©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Topological Analysis of Goldfish Kainate Receptors Predicts Three Transmembrane Segments (*)

(Received for publication, August 3, 1994; and in revised form, October 26, 1994)

Z. Galen Wo (§) Robert E. Oswald (¶)

From the Department of Pharmacology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Glutamate receptors are the most abundant excitatory neurotransmitter receptors in vertebrate brain. We have previously cloned cDNAs encoding two homologous kainate receptors (GFKARalpha, 45 kDa, and GFKARbeta, 41 kDa) from goldfish brain and proposed a topology with three transmembrane domains (Wo, Z. G., and Oswald, R. E.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7154-7158). These studies have been extended using an in vitro translation/translocation system in conjunction with site-specific antibodies and point and deletion mutations. We report here that the entire region between the previously proposed third and fourth transmembrane segments is translocated and likely to be extracellular in mature receptors. This was based on the following results. 1) The entire segment was protected from Proteinase K and trypsin digestion and could be immunoprecipitated by a site-specific antibody. 2) Functional sites for N-glycosylation are present in the C-terminal half of the segment, and 3) a mutation, constructed with an additional consensus site for N-glycosylation in the N-terminal half of the segment, was found to be glycosylated at that site. Given the fact that the N terminus of the protein is likely to be extracellular, this would place an even number of transmembrane segments between the extracellular N terminus and the glycosylated segment. In addition, results of N-glycosylation and proteolysis protection assays of GFKARalpha mutations indicated that the previously proposed second transmembrane segment is not a true transmembrane domain. These results provide further evidence in support of a topology with three transmembrane domains that has important implications for the relationship of structure to function in ionotropic glutamate receptors.


INTRODUCTION

Glutamate receptors are the primary excitatory neurotransmitter receptors in the vertebrate central nervous system and are of importance to a wide variety of normal and pathological processes. The major forms of glutamate receptors are ligand-gated ion channels that have been subdivided into AMPA(^1)/kainate and NMDA receptors according to their selective agonists(1) . Recent advances achieved using molecular cloning and functional analysis have revealed a high degree of molecular and functional complexity and diversity(2, 3, 4) . Based on binding and the activation of channel activity, the AMPA receptor subunits correspond to GluR1 to GluR4, the kainate receptor subunits are GluR5 to GluR7 and KA-1 to KA-2, and the NMDA receptor subunits are NMDAR1 and NMDAR2A to NMDA2D. The individual subunits of these receptors are approximately 100 kDa. In addition to these functional ionotropic glutamate receptors, lower molecular mass kainate subunits (40-50 kDa; also referred to as kainate binding proteins) have been cloned from nonmammalian vertebrate brain(5, 6, 7) . These low molecular weight forms exhibit significant sequence homologies with the C-terminal portions of the much larger 100-kDa mammalian brain AMPA/kainate subunits. The sequence homology of these 40-50-kDa receptors is highest (35-40%) with kainate receptors (GluR5, GluR6, GluR7, KA-1, and KA-2 (2, 3) based on a BLAST (Basic Local Alignment Search Tool) search(8) ). Although ion channel activity has yet to be demonstrated in expression systems for these nonmammalian kainate receptors, they have been implicated in the process of synapse formation in the avian brain, as indicated by the finding that kainate receptor expression is induced by an imprint stimulus in the duckling hyperstriatum ventral(9) . Also, a recent report indicated that the affinity column-purified lower molecular weight glutamate receptor protein(s) from Xenopus brain can function as ion channels (10) . This suggests that the related kainate receptors from other nonmammalian vertebrates may also be functional, but suitable reconstitution and/or expression conditions to demonstrate functional activity have not yet been found. The sequence homologies and similar hydropathy plots suggest that their structures will be similar to the C-terminal portion of the AMPA/kainate receptors(11) . Therefore, determining the transmembrane topology of the 40-50-kDa kainate receptors should shed light on the structure of the 100-kDa ionotropic glutamate receptors.

Ionotropic glutamate receptors have long been assumed to belong to the superfamily of ligand-gated ion channels(2, 4) , with nicotinic acetylcholine receptors as the prototype. The proposed structural features of this family are four putative transmembrane domains (TMs), with both the N and C termini located extracellularly and with a large cytoplasmic loop between the third (TMIII) and fourth (TMIV) transmembrane domains(12) . In the case of glutamate receptors, recent evidence suggests that the four-TM model may be incorrect. (^2)Like other ligand-gated ion channels, the N terminus of glutamate receptors is likely to be located extracellularly since the presence of the cleavable N-terminal signal sequence will translocate the N-terminal region across the membrane of the endoplasmic reticulum at the first step of the biosynthetic pathway (13) . This notion is supported by the fact that, in some ionotropic glutamate receptor (GluR) subtypes, potential N-glycosylation sites are present in the N-terminal half of the protein, and these sites are glycosylated(14, 15, 16) . Also, regions in the N-terminal domain have been implicated in the binding of agonist, polyamines, redox agents, and zinc(17, 18, 19) . On the other hand, the C-terminal tail of a subtype of the NMDA receptor (NMDAR1) was shown using protein kinase C phosphorylation to be located intracellularly(20) , which is consistent with the earlier observation that the C-terminal tail of an NMDA receptor subunit mediates the sensitivity of the response to the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (which activates intracellular protein kinase C, 21) and that antibodies raised against the C-terminal peptide stain the cytoplasmic side of cells expressing rat brain AMPA/kainate receptors(22, 23, 24) . These findings indicate that ionotropic glutamate receptors have a unique topology. The extracellular N terminus and cytoplasmic C terminus imply that an odd number of transmembrane domains must be present, and models with both three (6) and five (3, 20, 25) transmembrane domains have been proposed.

Previously we reported (6) the cloning of two goldfish kainate receptor subunits, GFKARalpha (45 kDa) and GFKARbeta (41 kDa). The N-glycosylation of these two goldfish brain kainate receptors was examined using a rabbit reticulocyte lysate in vitro translation/translocation system supplemented with canine pancreatic microsomal membranes derived from endoplasmic reticulum. Asn-307 of GFKARalpha, an amino acid residue lying between TMIII and TMIV, was found to be N-glycosylated. The site homologous to Asn-307 in GluR6 (Asn-720) was also shown to be N-glycosylated(25, 26) . Therefore, at least a portion of the region between the putative TMIII and TMIV, previously thought to be cytoplasmic, must be extracellular. We showed further that deletion of the putative TMII segment does not affect the N-glycosylation of GFKARalpha, suggesting that this segment may not be a true transmembrane domain. Given these results and consideration of the hydrophobicity pattern, we proposed a model with three transmembrane domains (TMI, TMIII, and TMIV) for goldfish kainate receptors. On the other hand, to account for the reported phosphorylation of GluR6 at Ser-684 by protein kinase A(27, 28) , a five-TM model has been proposed(3, 25, 26) . The N-glycosylation of Asn-720 of GluR6 constrains the possible location of the proposed fifth TM segment between Ser-684 and Asn-720 in GluR6. As we noted previously(6) , this region (36 amino acids) is hydrophilic, with numerous charged residues, and is, therefore, not a likely candidate for a true transmembrane domain. The sidedness of this region has become a key issue for solving the transmembrane topology of ionotropic glutamate receptors.

In this study, we have explored the transmembrane topology of GFKARalpha and GFKARbeta further using an in vitro translation/translocation system. Two salient features of this system, N-glycosylation and protection from protease digestion, were used in conjunction with site/subunit-specific antibodies and point and deletion mutations. The results are consistent with our previously proposed three-TM model (6) and specifically demonstrate 1) that the entire segment between TMIII and TMIV is extracellular and 2) that only TMI, TMIII, and TMIV are true membrane-spanning regions.


