed alone. XenU1 coexpression did not lead to an increase in XenNR1-4 plasma membrane expression or to a reduction in intracellular XenNR1-4 fluorescence (compare Fig. 6, A with B). XenNR1 and XenU1 subunits were colocalized in the plasma membrane (Fig. 6B). Thus, both subunits clearly are in a position to interact to form a heteromeric complex. Nevertheless, as described above, no indication of the unitary glutamate receptor pharmacology could be detected electrophysiologically.

View larger version (72K): [in this window] [in a new window]   Fig. 5. Western blot analysis of a crude membrane preparation (for details, see Materials and Methods) of HEK293 cells expressing XenU1-myc-His. For protein detection, a mouse anti-myc antibody was used.

View larger version (58K): [in this window] [in a new window]   Fig. 6. Subcellular localization of X. laevis glutamate receptor subunits in HEK293 cells. The white bars in the right corners of the pictures indicate 10 µm. A, representative laser scanning confocal pictures of HEK293 cells expressing XenU1-DsRed2, XenNR1-4a-EGFP, or XenNR1-4b-EGFP. B, representative laser scanning confocal pictures of HEK293 cells expressing the EGFP-tagged XenNR1 splice variants XenNR1-4a (top) or XenNR1-4b (bottom) together with XenU1-DsRed2. Colocalization is highlighted by white dots in the overlay pictures (right column).

If XenU1 does not interact with NR1 subunits, it is unlikely that XenU1 has the function of an NR2 substitute in X. laevis CNS as had been suggested previously (Soloviev and Barnard, 1997). This means that to generate functional NMDA receptors X. laevis has to express a functional equivalent to mammalian NR2 subunits. Therefore, we expected X. laevis to possess glutamate receptor subunits that are homologous to the rat NR2 subunits. To prove this hypothesis, we screened X. laevis brain cDNA for other glutamate receptors than XenNR1, particularly NR2 subunits. This was done by RT-PCR using the same degenerated primers we previously used for generating the XenNR1 probe. The amplified DNA fragments were subcloned and analyzed by sequencing. We identified fragments of X. laevis glutamate receptor subunits that were homologous to rat GluR1 (=XenGluR1; 489 bp), GluR2 (=XenGluR2; 492 bp), and NR2B (=XenNR2B; 480 bp). Compared with its rat homolog XenNR2B shows 81.7% sequence identity at the nucleotide level (Fig. 7) and 98% at the amino acid level. These data show that X. laevis actually possesses NR2 subunits and thus does not require XenU1 as an NR2 substitute. Therefore, XenU1 has to be classified as a X. laevis kainate binding protein of unknown function.

View larger version (62K): [in this window] [in a new window]   Fig. 7. Sequence alignment of a 443-bp X. laevis NR2B (XenNR2B) fragment with rat NR2B cDNA (GenBank accession no. U11419 [GenBank] ). The numbers given to the right of the sequences indicate the nucleotide positions of the line's last nucleotide in the rat NR2B complete coding region and of the amplified XenNR2B fragment, respectively. Sequence differences of XenNR2B compared with rat NR2B are highlighted with black boxes. Note that the XenNR2B sequence shown does not contain the regions complementary to the degenerated primers used for screening.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Coexpression of the X. laevis KBP XenU1 with Rat NR1 Splice Variants in X. laevis Oocytes Does Not Generate a Unitary Glutamate Receptor. When Green et al. (2002) re-examined the unitary glutamate receptor, they could not confirm its existence, although they used the same methods as Soloviev et al. (1996): ligand binding studies, electrophysiological analysis in HEK293 cells and additionally in X. laevis oocytes, and coimmunoprecipitation experiments. However, proponents of the unitary glutamate receptor concept could criticize the study by Green et al. (2002) based on the fact that they used exclusively rat NR1-1a, which is a different NR1 splice variant and originates from a different species compared with the one used in the original reports (Soloviev et al., 1996, 1998). To rule out the existence of any possible splice variant-dependent interaction between NR1 and XenU1, we tested all eight rat NR1 splice variants (Hollmann et al., 1993) in our attempts to re-examine the postulated unitary glutamate receptor. However, none of the splice variants showed a hint of the reported unique unitary glutamate receptor pharmacology. We only confirmed the well-known glutamate/glycine-induced currents and additionally observed tiny currents induced by glycine alone, the latter of which were never observed by Soloviev et al. (1996). Because we used rat NR1 subunits in this splice variant comparison, we could not rule out a possible species-dependent interaction at this point.

