GABAA Receptor Composition Is Determined by Distinct Assembly Signals within alpha  and beta  Subunits*

Karen BollanDagger , Dale KingDagger , Laura A. RobertsonDagger , Kenneth BrownDagger , Pamela M. Taylor§, Stephen J. Moss§, and Christopher N. ConnollyDagger

From the Dagger  Department of Pharmacology and Neuroscience, Ninewells Medical School, University of Dundee, Dundee DD1 9SY, Scotland and § Medical Research Council Laboratory for Molecular Cell Biology, University College London, Gordon St., London WC1E 6BT, United Kingdom

Received for publication, October 7, 2002, and in revised form, December 4, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Key to understanding how receptor diversity is achieved and controlled is the identification of selective assembly signals capable of distinguishing between other subunit partners. We have identified that the beta 1-3 subunits exhibit distinct assembly capabilities with the gamma 2L subunit. Similarly, analysis of an assembly box in alpha 1-(57-68) has revealed an absolute requirement for this region in the assembly of alpha beta receptors. Furthermore, a selective requirement for a single amino acid (Arg-66), previously shown to be essential for the formation of the low affinity GABA binding site, is observed. This residue is critical for the assembly of alpha 1beta 2 but not alpha 1beta 1 or alpha 1beta 3 receptors. We have confirmed the ability of the previously identified GKER signal in beta 3 to direct the assembly of beta gamma receptors. The GKER signal is also involved in driving assembly with the alpha 1 subunit, conferring the ability to assemble with alpha 1R66A on the beta 2 subunit. Although this signal is sufficient to permit the formation of beta 2gamma 2 receptors, it is not necessary for beta 3gamma 2 receptor formation, suggesting the existence of alternative assembly signals. These findings support the belief that GABAA receptor assembly occurs via defined pathways to limit the receptor diversity.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

gamma -Aminobutyric acid, type A (GABAA)1 receptors are the major sites of fast synaptic inhibition in the brain. In mammals, they are constructed as pentameric structures from multiple subunits selected predominantly from the following distinct classes: alpha  (1-6), beta  (1-3), gamma  (1-3), delta , epsilon , theta , and sigma , creating an incredible (165) potential for structural diversity.

However, relatively few functionally distinct receptor compositions are thought to exist in vivo (1). It is possible that multiple receptor types may exist that are functionally equivalent. Their distinct subunit compositions may provide subtle functions such as modulation by endogenous ligands such as neurosteroids (2) or second messenger systems (3, 4), subcellular localization (5), or long term differences in the regulation of receptor surface expression (6, 7). Despite these caveats, GABAA receptor heterogeneity occurs via defined pathways to limit receptor diversity (3, 6). Possible mechanisms include brain region-specific (8) and temporal expression (9). However, many neuron types often express multiple receptor subunit mRNAs simultaneously (8), suggesting that subcellular mechanisms for differential receptor assembly may also exist. Potential processes could include the discrete sites of subcellular receptor assembly (10, 11) and/or the presence of assembly signals capable of differential interaction with other subunits.

In support of the existence of differential assembly signals, GABAA receptor assembly appears to be strictly controlled, producing receptors with a fixed stoichiometry of 2alpha , 2beta , and 1gamma (12-17). Furthermore, GABAA receptor assembly signals have been identified in the alpha 1 (18, 19), beta 2/3 (19, 20), and gamma 3 (21) subunits. Although the regions identified in these studies may exhibit subunit class-specific interactions, to date no studies have investigated the ability of GABAA receptors to discriminate between subunits of the same class.

Consistent with the location of these assembly signals to intersubunit contact points, the alpha /gamma signals (18, 19, 21) are located proximal to the GABA and benzodiazepine binding sites (22, 23) formed at subunit interfaces between the alpha -beta and alpha gamma subunits, respectively, and also the beta 2 high affinity GABA site (24). Similarly, the homologous region in rho 1 is an important component of the GABA binding domain (25).

Given the high degree of homology between the alpha  and gamma  subunits in this region combined with their differential ability to assemble with beta  subunits (alpha 1 with beta 1-3, gamma 2 with beta 3, possibly beta 1, but not beta 2) (20), we sought to investigate the role of these sequences in the differential assembly of alpha /gamma subunits with beta  subunits. Using site-directed mutagenesis, we have determined that the conversion of a single amino acid in alpha 1 to that of gamma 2 (R66A) is sufficient to alter the assembly profile of the alpha 1 subunit to that of the gamma 2, as determined by immunofluorescence and cell surface ELISA. These results demonstrate that the identity of a single amino acid may be critical in determining assembly with particular receptor subunits and may identify a basis for subunit-specific GABAA receptor assembly. Furthermore, we present evidence for the existence of alternative assembly signals, which may permit the formation of diverse receptor types, dependent upon subunit availability.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture and Transfection-- COS 7 cells (ATCC CRL 1651) were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, 100 µg/ml streptomycin, and 100 units/ml penicillin in an atmosphere of 5% CO2. Exponentially growing cells were transfected by electroporation (400 V, infinity resistance, 125 microfarads, Bio-Rad Gene Electropulser II). 10 µg of DNA was used per transfection (2 × 106 cells) using equimolar ratios of expression constructs. Cells were analyzed 12-48 h after transfection.