EXPERIMENTAL PROCEDURES

Materials

L-[S]Methionine (1100 Ci/mmol) was purchased from Amersham Corp. Renaissance(TM) Western blot chemiluminescence reagent and Enhancer for fluorography were obtained from DuPont NEN. Protein A-Sepharose 4B was purchased from Sigma. Endo-beta-N-acetylglucosaminidase H (Endo H) was obtained from Boehringer Mannheim. Custom oligonucleotides were prepared by the Cornell Biotechnology Institute.

Peptide Synthesis and Antipeptide Antibody Production

Peptides and corresponding antipeptide antibodies were commercially prepared (Research Genetics, Huntsville, AL). The peptides corresponded to GFKARalpha residues 255-271 (SDNPTHHRIYEHIKNAQ) and GFKARbeta residues 256-272 (NPSYRRIYEHMERRKSF) and were synthesized with the Fmoc solid phase method using the multiple antigen peptide resin technology(29, 30) . Rabbit polyclonal antibodies were raised to these peptides.

In Vitro Transcription/Translation and Immunoprecipitation

Promega's TNT coupled rabbit reticulocyte lysate system was used as described previously(6) . In order to immunoprecipitate translated proteins, approximately 3 µl of translation reaction was solubilized in 22 µl of 2% SDS, boiled for 3 min and diluted in 125 µl of 0.1% Triton X-100 TSA buffer (100 mM Tris-HCl, 0.02% sodium azide, and 2 mM EDTA, pH 7.5). 5 µl of antiserum were added, and the mixture was incubated for at least 4 h at 4 °C with agitation. At the end of this period, 30 µl of a 1:1 mixture of Sepharose-coupled Protein A (prewashed with TSA buffer) was added, and the incubation was continued at 4 °C for at least 2 h with agitation. The Sepharose pellet was washed 3 times with 1 ml of wash buffer and aspirated to dryness. 20 µl of SDS-polyacryamide gel electrophoresis loading buffer with 10% beta-mercaptoethanol was added, and the sample was boiled for 3 min and analyzed on an SDS-polyacrylamide gel. Radioactivity was detected with fluorography using Enhancer and exposure to Kodak XAR film.

COS Cell Transfection and Immunoblotting

A modified DEAE-dextran protocol (31) was used for transient transfection of COS-7 cells, which were plated and grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. The cDNAs encoding GFKARalpha and GFKARbeta used in the transfection were in the pcDM8 vector (Invitrogen, CA). 48 h after the transfection, cells were incubated in 0.5 mM EDTA/PBS (PBS: 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na(2)HPO(4), 1.4 mM KH(2)PO(4), pH 7.3) at 37 °C for 15 min and harvested with a Pasteur pipette. After a 10-s homogenization, membrane pellets were centrifuged at 35,000 times g for 20 min. Pellets were washed by resuspension and centrifugation in 50 mM Tris-HCl, pH 7.5, followed by resuspension and storage at -20 °C. Western blotting was used to detect the expressed GFKARalpha and GFKARbeta proteins(32) . COS cell membrane pellets were solubilized in SDS and separated on 10% SDS-polyacryamide gel electrophoresis minigels; beta-mercaptoethanol (8%) was included in all samples. Proteins were then electrophoretically transferred to an Immobilon-P membrane (Millipore Corp., Bedford, MA). The membrane was incubated for 1 h at room temperature in blocking buffer (PBS, pH 7.2, containing 0.1% Tween-20 and 4.5% (w/v) instant powdered milk). The membrane was incubated overnight with a 1:3000 dilution of the antiserum in the same buffer. After two 15-min washes in PBS, pH 7.2, containing 0.1% Tween 20 and one wash in blocking buffer, the membrane was incubated for 2 h with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody, diluted 1:5000 in blocking buffer. Polypeptides recognized by the antipeptide antibodies were visualized using enhanced chemiluminescence.

Proteolysis of in Vitro Translated Proteins

Aliquots from the in vitro translation were prepared for digestion with Proteinase K (Sigma) or trypsin (N-tosyl-L-phenylalanyl chlormethyl ketone-treated; Calbiochem). To 9 µl of translation mixture, either 1 µl of water, 1 µl of 100 µg/ml Proteinase K (predigested for 30 min at 37 °C), or 1 µl of 100 µg/ml Proteinase K (or 1 mg/ml trypsin) plus 1.2 µl 10% Triton X-100 was added, and the digests were incubated on ice for 1 h. At the end of the digestion, phenylmethylsulfonyl fluoride was added to the reaction mixture to a concentration of 2 mM. The reactions with trypsin were terminated by the addition of 1 µl of 10 mg/ml soybean trypsin inhibitor. These mixtures were used directly for immunoprecipitation or were treated with Endo H prior to immunoprecipitation as described previously(6) .

Mutagenesis

Point and deletion mutations were introduced using PCR. 1) Preparation of the deletion mutant, alphaDeltaTMII, lacking the proposed TMII (FTLSHSFWYTMGAMTLQGA) was described previously(6) . 2) To construct a mutant lacking the proposed TMI (alphaDeltaTMI), 3 oligonucleotides were synthesized: GF10 (antisense, 5`-GGGAATTCAGTGGAGAACAGGCTC-3`), GF11 (sense, 5`-GGGAATTCTCACGGATTAGCCCGTGTG-3`), and GF12 (antisense, 5`-CGCCTTGGGGTGAGGACC-3`). The SP6/GF10 pair and the GF11/GF12 pair were used to amplify GFKARalpha cDNA by PCR. The SP6/GF10 PCR fragment (0.5 kb) was digested with BamHI and EcoRI; the GF11/GF12 PCR fragment (1.6 kb) was digested with EcoRI and StyI. These two PCR fragments were ligated back into the pcDNAII vector containing the remainder of the GFKARalpha cDNA with BamHI and StyI ends, creating the deletion of 19 amino acid residues (MWMGVLVAYLLTSVCIFLV, the proposed TMI). 3) To construct the deletion mutant of the proposed TMIII from GFKARalpha (alphaDeltaTMIII), an oligonucleotide (sense, 5`-GGCCAAGGCCAGTTTATGGCTGCATTC-3`) was synthesized and used with T7 primer to amplify the GFKARalpha cDNA by PCR. The PCR fragment (1.3 kb) was digested by StyI and XhoI and ligated into the pcDNAII vector containing the N-terminal portion of the GFKARalpha cDNA (XhoI digest and StyI partial digest), generating a mutant lacking 25 amino acid residues (LSGRVITSIWWLFSLVLLACYFANL). 4) To construct the alphabeta(N279GT) and alphabeta(NKT439) mutations, an alphabeta chimeric cDNA was first prepared. The SP6 primer and an antisense oligonucleotide (5`-GGTGAATTCAGTGAATAGCTCTGG-3`) were used to amplify the N-terminal portion of GFKARalpha cDNA by PCR, and the resulting PCR fragment (0.9 kb) was digested with BamHI and EcoRI. A sense oligonucleotide (5`-CGGAATTCGGATGAGGGTGTCCAG-3`) and T7 primer were used to PCR amplify the C-terminal portion of GFKARbeta cDNA, and the PCR fragment (1.1 kb) was digested by EcoRI and XhoI. The pcDNAII vector with BamHI and XhoI ends was ligated with the two prepared PCR fragments, generating a chimeric construct encoding alpha(1-276)beta(277-438). To introduce a new consensus N-glycosylation site at beta279, a sense oligonucleotide (5`-CGGAATTCGGATAACGGTACCCAGAAAGCA-3`) was paired with T7 primer to amplify the C-terminal portion of GFKARbeta cDNA. The PCR fragment (1.1 kb) was digested with EcoRI and XhoI, ligated into the pcDNAII vector containing the N-terminal portion of the alphabeta chimeric cDNA. To introduce a new consensus N-glycosylation site at beta439, a antisense oligonucleotide (5`-GGCTCGAGTTATGTCTTGTTTTTGTC-3`) was paired with GF8 primer to PCR amplify the C-terminal portion of GFKARbeta cDNA, and the PCR fragment (500 base pairs) was digested with EcoRI and XhoI. This PCR fragment was used to replace the C-terminal beta-portion of alphabeta chimera cDNA, generating Ala-439 Thr mutation (a consensus N-glycosylation site NKT439). 5) To construct beta(K186A) mutant, two sequential PCR steps were used. Two oligonucleotides (GF21 (antisense), 5`-TCCTGACACAGCTGCAGGGTG-3` and GF22 (sense) 5`-CCTCACCCTGCAGCTGTGTCA-3`) were synthesized. SP6/GF21 and GF22/T7 primer pairs were used to amplify GFKARbeta cDNA separately. The two PCR fragments (720 and 1320 base pairs) were purified and then placed in the same tube and amplified in a second PCR step (using SP6 and T7 primers only). The full-length fragment (2 kb, with Lys-186 Ala mutation) generated was digested with BamHI and XhoI and subcloned into pcDNAII vector. Note that the primers GF21 and GF22 overlap by 18 bases. In each case above, the restriction enzyme site in oligonucleotides used for ligation after PCR amplification is indicated by an underline. All mutations were confirmed by DNA sequencing.