Structural, Functional, and Subcellular Localization Analysis of XenNR1-4a and XenNR1-4b with and without XenU1 Fails to Detect Evidence for the Existence of the Unitary Glutamate Receptor. To investigate the exact same subunit combinations from the exact same species as used by Soloviev et al. (1996), we cloned two X. laevis NR1 splice variants: XenNR1-4a and XenNR1-4b. These subunits were identical to the published XenNR1 (Soloviev et al., 1996), except for one amino acid in position 166 in the N-terminal domain. The additional construction and electrophysiological investigation of XenNR1-4b(E166G), which is a clone encoding the exact same amino acid sequence as was used by Soloviev et al. (1996), showed that this single-amino acid deviation does not alter any pharmacological properties and did not lead to the formation of a unitary glutamate receptor when coexpressed with XenU1. Contrary to Soloviev et al. (1996), we found the a splice variant to be more abundant in X. laevis CNS than the b variant. A similar relationship had previously been shown for mammalian NR1 subunits (Sugihara et al., 1992; Hollmann and Heinemann, 1994). We confirmed the finding of Soloviev et al. (1996) that the N terminus of XenNR1 is 13% different from rat NR1. This sequence difference was suggested to alter the functional properties of the NR1 subunit, thus enabling XenNR1 to interact efficiently with XenU1 (Soloviev et al., 1996). Because a full characterization of XenNR1 has not been reported, we extensively characterized the functional properties of XenNR1-4a and XenNR1-4b. We found no functional differences between the X. laevis NR1 subunits and their rat homologs. The XenNR1 splice variants in our hands showed all the properties reported for rat NR1 subunits, such as activation by glutamate/glycine, NMDA/glycine, and glycine alone (Moriyoshi et al., 1991); glycine dependence of the induced currents (Kleckner and Dingledine, 1988; Laube et al., 1993); interaction with NR2 subunits (Ikeda et al., 1992; Kutsuwada et al., 1992; Meguro et al., 1992); similar EC₅₀ values; linear I/V curves in the absence of Mg²⁺; block by extracellular Mg²⁺ ions; and block by MK-801 (Hollmann et al., 1993). In addition, we found two alleged unique unitary glutamate receptor properties (Soloviev et al., 1996) to occur in both X. laevis and rat NMDA receptors in the absence of XenU1: although AMPA and DNQX indeed antagonize agonist-induced currents at XenNR1 plus XenU1, the same effect can be demonstrated for XenNR1 expressed alone as well as in coexpression with rat NR2B. We never saw current upon application of non-NMDA receptor agonists in oocytes expressing XenNR1 with or without XenU1. In HEK293 cells, we never detected currents upon application of glutamate/glycine, although the cells were transfected with the same subunit combination, XenNR1-4b plus XenU1, which Soloviev et al. (1996) had used in their studies in HEK293 cells where they had observed currents. We also recorded from cells transfected separately with XenNR1 (n = 31) and XenU1 (n = 13) as negative controls and never obtained currents. By contrast, Soloviev et al. (1996) detected in one of 27 and two of 89 cells unexplained currents for "homomeric" XenNR1 and XenU1 receptors, respectively (Soloviev et al., 1996). This is surprising because NR1 subunits expressed homomerically in mammalian cell lines in other studies have not been reported to form functional ion channels (Dingledine et al., 1999). Likewise, it had been reported that XenU1 does not form functional ion channels alone (Ishimaru et al., 1996), a property that is matched by the other five known KBPs (Henley, 1994; Hollmann, 1999). Using confocal microscopy, we showed that the lack of function in our experiments is not caused by a lack of expression. We demonstrated that XenNR1 and XenU1 are expressed in the plasma membrane and thus are in a position to interact to form the postulated unitary glutamate receptor. Therefore, as the subunit combinations XenNR1-4a plus XenU1 and XenNR1-4b plus XenU1 used by Soloviev et al. (1996) fail to generate a unitary glutamate receptor pharmacology despite proven membrane expression of all three subunits and proven functionality of XenNR1-4a and XenNR1-4b, we conclude that NR1 subunits do not form the postulated unitary glutamate receptor upon coexpression with XenU1.