DNA Constructions-- Murine alpha 1, beta 1-3, and gamma 2L subunit cDNAs containing either the Myc or FLAG epitope tags (between amino acids 4 and 5 of the mature polypeptide) have been described previously and shown to be functionally silent with respect to receptor pharmacology and physiology (5, 18, 20, 26). The mutant constructs alpha 1S, alpha 1(rho 1), beta 2GKER, and beta 3DNTK were generated by site-directed mutagenesis as reported previously (18, 20). The remaining mutant alpha 1/gamma 2 constructs were generated by site-directed mutagenesis using the oligonucleotides: alpha 1(gamma 2), 5'-CATCCTTCCAAGTTTGAGCGAAAAAAATATCTATTGTATACTC-3'; alpha 1(R-A), 5'-CTTCCAACTTTGAGCGAAAAACACATC-3'; gamma 2(alpha 1), 5'-TGTCATACCAGGATTGGCGAAAAAAAACATCAATTGTATATTC-3', gamma 2(A-R), 5'-CATACCAGGTTTGGCGAAAAAAAATATC-3'. The fidelity of the final expression constructs was verified by DNA sequencing.

Antibodies-- The 9E10 antibody was obtained from 9E10 hybridoma cells (27) and used directly as supernatant without purification. The secondary antibodies, goat anti-mouse Alexa Fluor 568 and goat anti-mouse Alexa Fluor 488, were purchased from Molecular Probes and goat anti-mouse horseradish peroxide was from Amersham Biosciences.

Immunofluorescence-- COS7 cells were fixed in 3% paraformaldehyde (in PBS), washed twice in 50 mM NH4Cl (in PBS), and blocked (10% fetal bovine serum, 0.5% bovine serum albumin in PBS) for 30 min. Subsequent washes and antibody dilutions were performed in PBS containing 10% fetal bovine serum and 0.5% bovine serum albumin. After surface immunofluorescence cells were permeabilized by the addition of 0.5% Triton X-100 (10 min), and the immunofluorescence protocol was repeated from the NH4Cl step. Cells were examined using a confocal microscope (Zeiss LSM510).

Quantification of Cell Surface Expression-- COS7 cells were plated onto 3-cm wells of a 6-well dish. Two transfections were pooled and used to seed 6 wells ("surface" and "total" in triplicate). Cells were fixed in 3% paraformaldehyde (in PBS). Cell surface detection was performed in the absence of detergent, and total expression levels were determined following Triton X-100 (0.5%, 15 min) treatment. Cells were washed twice in 50 mM NH4Cl (in PBS) and blocked (5% fat-free powdered milk (Marvel), 10% fetal bovine serum , 0.5% bovine serum albumin in PBS) for 1 h. Subsequent washes were performed in block. Receptor expression was determined using a horseradish peroxide-conjugated secondary antibody and assayed using 3,3',5,5'-tetramethylbenzidine (Sigma) as the substrate, with detection at 450 nm after 30 min, after the addition of 0.5 M H2SO4. The reaction rate was determined to remain linear for up to 1 h (results not shown).

Immunoprecipitation-- Cells were L-methionine-starved for 30 min before labeling with [35S]methionine (0.5 mCi/10-cm dish, Translabel ICN/Flow) for 4 h. Cells were lysed in 10 mM sodium phosphate buffer containing 5 mM EDTA, 5 mM EGTA, 50 mM sodium fluoride, 50 mM sodium chloride, 1 mM sodium orthovanadate, 5 mM sodium pyrophosphate, 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml antipain, 10 µg/ml pepstatin, 0.1 mg/ml aprotinin, and 2% Triton X-100 (lysis buffer). Post-nuclear supernatants were preabsorbed with protein A-Sepharose and immunoprecipitated with 200 µl of 9E10 supernatant in the presence of protein A-Sepharose. Pellets were washed in lysis buffer (containing 0.5% deoxycholate and 0.2% SDS) and centrifuged through a 30% sucrose cushion followed by 3 additional washes in buffer supplemented with 0.5 M NaCl and a final wash without NaCl. Pellets were then resuspended in reducing sample buffer (2% SDS, 5% beta -mercaptoethanol in 0.68 M Tris, pH 6.8) and analyzed by 8% SDS-polyacrylamide gel electrophoresis and autoradiography.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Previous studies have revealed that the subunits of the most commonly expressed GABAA receptor (alpha 1beta 2gamma 2) cannot reach the cell surface when expressed alone nor when alpha 1gamma 2L or beta 2gamma 2L combinations are co-expressed. Only when alpha 1beta 2 or alpha 1beta 2gamma 2L subunits are co-expressed are functional cell surface receptors produced (5). Non-productive subunit monomers or alpha 1gamma 2L or beta 2gamma 2L dimers are retained in the endoplasmic reticulum (ER) by interactions with BiP or calnexin.