RESULTS

Design of the Internal Peptide

Our recently described three-TM model places the region between the proposed TMIII and TMIV on the extracellular side of the membrane. On the other hand, the reported phosphorylation of Ser-684 of GluR6 (which corresponds to Ser-272 in GFKARalpha and Ser-271 in GFKARbeta) by protein kinase A suggests that the region around the Ser-684 of GluR6 is on the cytoplasmic side. Given the significant sequence homologies among GFKARalpha, GFKARbeta, and GluR6, one would expect the same topological location of this region in both types of receptor protein. Therefore, determination of the location of this region (Fig. 1) has become a key issue in distinguishing between the proposed three-TM and five-TM models. Based on the amino acid sequence, the method of Hopp and Wood(33, 34) predicts that the region between residues 250 and 275 in GFKARalpha and GFKARbeta has strong antigenicity. Therefore, two peptides containing 17 amino acid residues from the deduced sequences of GFKARalpha and GFKARbeta were synthesized using the multiple antigen peptide system, and antibodies were raised against these sequences. This region is not conserved between GFKARalpha and GFKARbeta; thus, corresponding antipeptide antibodies would be expected to be both site- and subunit-specific.


Figure 1: Amino acid sequence alignment of GFKARalpha and GFKARbeta with rat 100-kDa ionotropic AMPA (GluR1) and kainate receptor (GluR6) subunits in a key region between the proposed TMIII and TMIV. Conserved residues are shown white on black. Consensus sites for N-glycosylation (g), Asn-307 of GFKARalpha, and Asn-720 of GluR6, are in boldface. The reported phosphorylation site (p) of GluR6, Ser-684, is shown in italic. E279GV of the alphabeta chimera was mutated to N279GT to introduce a consensus N-glycosylation site (g(new)) and is shadowed. The underlined sequences of GFKARalpha and GFKARbeta were used to design the synthetic peptides.



Characterization of Site-specific Antibodies

Antiserum (termed Ab-alpha1 and Ab-beta1) raised against the two synthetic peptides were initially evaluated by enzyme-linked immunosorbent assay. Each peptide antibody binds to the corresponding peptide and not the other peptide. In contrast, preimmune serum did not bind significantly to either peptide (data not shown). The site-specific antibodies thus exhibited a high titer and specificity for recognition of their corresponding target peptides.

Binding of the site-specific antibodies to in vitro translated GFKARalpha and GFKARbeta proteins was examined using immunoprecipitation. We had reported previously that GFKARalpha and GFKARbeta, translated in vitro, are 41-kDa proteins in the absence of microsomal membranes. In the presence of microsomal membranes, two N-glycosylated GFKARalpha species of approximately 45 kDa are routinely observed. The in vitro translation products from both subunits could be effectively immunoprecipitated by the corresponding antipeptide antiserum. Ab-alpha1 recognizes only the nonglycosylated and N-glycosylated GFKARalpha (Fig. 2A, lanes1 and 2) but not GFKARbeta (Fig. 2A, lanes5 and 6). Ab-beta1 recognizes only the translated GFKARbeta in the absence and presence of microsomal membranes (Fig. 2A, lanes9 and 10). No detectable translated products were immunoprecipitated in the presence of corresponding peptide immunogen (Fig. 2A, lanes3 and 4 for Ab-alpha1 and lanes11 and 12 for Ab-beta1).


Figure 2: A, immunoprecipitation of GFKARalpha and GFKARbeta proteins translated in vitro in the absence or presence of microsomal membranes (M.M.). The asterisk indicates a glycosylated species. Peptide immunogen refers to the addition of an excess of the multiple antigen peptide peptide used in the production of the corresponding antibodies. B, immunoblotting of GFKARalpha (45 kDa) and GFKARbeta (41 kDa) from goldfish brain (GF) and from COS cells transiently expressing GFKARalpha (Calpha) or GFKARbeta (Cbeta). The asterisk indicates a glycosylated species expressed in COS cells. Detectable signals are seen with the corresponding antiserum (Ab-alpha1 or Ab-beta1) diluted 1:3000.



The specificity of the two antipeptide antibodies for GFKARalpha and GFKARbeta was also established with an immunoblotting assay. Membranes were prepared from goldfish brain and COS cells transfected with GFKARalpha and GFKARbeta. When these membrane proteins were probed with Ab-alpha1 and Ab-beta1 antiserum (1:3000 dilution), specific immunoreactivity was detected. Ab-alpha1 recognizes the 45-kDa protein in both goldfish brain membranes and GFKARalpha-transfected COS cells (Fig. 2B, lanes1 and 2). Ab-beta1 recognizes the 41-kDa protein in both goldfish brain membranes and GFKARbeta-transfected COS cells (Fig. 2B, lanes4 and 6). The doublet observed for transfected COS cells is probably the result of different degrees of glycosylation because under-glycosylation sometimes occurs in COS cells(35) . These specific immunoreactivities were abolished using corresponding competing peptide (Fig. 2B, lanes4-6 and 10-12). Thus, Ab-alpha1 and Ab-beta1 could be used selectively to identify the 45- and 41-kDa kainate receptor subunits from goldfish brain, transfected COS cells, and in vitro translation. We demonstrated, by comparison of the protein sequence and DNA sequence(6, 32) , that GFKARbeta encodes the 41-kDa kainate receptor subunit and suggested that GFKARalpha is likely the cDNA for the 45-kDa kainate receptor subunit from goldfish brain. The observation that Ab-alpha1 indeed recognizes specifically a 45-kDa protein of goldfish brain membrane and GFKARalpha-transfected COS cells and that the 45-kDa protein is an N-glycoprotein both in vivo and in vitro further supports the assertion that GFKARalpha encodes the previously purified 45-kDa kainate receptor subunit from goldfish brain.