The X. laevis KBP XenU1 Does Not Replace NR2 Subunits in the CNS of X. laevis. After Soloviev et al. (1996) postulated that XenU1 can interact with NR1 subunits to generate the unitary glutamate receptor, it was suggested that XenU1 may be a substitute for NR2 subunits in X. laevis CNS (Soloviev and Barnard, 1997). This hypothesis was supported by PCR-mediated screening experiments for X. laevis NR2 subunits, which did not lead to the identification of any such subunits (Soloviev and Barnard, 1997), despite evidence for their existence from the observed crossreactivity of an anti-rat NR2 antibody with X. laevis CNS proteins (Soloviev et al., 1996). However, when we used the same strategy as Soloviev and Barnard (1997) in screening experiments to identify an NR2 homolog in X. laevis brain, we did identify a fragment of a X. laevis NR2B subunit, which we termed XenNR2B. Likewise, in contrast to the report by Soloviev et al. (1996), our screening experiments revealed additional XenNR1 splice variants: a X. laevis homolog of the alternatively spliced exon 21 was identified, which proved the existence of XenNR1-3 splice variants. This indicates that X. laevis NR1 is likely to have the same gene structure and probably undergoes the same splicing events as known from rat NR1 (Hollmann et al., 1993). In addition, the existence of several other X. laevis glutamate receptor subunits has been verified: XenGluR1 and XenGluR2 (both in our laboratory; R. Trippe and M. Hollmann, unpublished observations); XenGluR5, and XenGluR6 (Ishimaru et al., 1996). Thus, it is possible to classify functional X. laevis glutamate receptors just like mammalian receptors into AMPA, kainate, and NMDA receptor subfamilies (Hollmann, 1999). XenU1 belongs to the subfamily of the KBPs, known from non-mammalian vertebrates such as fish, birds, and amphibians. This is supported by several distinct properties that XenU1 shares with other KBPs (Henley, 1994), such as the high affinity for kainate and AMPA (Ishimaru et al., 1996; Soloviev et al., 1996), a glutamate receptor-untypical short N terminus (Ishimaru et al., 1996), low sequence homology with other glutamate receptor subunits (Hollmann, 1999), and functional ion pore domains (Villmann et al., 1997), despite the fact that full-length KBPs apparently do not form functional homomeric ion channels. The fact that sequence identity of XenU1 is only 67.9% compared with another amphibian KBP (from Rana pipiens berlandieri) is not surprising because the sequence identities of KBPs in general are very low; even the two KBPs known from one species (goldfish) only share 60.9% sequence identity (Wo and Oswald, 1994; Hollmann, 1999). Therefore, the low sequence homology of XenU1 compared with other KBPs is no reason to classify XenU1 differently as postulated in Ishimaru et al. (1996) and Soloviev et al. (1996). In addition, if a unitary glutamate receptor exists, it is very unlikely that XenU1 is involved in its formation because most experiments that provided evidence for such a receptor type were done in the mammalian CNS (Jahr and Stevens, 1987) where no KBPs or XenU1 homologs have ever been identified (Hollmann, 1999). Thus, although the X. laevis KBP XenU1 is definitely not involved in the formation of a unitary glutamate receptor, its physiological role still poses an interesting question and remains to be elucidated.

	Acknowledgements

 
We thank Prof. Dr. Bernd-Joachim Benecke for providing the mouse anti-myc antibody, Christina Klein for expert technical help with HEK293 cell culture, and Björn Peters for oocyte preparations.

	Footnotes

 
This work was supported by the Deutsche Forschungsgemeinschaft Graduate School GRK 736.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.105.016840.

ABBREVIATIONS: iGluR, ionotropic glutamate receptor; CNS, central nervous system; NMDA, N-methyl-D-aspartate; AMPA, -amino-3-hydroxy-5-methyl-4-isoxazole propionate; KA, kainate; KBP, kainate binding protein; HEK, human embryonic kidney; PCR, polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction; bp, base pair(s); EGFP, enhanced green fluorescent protein; NFR, normal frog Ringer; I/V, current-voltage; MK-801, (–)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate; DNQX, 6,7-dinitrochinoxaline-2,3-dione.

Address correspondence to: Prof. Dr. M. Hollmann, Department of Biochemistry I-Receptor Biochemistry, Ruhr University Bochum, Universitaetsstrasse 150, Building NC, Level 6, Room 170, D-44789 Bochum, Germany. E-mail: michael.hollmann{at}rub.de

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