Number of Functional N-Glycosylation Sites in GFKARalpha and GFKARbeta

The uniform core oligosaccharides transferred from a lipid donor to acceptor Asn residues contains 3 glucose, 9 mannose, and 2 N-acetylglucosamine residues(36) . Each oligosaccharide chain has an apparent molecular mass of approximately 3 kDa(37) . Using the improved resolution of 8 versus 10% SDS-polyacrylamide gels, we were able to separate nonglycosylated, partially glycosylated, and fully glycosylated species of in vitro translated proteins. We showed previously that Asn-307 of GFKARalpha is a functional N-glycosylation site(6) . We have now determined that GFKARalpha has three oligosaccharide chains, whereas, GFKARbeta is actually N-glycosylated, with one oligosaccharide chain (Fig. 3A, lanes1 and 3). In the GFKARalpha(N307D) mutant, two functional sites are left (Fig. 3A, lane2). This would suggest that, in addition to Asn-307, Asn-333 and Asn-340 are glycosylated in GFKARalpha, and Asn-332 is glycosylated in GFKARbeta. Asn-212 is not a functional glycosylation site because truncated GFKARalpha translated from BstNI-cut cDNA (i.e. truncation just before Asn-307 at position 299) was not glycosylated(6) . The most likely reason for this is that Asn-212 is very near the end of TMIII, and a minimum distance of 12-14 residues from the lumenal surface of the endoplasmic reticulum membrane is required for efficient glycosylation(38, 39) . The glycosylation efficiency of Asn-307 seems to be much higher than that of Asn-333 and Asn-340 because no g(1) species were ever observed for GFKARalpha, but when Asn-307 was deleted (alpha(N307A) mutant), more g(1) species were observed than g(2) species (see Fig. 3A). This is consistent with the observation that most N-glycosylated sites are in the N-terminal part of an extracellular region(38, 40) .


Figure 3: A, Synthesis and N-glycosylation of native GFKARalpha and GFKARbeta as well as GFKARalpha(N307D). The notation, g(0)-g(3), denotes 0-3 oligosaccharide chains attached to the protein. B, synthesis and N-glycosylation of GFKARalpha, the alphabeta chimera, the alphabeta(N278GT) mutant, and the alphabeta(NKT439) mutant in the presence and absence of microsomal membranes (MM). The consensus N-glycosylation sites, N278GT and NKT439, were introduced to determine which would be glycosylated (translocated). Endo H treatment removes attached oligosaccharide chains. In B, asterisks indicate the glycosylated species.



N-Glycosylation of alphabeta(N279GT) Mutant

An alphabeta chimera was initially constructed as the reciprocal mutation of the betaalpha chimera(6) . The alphabeta chimera has the N-glycosylation pattern of GFKARbeta, and likewise the betaalpha chimera has the glycosylation pattern of GFKARalpha. These two chimeras thus provided further evidence that the functional N-glycosylation sites reside in the C-terminal half of GFKARalpha and GFKARbeta rather than in the N-terminal extracellular domain. For convenience, the alphabeta chimera was used to add an additional consensus site for N-glycosylation at the beta279 site (Fig. 1). This site is 6 amino acids from Ser-272, which is homologous to Ser-684 of GluR6 (i.e. the site assumed to be phosphorylated by protein kinase A). Using the in vitro system in the presence of microsomes, both constructs were glycosylated, with the alphabeta chimera having a mobility consistent with the attachment of one oligosaccharide chain and the alphabeta(N279GT) mutation mobility consistent with the attachment of two chains (Fig. 3C, lanes5 and 8). This indicates that the consensus site introduced at alphabeta279 is N-glycosylated and likewise, the segment surrounding this site must be translocated into the microsomes. In addition, the lack of a species with one glycosylation for the alphabeta(N279GT) mutant (Fig. 3, lane5) indicated that this newly introduced consensus site is very efficient. A new glycosylation site (NKT439) was introduced near the C terminus of the alphabeta chimera by mutation (Ala-439 Thr). In vitro translation of the alphabeta(NKT439) mutant in the presence of microsomes resulted in a protein of the same molecular weight as the alphabeta chimera (Fig. 3, lane11), indicating NKT439 is not used, consistent with the predicted cytoplasmic location of the C terminus.

Protease Digestion of Expressed GFKARalpha and GFKARbeta

In order to determine the transmembrane location of the epitopes recognized by the antibodies, the microsomes containing the newly synthesized polypeptides were subject to proteolytic digestion with Proteinase K and immunoprecipitated with corresponding antiserum. When GFKARalpha was treated with Proteinase K, two peptides (30 and 27 kDa) were specifically recovered by Ab-alpha1 (Fig. 4A, lane4). These two peptides were converted into a single species of 20 kDa by Endo H (Fig. 4A, lane5), indicating that the two peptides are N-glycosylated. Likewise the alpha(N307D) mutant shows two glycosylated fragments consistent with one fewer oligosaccharide chain than GFKARalpha (Fig. 4A, lane1). When GFKARbeta was treated with Proteinase K, a peptide of 24 kDa was recovered by Ab-beta1 (Fig. 4A, lane8). This peptide was converted into a 20-kDa species by Endo H (Fig. 4A, lane9), indicating that it is also N-glycosylated. The observed mobility differences between these peptides are consistent with the number of attached oligosaccharide chains in GFKARalpha and GFKARbeta. No immunoreactive peptides were recovered (Fig. 4A, lanes6 and 10) upon the addition of Triton X-100 prior to digestion when the microsomes are permeable to proteases. The same patterns of proteolytic peptides were recovered when GFKARalpha and GFKARbeta were treated with trypsin rather than Proteinase K (data not shown). The size of the fragments recovered from proteolysis of GFKARalpha and GFKARbeta, the observation that the protected fragment carries the expected oligosaccharide chains, and the fact that the alphabeta(N279GT) mutant is glycosylated together strongly indicate that the epitope specifically recognized by antipeptide antibodies is translocated extracellularly, consistent with prediction of our proposed three-TM model.


Figure 4: A, proteolysis and immunoprecipitation of native GFKARalpha and GFKARbeta proteins and the alpha(N307D) mutant. Proteins were expressed in vitro in the presence of microsomes. The translation mixtures were either untreated or digested with Proteinase K in the absence or presence of Triton X-100. Proteinase K-treated samples were divided for treatment with Endo H as described under ``Experimental Procedures.'' Samples were then immunoprecipitated with the corresponding antiserum (Ab-alpha1 for GFKARalpha and alpha(N307D) mutant, Ab-beta1 for GFKARbeta). The asterisk indicates a glycosylated species, and the arrow indicates a deglycosylated species. B, proteolysis and immunoprecipitation of native GFKARbeta and the beta(K186A) mutant.



The above proteolytic patterns suggest that the region between the TMI and TMIII may be the target of Proteinase K and trypsin. Proteinase K primarily cleaves following the carboxyl group of hydrophobic aliphatic and aromatic amino acids; whereas, trypsin specifically catalyzes the hydrolysis of peptide bonds at the carbonyl group of the basic amino acids, Arg and Lys. In GFKARbeta, only three basic residues are present between TMI and TMIII: Arg-148, Lys-186, and Arg-191. Arg-148 is located at the end of TMI and Arg-191 is present at the beginning of TMIII, placing them close to the surface of the membrane and possibly not easily accessible to trypsin. Therefore, we reasoned that Lys-186 could be a primary target for trypsin and constructed a mutant to remove this potential site for trypsin action. The beta(K186A) mutant is expressed and glycosylated in a manner indistinguishable from native GFKARbeta. Also, proteolysis by Proteinase K is identical to the wild-type protein (Fig. 4B, lane2versus6). In the case of trypsin, however, the pattern is changed (Fig. 4B, lane3versus7). More than 50% of the protein is essentially intact (approximately 42 kDa), with the remainder of the protein present as two smaller bands (20 and 24 kDa). The most likely interpretation of these results is that Lys-186 is the primary site of trypsin digestion but that Arg-148 and Arg-191 may also be accessible to trypsin. This would suggest that Lys-186 is present on the cytoplasmic side of the membrane and that TMIII traverses the membrane from the intracellular to the extracellular side.

Expression and N-Glycosylation of GFKARalpha Deletion Mutants

The Proteinase K digestion and immunoprecipitation by site-specific antibodies as well as the observation that alphabeta(N279GT) mutant forms an additional functional N-glycosylation site indicate that the whole region between the proposed TMIII and TMIV is translocated at the endoplasmic reticulum (extracellular in mature protein). Therefore, an odd number of transmembrane segment(s) exist between the extracellular N terminus and the proposed TMIII. Our previous observations indicate that the proposed TMII may not be a true transmembrane domain. We extended this finding by deleting the proposed TMI and TMIII from GFKARalpha. Results of the N-glycosylation of in vitro synthesized GFKARalpha deletion mutations are shown in Fig. 5A. Deletion of the proposed TMI or TMIII rendered the proteins largely nonglycosylated (although some glycosylated forms remained at least in the case of alphaDeltaTMIII; Fig. 5A, lanes3 and 9); whereas, deletion of the proposed TMII retained the N-glycosylation pattern of wild-type GFKARalpha (Fig. 5A, lane6). This suggests that the deletion of TMI and TMIII, but not TMII, changed the transmembrane topology of the mutant proteins.


Figure 5: A, in vitro synthesis and N-glycosylation of three GFKARalpha deletion mutations (alphaDeltaTMI, alphaDeltaTMII, alphaDeltaTMIII) in the presence or absence of microsomal membranes (MM). Endo H treatment removes attached oligosaccharide chains. Positions of glycosylated subunits are indicated by asterisks. B, proteolysis (trypsin, Proteinase K) and immunoprecipitation of the GFKARalpha deletion mutations with Ab-alpha1. Positions of the protected proteolytic fragments are indicated by asterisks. C, schematics showing the possible transmembrane arrangements of the deletion mutations consistent with the N-glycosylation and proteolytic results. Glycosylated consensus sites are indicated by circle and those consensus sites that are not glycosylated are shown by . Arrangements for which the epitope recognized by Ab-alpha1 is proteolyzed are indicated by an opentriangle and those for which the epitope is protected are shown with a filledtriangle.



Protease Protection of GFKARalpha Deletion Mutants

The deletion mutants were studied further using protease protection assays. As described above, wild-type GFKARalpha exhibits immunoprecipitated peptides of 27 and 30 kDa following Proteinase K or trypsin treatment (Fig. 5B, lane2). In the case of alphaDeltaTMI, no immunoprecipitated fragment was observed following treatment with either Proteinase K or trypsin (Fig. 5B, lanes4 and 5). This is consistent with the finding that this deletion mutant is largely nonglycosylated and further supports the notion that TMI is a topogenic element, the deletion of which prevents the translocation of the consensus sites for N-glycosylation and exposes the epitope to the protease. The deletion mutant alphaDeltaTMII exhibits a pattern for Proteinase K that is different from trypsin digestion. In the case of Proteinase K, the 27-30-kDa fragments were not observed (Fig. 5B, lane7). Trypsin, on the other hand, produces fragments of alphaDeltaTMII (Fig. 5B, lane9) that are similar to those obtained for native GFKARalpha (data not shown). The trypsin fragments for alphaDeltaTMII and native GFKARalpha are slightly smaller than those obtained with Proteinase K, probably as a result of the complete cleavage of the C-terminal fragment by trypsin (the C-terminal region is rich in Arg and Lys residues). The simplest interpretation of these results is that in the region between TMI and TMIII only TMII is sensitive to Proteinase K treatment so that removal of TMII renders this region insensitive to the protease. This would suggest that at least a portion of TMII is cytoplasmic and accessible to protease when translated in vitro. The alphaDeltaTMIII mutant is somewhat more complicated. Approximately 60% of the protein is nonglycosylated. Treatment with Proteinase K or trypsin removes the nonglycosylated forms, as would be expected if they were not translocated, but a fraction that is glycosylated remains. Interestingly, Proteinase K treatment did not result in the 27- and 30-kDa fragments but left essentially full-length glycosylated forms that are only slightly decreased in size (Fig. 5B, lane12). Trypsin produced minor glycosylated fragments slightly larger than those produced with GFKARalpha (Fig. 5B, lane14). This suggests that in the majority of cases, deletion of TMIII stops the translocation of the region between TMIII and TMIV and, thus, is a topogenic element. However, in some cases, this region is translocated, possibly by having TMII, or some other region, serve anomalously as a TM segment (see ``Discussion''), consistent with the lack of sensitivity to Proteinase K.


DISCUSSION

We have extended our analysis of transmembrane topology of the goldfish kainate receptors(6) . The two major conclusions are that the 166-amino acid region between the proposed TMIII and TMIV is entirely translocated across the endoplasmic reticulum membrane and that the previously proposed TMII is not likely to be a transmembrane helix. The first conclusion is based on the following observations. 1) Three consensus sites for N-glycosylation are present in the C-terminal half of this segment for GFKARalpha and one is present in GFKARbeta. All of these consensus sites are glycosylated. 2) A site engineered into the N-terminal half of an alphabeta chimera (N279GT) can also be N-glycosylated. 3) In an in vitro translation/translocation system, site-specific antibodies raised against peptides in the middle of this segment can, following proteolysis with Proteinase K or trypsin, immunoprecipitate N-glycosylated 24-30-kDa peptides. This would be the size expected if the segment between TMIII and TMIV were entirely translocated into the lumen of the endoplasmic reticulum and thus protected from proteolysis. The second conclusion, concerning the nature of TMII, is based on the following observations. 1) The finding that the segment between TMIII and TMIV is extracellular would suggest that an even number of transmembrane segments (probably 2 or 4) exists between the extracellular N terminus and that segment. This would mean either that one of the originally proposed TM segments does not traverse the membrane or that an additional TM segment is present. 2) Deletion of TMII has no effect on the N-glycosylation pattern, suggesting that it is not a topogenic element, and 3) the deletion of either TMI or TMIII affects the translocation of the region between TMIII and TMIV and, thus, changes the N-glycosylation pattern. 4) Lys-186 of GFKARbeta is the primary site of trypsin digestion. These results lend further evidence in support of the three-TM model (Fig. 6), which we proposed previously(6) .


Figure 6: Schematic depiction of the proposed transmembrane topology (3-TM model) of GFKARalpha and GFKARbeta. The entire region between the TMIII and TMIV is on the extracellular side of the plasma membrane because it is protected from protease digestion and the four marked N-glycosylation sites are functional. The three native consensus sites are indicated by circle and the site introduced by the mutation at position 279 is indicated by bullet. The sequence from which the antibodies were raised is indicated with the boldfaceline and indicated by Ab. We postulate that in vivo after subunit folding and assembly of the receptor, the TMII region may be associated with the ion channel pore but not necessarily in contact with the membrane.



The finding that the entire segment between TMIII and TMIV is extracellular is not consistent with the reported phosphorylation of Ser-684 in GluR6. In particular, the newly engineered consensus site, only 6 amino acids C-terminal to the amino acid corresponding to Ser-684, was found to be N-glycosylated. Raymond and co-workers (27) indicate that phosphorylation of Ser-648 alone is sufficient to enhance responses to glutamate, whereas Wang and co-workers (28) suggest that phosphorylation at both Ser-684 and Ser-666 is required. The reasons for the discrepancy between the phosphorylation and glycosylation results are unclear; however, it is noteworthy that in the case of the beta-amyloid precursor protein, phosphorylation at an extracellular site in a transfected system has been observed(41) . Also, the potential role of the consensus protein kinase A sites in the intracellular C-terminal domain of GluR6 has not been investigated. Several lines of evidence, however, argue against the intracellular location of Ser-684 in GluR6 and the corresponding positions in GFKARalpha and GFKARbeta. 1) To account for the N-glycosylation of Asn-720 (extracellular) and the reported phosphorylation of Ser-684 (assumed to be cytoplasmic) of GluR6, the proposed five-TM model must place a transmembrane domain between Ser-684 and Asn-720(25, 26) . A membrane-spanning domain in this portion of the protein is unlikely because this region (35 amino acids) is very hydrophilic, with 7 charged amino acids. 2) The length of a transmembrane helix is approximately 20 amino acids. If this region contains a true transmembrane domain, Ser-684 and Asp-720 of GluR6 would be very close to each side of the membrane surface. However, a minimum distance of 12-14 residues from the lumenal surface of the endoplasmic reticulum membrane is required for efficient glycosylation (39) . 3) Transmembrane domains are important structural components, and insertions or deletions in transmembrane domains have not been observed within the same family, including other ligand-gated ion channels(42, 43) . However, between the residues corresponding to Ser-684 and Asn-720, a KG sequence in GluR1-GluR4 is absent in GluR5-GluR7, KA-1 and -2, and the 40-50-kDa kainate receptors. The KG insertion is 16 residues C-terminal to Ser-684 and 19 residues N-terminal to Asn-720. 4) At least in the goldfish receptors, a mutation (alphabeta(N279GT)) that creates a consensus site for N-glycosylation only 6 amino acids C-terminal to the position corresponding to Ser-684 is in fact glycosylated, suggesting that it is extracellularly located. Likewise, the region just before TMIII is likely to be cytoplasmic based on the beta(K186) mutant, which modifies the susceptibility to trypsin. Based on these points, we propose that the entire region between TMIII and TMIV is likely to be located on the extracellular side of the membrane.

The central reason for studying transmembrane topology is to provide insights into the structure of the receptors and the relationship between structure and function. Our proposed transmembrane topology (three-TM model) for ionotropic glutamate receptors is related to the formation of the channel pore, the intracellular regulatory sites, and the structure of the ligand binding site of this class of receptors. For other ligand-gated ion channels (e.g. the nicotinic acetylcholine receptor), significant divergence in sequence and length is observed in the cytoplasmic loop separating TMIII and TMIV. In contrast, this region is of fixed length and shows greater than 80% sequence identity among 100-kDa glutamate receptor subunits and 35% sequence identity between the 40-50-kDa kainate receptors and 100-kDa ionotropic glutamate receptors. This striking conservation unique to glutamate receptors indicates that this region may be structurally constrained. The residues in the N-terminal extracellular domain and in the loop between TMIII and TMIV are homologous to the Escherichia coli periplasmic glutamate binding protein(11, 44) ; and a two-domain glutamate binding site has been suggested by us and others (3, 6, 44, 45) . Interaction with the same endogenous ligand (glutamate) might be the structural constraint necessary to keep this region conserved. Strong support for this model comes from the analysis of the glycine binding site on the NMDA receptor, which showed that mutations both in the N-terminal extracellular domain and in the region between TMIII and TMIV can affect the action of glycine(45) .

Based on hydropathy analysis (46) and presumed homologies to other ligand-gated ion channels, cloned GluR subunits have been assumed, until recently, to have five TM segments (i.e. the signal sequence plus four membrane spanning regions found in the mature protein). Inspection of the hydropathy plots for ionotropic glutamate receptor subunits in many cases reveals only four regions of high hydrophobicity comprised of the N-terminal signal sequence and three regions of sufficient length to form alpha-helical transmembrane domains (i.e. the proposed TMI, TMIII, and TMIV). The proposed TMII in iGluRs is often only weakly hydrophobic. In fact, as pointed out by Hollmann and Heinemann(2) , a segment preceding TMI is as hydrophobic as the proposed TMII. One can argue, based on the hydropathy analysis alone, that: 1) both the TMII segment and the segment before the proposed TMI act as transmembrane domains, or 2) neither are transmembrane domains (3-TM model) because of their low hydrophobicity. Thus, no definitive statement can be made without the benefit of additional experimental data.

We have used an in vitro translation system to analyze the transmembrane orientation of the polypeptide chain. In an in vitro translation system supplemented with microsomes, previous results have shown that receptor subunits are synthesized, inserted into the membrane, glycosylated, and undergo signal sequence processing in a manner similar to that observed in mammalian cells(47) . The transmembrane orientation assumed by the newly synthesized subunits, at least for native proteins, is generally identical to that of the subunits in the mature receptor complex. Where tested, the pattern of glycosylation of GFKARalpha and GFKARbeta that we observe is the same in the in vitro translation system as it is for the protein transiently expressed in COS cells. It has also been demonstrated that, in the case of the nicotinic acetylcholine receptor, the introduction of new glycosylation sites can provide valuable information concerning the topographical location of particular sites in the primary sequence of transmembrane proteins (48

Our use of the native GFKARalpha and GFKARbeta proteins, in conjunction with immunoprecipitation, for the protease protection experiments is a variant of the widely used reporter domain technique for studying transmembrane topology(47) . The region in GFKARalpha and GFKARbeta containing the sequence of synthetic peptide to which antibodies were raised is, in effect, a reporter domain specifically recognized by its corresponding antibody. In this way, we avoided the concern that a foreign reporter domain might disrupt the normal topology of the protein(49) , which is a reasonable concern for artificial constructs (see below). Our three-TM model places TMII on the cytoplasmic side of the membrane so that the region between TMI and TMIII is not translocated and may be a target for protease digestion. The observed patterns of Proteinase K and trypsin digestion in GFKARalpha and alphaDeltaTMII were consistent with this arrangement. Both Proteinase K and trypsin digestion of GFKARalpha gave a similar pattern of protected peptides, but alphaDeltaTMII is resistant to Proteinase K and sensitive to trypsin. One possibility is that TMII of GFKARalpha is the target of Proteinase K digestion. When it is deleted, the loop between TMI and TMIII is shortened to 28 amino acids. This region is very hydrophilic, with 5 Arg/Lys residues, making it susceptible to trypsin but possibly not Proteinase K. It may become resistant to Proteinase K either due to the lack of hydrophobic residues or potentially to the shortening in length, as has been observed previously(47) . Inspection of the two short hydrophilic segments flanking TMII may also yield clues to its transmembrane orientation in that the surrounding charges can affect the transmembrane behavior of a hydrophobic segment(50) . In general, positive charges (Lys, Arg) tend to remain in nontranslocated regions. Particularly for proteins with multiple transmembrane domains, the charge bias is apparent for hydrophilic segments shorter than 70 or 80 residues(13) . The small polar segment (approximately 11 amino acids) between TMII and TMIII is positively charged, with 2 or 3 Arg or Lys residues. Both the four-TM and five-TM models place it extracellularly (i.e. the models assume that it is translocated). Our observation that TMII seems not to act as a true membrane spanning region is consistent with the distribution of charges. Perhaps, the combination of the low hydrophobicity and the distribution of surrounding charges prevent TMII from functioning as a true transmembrane domain in the coupled translation/translocation step.

Unlike native proteins, deletion of the proposed TMI, TMII, or TMIII from native GFKARalpha-generated artificial constructs. The region carrying the previously identified functional N-glycosylation sites becomes the endogenous reporter domain, and its membrane orientation, and thus N-glycosylation, is determined by topogenic elements preceding it in the sequence. Generally, the removal of upstream topogenic domains affects the downstream region, whereas the removal of downstream topogenic elements does not affect the topology of upstream regions(47) . Our results showed that the region between the proposed TMIII and TMIV is translocated (i.e. it corresponds to an extracellular domain); therefore, an even number of transmembrane segments must exist between this region and the N-terminal extracellular domain (either two- or four-TM segments). Deletion of TMII does not affect the N-glycosylation; however, deletion of TMI or TMIII does affect the N-glycosylation of the mutant proteins. This would suggest that TMII may not be a true transmembrane segment in the native proteins, but that TMI and TMIII are true transmembrane segments. Nevertheless, the cases for alphaDeltaTMI and alphaDeltaTMIII are somewhat complicated in that, particularly for alphaDeltaTMIII, a mixture of forms was present (Fig. 5A, lane11). In general, topogenic sequences act in an absolute manner, directing the nascent polypeptide to a single topologic form. However, in a number of artificial protein constructs, topogenic elements may act in a less than absolute fashion, producing more than one topogenic form(51, 52, 53, 54) . Thus, although the major species in each case was consistent with the prediction of the three-TM model, alternative forms may have arisen since the deletions produced artificial constructs.

Membrane-associated regions that arise from subsequent rearrangements during subunit folding and assembly, however, may not be detected in the above approaches. The P-segment of voltage-gated K channels (55, 56, 57) and several membrane segments (M5, M6, and M7) of the H,K-ATPase (58) are such examples. To account for its role in the ion permeation process, we previously speculated that the proposed TMII, although not a true transmembrane domain, may insert itself into the channel pore of the receptor complex(6) . However, this type of ``membrane domain'' is distinct from a true transmembrane-spanning segment in the following ways. 1) The two types of structure arise differently. True transmembrane domains of eukaryotic membrane proteins are inserted in the coupled translation/translocation process. The insertion occurs in a sequential fashion from the N-terminal to the C-terminal(37) . A ``membrane domain'' such as the P-segment in the K channel arises from subunit folding and assembly. It may not have any contact with the membrane lipid bilayer at all. 2) True transmembrane-spanning regions are topogenic elements that exert their effects on downstream regions of the polypeptide. In contrast, the second type of ``membrane domain'' has no topological effects on the sequences following it. Therefore, identification of the second type of ``membrane domain'' is an issue of protein folding/assembly rather than translocation and transmembrane topology.

The RNA editing site (the Gln/Arg site in the proposed TMII) suggests that the proposed TMII segment is involved in the formation of the channel pore. This postulated position of TMII brings the identified critical site for channel properties (the Gln/Arg/Asn site) closer to the midpoint of the channel. This position is more consistent with the measured electrical distance (approximately 70% into the electric field from the extracellular side) of the Mg blockade site (the Asn in TMII) of NMDA receptors. It is difficult for the proposed four-TM and five-TM models to accommodate this constraint(2) . The outer mouth of the channel may be formed in part by the N-terminal portion of the proposed TMI, as indicated by the finding that RNA editing (generating Ile Val and Tyr Cys) of TMI of GluR6 also affects Ca permeability(59) . These two amino acid side chains modified by RNA editing would lie on the same side of the transmembrane alpha-helix, exactly one turn apart. The argument that ionotropic glutamate receptors have a distinct channel pore structure not shared with nicotinic acetylcholine receptors is also consistent with the finding that the serine residues in TMII of GluR1 to GluR4 appear not to be involved in channel function, unlike their role in nicotinic acetylcholine receptors(60) . Finally, our three-TM model predicts that the C-terminal tail is located on the cytoplasmic side, which is consistent with antibody staining of AMPA-kainate receptors (22, 24, 61) and phosphorylation of an NMDA receptor subunit(20, 21) . The C-terminal tails of GluR6 and other kainate receptor subtypes are rich in consensus protein kinase A sites, whether these sites have any physiological role in protein kinase A-mediated phosphorylation of GluR6 remains to be determined experimentally.

The fact that the ionotropic glutamate receptor family lacks sequence homology with other ligand-gated ion channels (i.e. those for which the nicotinic AChRs are the prototypes) and now seems to have a different transmembrane topology suggests that the glutamate receptor family may arise from different ancestral genes and consequently has its own unique structural design. Thus, ligand-gated ion channels very likely do not consist of a single superfamily of receptors. This is consistent with the recent cloning of an extracellular ATP-activated cation-selective ion channel, which defines a new family of ligand-gated ion channels with its pore-forming motif resembling that of potassium channels(62, 63) . The results described here are consistent with a three-TM model of transmembrane topology, but the structures of the ligand binding site, the channel pore, and the intracellular regulatory sites remain to be determined.


FOOTNOTES

*
This work was supported in part by a Grant IBN-9309480 from the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by National Institutes of Health predoctoral training Grant T32-GM08210.

To whom correspondence should be addressed: Dept. of Pharmacology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853. Tel.: 607-253-3877; Fax: 607-253-3659; reo1{at}cornell.edu.

(^1)
The abbreviations used are: AMPA, alpha-amino-3-hydroxy-5-methylisoxazolepropionate; NMDA N-methyl-D-aspartate; PCR, polymerase chain reaction; Endo H, endo-beta-N-acetylglucosaminidase H; GFKARalpha, 45-kDa kainate receptor subunit from goldfish brain; GFKARbeta, 41-kDa kainate receptor subunit from goldfish brain; GluR, ionotropic glutamate receptor; TM, transmembrane domain; TMI, TMII, TMIII, and TMIV, first, second, third, and fourth putative transmembrane domain as originally defined by hydropathy plots.

(^2)
Despite the fact that the evidence in this paper does not support the four-TM model, for consistency with the literature we will refer to the four putative transmembrane domains (TMI, TMII, TMIII, and TMIV; Fig. 1) that have been proposed previously for ionotropic glutamate receptors based on hydropathy plots and the assumed homologies with other ligand-gated ion channels.


ACKNOWLEDGEMENTS

We thank Prof. J. L. Guan for helpful advice and discussion on the COS cell transfections and Prof. G. A. Weiland for critical reading of the manuscript.


REFERENCES

  1. Monaghan, D. T., Bridges, R. J., and Cotman, C. W. (1989) Annu. Rev. Pharmacol. Toxicol. 29, 365-402 [CrossRef][Medline] [Order article via Infotrieve]
  2. Hollmann, M., and Heinemann, S. (1994) Annu. Rev. Neurosci. 17, 31-108 [CrossRef][Medline] [Order article via Infotrieve]
  3. Seeburg, P. H. (1993) Trends Neurosci. 16, 359-365 [CrossRef][Medline] [Order article via Infotrieve]
  4. Nakanishi, S., and Masu, M. (1994) Annu. Rev. Biophys. Biomol. Struct. 23, 319-348 [CrossRef][Medline] [Order article via Infotrieve]
  5. Gregor, P., Mano, I., Maoz, I., McKeown, M., and Teichberg, V. (1989) Nature 342, 689-692 [Medline] [Order article via Infotrieve]
  6. Wo, Z. G., and Oswald, R. E. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7154-7158
  7. Wada, K., Dechesne, C. J., Shimasaki, S., King, R. G., Kusano, K., Buonanno, A., Hampson, D. R., Banner, C., Wenthold, R. J., and Nakatani, Y. (1989) Nature 342, 684-689 [Medline] [Order article via Infotrieve]
  8. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215, 403-410 [CrossRef][Medline] [Order article via Infotrieve]
  9. Kimura, N., Kurosawa, N., Kondo, K., and Tsukada, Y. (1993) Mol. Brain Res. 17, 351-355 [Medline] [Order article via Infotrieve]
  10. Kerry, C. J., Sudan, H. L., Abutidze, K., Mellor, I. R., Barnard, E. A., and Usherwood, P. N. (1993) Mol. Pharmacol. 44, 142-152 [Abstract]
  11. Nakanishi, N., Schneider, N. A., and Axel, R. (1990) Neuron 5, 569-581 [Medline] [Order article via Infotrieve]
  12. Changeux, J. P., Galzi, J. L., Devillers-Thiéry, A., and Bertrand, D. (1992) Q. Rev. Biophys. 25, 395-432 [Medline] [Order article via Infotrieve]
  13. von Heijne, G., and Gavel, Y. (1988) Eur. J. Biochem. 174, 671-678 [Abstract]
  14. Rogers, S. W., Hughes, T. E., Hollmann, M., Gasic, G. P., Deneris, E. S., and Heinemann, S. (1991) J. Neurosci. 11, 2713-2724 [Abstract]
  15. Hullebroeck, M. F., and Hampson, D. R. (1992) Brain Res. 590, 187-192 [Medline] [Order article via Infotrieve]
  16. Blackstone, C. D., Moss, S. J., Martin, L. J., Levey, A. I., Price, D. L., and Huganir, R. (1992) J. Neurochem. 58, 1118-1126 [Medline] [Order article via Infotrieve]
  17. Uchino, S., Sakimura, K., Nagahari, K., and Mishina, M. (1992) FEBS Lett. 308, 253-257 [CrossRef][Medline] [Order article via Infotrieve]
  18. Zheng, X., Zhang, L., Durand, G. M., Bennet, M. V. L., and Zukin, R. S. (1994) Neuron 12, 811-818 [Medline] [Order article via Infotrieve]
  19. Köhr, G., Eckardt, S., Luddens, H., Monyer, H., and Seeburg, P. H. (1994) Neuron 12, 1031-1040 [Medline] [Order article via Infotrieve]
  20. Tingley, W. G., Roche, K. W., Thompson, A. K., and Huganir, R. L. (1993) Nature 364, 70-73 [CrossRef][Medline] [Order article via Infotrieve]
  21. Mori, H., Yamakura, T., Masaki, H., and Mishina, M. (1993) Neuroreport 4, 519-522 [Medline] [Order article via Infotrieve]
  22. Martin, L. J., Blackstone, C. D., Levey, A. I., and Huganir, R. L. (1993) Neuroscience 53, 327-358 [CrossRef][Medline] [Order article via Infotrieve]
  23. Molnár, E., McIlhinny, R. A. J., and Somogyi, P. (1994) J. Neurochem. 63, 683-693 [Medline] [Order article via Infotrieve]
  24. Petralia, R. S., and Wenthold, R. J. (1992) J. Comp. Neurol. 318, 329-354 [Medline] [Order article via Infotrieve]
  25. Taverna, F. A., Wang, L. Y., MacDonald, J. F., and Hampson, D. R. (1994) J. Biol. Chem. 269, 14159-14164 [Abstract/Free Full Text]
  26. Roche, K. W., Raymond, L. A., Blackstone, C., and Huganir, R. L. (1994) J. Biol. Chem. 269, 11679-11682 [Abstract/Free Full Text]
  27. Raymond, L. A., Blackstone, C. D., and Huganir, R. L. (1993) Nature 361, 637-641 [CrossRef][Medline] [Order article via Infotrieve]
  28. Wang, L. Y., Taverna, F. A., Huang, X. P., MacDonald, J. F., and Hampson, D. R. (1993) Science 259, 1173-1175 [Medline] [Order article via Infotrieve]
  29. Posnett, D. N., McGrath, H., and Tam, J. P. (1988) J. Biol. Chem. 263, 1719-1725 [Abstract/Free Full Text]
  30. Tam, J. P. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5409-5423 [Abstract]
  31. Ausubel, F. M., Brent, R., Kingston, R. E., Smith, J. A., and Struhl, K. (1991) Current Protocols in Molecular Biology , p. 16.13.1, Greene Publishing Associates and Wiley-Interscience, New York
  32. Ziegra, C. J., Willard, J. M., and Oswald, R. E. (1992) Mol. Pharmacol. 42, 203-209 [Abstract]
  33. Hopp, T. P., and Woods, K. R. (1983) Mol. Immunol. 20, 483-489 [CrossRef][Medline] [Order article via Infotrieve]
  34. Hopp, T. P., and Woods, K. R. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 3824-3828 [Abstract]
  35. Aruffo, A., and Seed, B. (1987) EMBO J. 6, 3313-3316 [Abstract]
  36. Rothman, J. E., and Lodish, H. F. (1977) Nature 269, 775-780 [Medline] [Order article via Infotrieve]
  37. Wessels, H. P., and Spiess, M. (1988) Cell 55, 61-70 [CrossRef][Medline] [Order article via Infotrieve]
  38. Landolt-Marticorena, C., and Reithmeier, R. A. (1994) Biochem. J. 302, 253-260 [Medline] [Order article via Infotrieve]
  39. Nilsson, I., and von Heijne, G. (1993) J. Biol. Chem. 268, 5798-5801 [Abstract/Free Full Text]
  40. Gavel, Y., and von Heijne, G. (1990) Protein. Eng. 3, 433-442 [Abstract]
  41. Hung, A. Y., and Selkoe, D. J. (1994) EMBO J. 13, 534-542 [Abstract]
  42. Engelman, D. M., Steitz, T. A., and Goldman, A. (1986) Annu. Rev. Biophys. Biophys. Chem. 15, 321-353 [CrossRef][Medline] [Order article via Infotrieve]
  43. Jennings, M. L. (1989) Annu. Rev. Biochem. 58, 999-1027 [CrossRef][Medline] [Order article via Infotrieve]
  44. O'Hara, P. J., Sheppard, P. O., Thøgersen, H., Venezia, D., Haldeman, B. A., McGrane, V., Houamed, K. M., Thomsen, C., Gilbert, T. L., and Mulvihill, E. R. (1993) Neuron 11, 41-52 [Medline] [Order article via Infotrieve]
  45. Kuryatov, A., Laube, B., Betz, H., and Kuhse, J. (1994) Neuron 12, 1291-1300 [Medline] [Order article via Infotrieve]
  46. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132 [Medline] [Order article via Infotrieve]
  47. Chavez, R. A., and Hall, Z. W. (1992) J. Cell Biol. 116, 385-393 [Abstract]
  48. Chavez, R. A., and Hall, Z. W. (1991) J. Biol. Chem. 266, 15532-15538 [Abstract/Free Full Text]
  49. Ewalt, K. (1994) Biochemistry 33, 5077-5088 [Medline] [Order article via Infotrieve]
  50. High, S., and Dobberstein, B. (1992) Curr. Opinion Cell Biol. 4, 581-586 [Medline] [Order article via Infotrieve]
  51. Coleman, J., Inukai, M., and Inouye, M. (1985) Cell 43, 351-360 [Medline] [Order article via Infotrieve]
  52. Akiyama, Y., and Ito, K. (1987) EMBO J. 8, 3465-3470
  53. Nakahara, D. H., Lingappa, V. R., and Chuck, S. L. (1994) J. Biol. Chem. 269, 7617-7622 [Abstract/Free Full Text]
  54. Lipp, J., Flint, N., Haeuptle, M. T., and Dobberstein, B. (1989) J. Cell Biol. 109, 2013-2022 [Abstract]
  55. Hartmann, H. A., Kirsch, G. E., Drewe, J. A., Joho, R. H., and Brown, A. M. (1991) Science 251, 942-944 [Medline] [Order article via Infotrieve]
  56. Yellen, G., Jurman, M., Abramson, T., and MacKinnon, R. (1991) Science 251, 939-942 [Medline] [Order article via Infotrieve]
  57. Yool, A. J., and Schwarz, T. L. (1991) Nature 349, 700-704 [CrossRef][Medline] [Order article via Infotrieve]
  58. Bamberg, K., and Sachs, G. (1994) J. Biol. Chem. 269, 16909-16919 [Abstract/Free Full Text]
  59. Köhler, M., Burnashev, N., Sakmann, B., and Seeburg, P. H. (1993) Neuron 10, 491-500 [Medline] [Order article via Infotrieve]
  60. Dingledine, R., Hume, R. I., and Heinemann, S. F. (1992) J. Neurosci. 12, 4080-4087 [Abstract]
  61. Molnár, E., Baude, A., Richmond, S. A., Patel, P. B., Somogyi, P., and McIlhinney, R. A. J. (1993) Neuroscience 53, 307-326 [Medline] [Order article via Infotrieve]
  62. Brake, A. J., Wagenbach, M. J., and Julius, D. (1994) Nature 371, 519-523 [CrossRef][Medline] [Order article via Infotrieve]
  63. Valera, S., Hussy, N., Evans, R. J., Adami, N., North, R. A., Surprenat, A., and Buell, G. (1994) Nature 371, 516-519 [CrossRef][Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.