Receptor Homology within the Region of a Putative Assembly Signal-- Recently, a putative assembly signal within the alpha 1 and alpha 6 subunits (see alpha 6S, Fig. 1A) was identified as being required for assembly and cell surface expression with beta 3 (18). This region exhibits homology between all the GABAA receptor subunits (Fig. 1). The highest homology exists within subunit classes (alpha  values = 63.2%, beta  values = 78.9%, and gamma  values = 71.1%), whereas homology between the subunit classes (including delta  and epsilon ) is 26.3%. Within this region, both the glutamine (Gln-67) and the tryptophan (Trp-69) have been shown to be essential for assembly (18, 28). However, the glutamine is completely conserved among all GABAA receptors, and the tryptophan is completely conserved among all members of the ligand-gated ion channel superfamily, indicating a general role in subunit architecture or folding.


View larger version (44K):
[in this window]
[in a new window]
 
Fig. 1.   Sequence alignment of GABAA receptor subunits between amino acids 57 and 94 (alpha 1 mouse numbering). Sequence alignments over this region of alpha  (A), beta  (B), and gamma  (C) subunits are shown. Consensus sequences are shown for each individual subunit class. An overall consensus sequence is shown for alpha , beta , and gamma  subunits (D) along with the sequences of delta  and epsilon  subunits. The amino acid underlined (in bold) identifies a single residue completely conserved within each subunit class but absent from all other subunits. Completely conserved tryptophans (in all members of the ligand-gated superfamily) are illustrated in bold.

To implicate residues that may be involved in subunit class-specific assembly of GABAA receptors, we examined the sequences in this region to identify residues completely conserved within a subunit class but absent from the other subunit classes. Surprisingly, despite the high degree of homology evident, only one residue (Arg-66 in alpha 1) fits this criterion. At this position, an arginine (alpha s), glutamine (beta s), or alanine (gamma s), is always present (histidine in delta , serine in epsilon ). Interestingly, this residue in alpha 1 has been shown to be critical for GABA binding (22).

To assess the role of this region in GABAA receptor assembly we investigated alpha 1/gamma 2 subunit chimeras and mutants for their ability to assemble with the beta  subunits. The constructs used are alpha 1myc, alpha 1Smyc (lacking residues 57-68), alpha 1(gamma 2)myc (residues 57-68 replaced with the homologous residues from gamma 2), alpha 1(R-A)myc (arginine at position 66 mutated to alanine), gamma 2Lmyc, gamma 2L(alpha 1)myc (residues replaced with homologous residues from alpha 1), and gamma 2L(A-R)myc. Subunit detection was performed using 9E10 antibodies that recognize the Myc epitope present in all the alpha 1/gamma 2L subunits. All beta  subunits used were epitope-tagged with the FLAG epitope. When expressed alone, all the above alpha 1/gamma 2L subunits do not reach the cell surface but are retained within the ER (5) (results not shown). Thus, we measure the ability of the alpha 1/gamma 2L subunits to be rescued from the ER and expressed on the surface as heteromeric receptors with beta  subunits.

Cell Surface Expression of alpha 1/gamma 2 Subunits with beta 2-- COS7 cells were transfected with alpha /gamma mycbeta 2FLAG, and immunofluorescence was performed using 9E10 antibodies. As shown previously (5, 26), alpha 1 and beta 2 subunits are able to assemble and form heteromeric receptors on the cell surface (Fig. 2A). Consistent with the presence of an assembly signal existing between residues 57 and 68 of the alpha 1 subunit, alpha 1S (lacking this region) cannot reach the cell surface despite the presence of the beta 2 subunit. The alpha 1S is retained within the ER, as evidenced by the classic reticular staining pattern observed (Fig. 2A). To address the possibility that this subunit might misfold, we examined an alpha 1(rho 1) chimera in which residues 57-68 in the alpha 1 were replaced with the homologous residues from GABAC receptor rho 1 subunit (18). Given the expected structural similarities between rho 1 and GABAA receptor subunits combined with the recent observation that this region in rho 1 also contributes to the GABA binding site (25), this chimera would be less likely to misfold. In keeping with the inability of the rho 1 subunit to assemble with the GABAA receptor beta  subunits (29, 30), coexpression of alpha 1(rho 1) with beta 2 did not lead to cell surface expression (results not shown).


View larger version (45K):
[in this window]
[in a new window]
 
Fig. 2.   Surface expression of beta 2 recombinant GABAA receptors requires the presence of the alpha 1 subunit. COS 7 cells coexpressing beta 2FLAG with either alpha 1myc, alpha 1Smyc, gamma 2Lmyc, alpha 1(gamma 2)myc, alpha 1(R-A)myc, gamma 2L(alpha 1)myc, or gamma 2(A-R)myc were examined by immunofluorescence (A) for the presence of the Myc-tagged subunits at the surface and intracellularly. Quantification of surface expression levels (B) were performed by cell ELISA in the absence (Surface) or presence (total) of detergent and normalized to alpha 1 levels. COS 7 cells were coexpressing beta 2FLAG with either alpha 1myc (lane 1), alpha 1Smyc (lane 2), gamma 2Lmyc (lane 3), alpha 1(gamma 2)myc (lane 4), alpha 1(R-A)myc (lane 5), gamma 2L(alpha 1)myc (lane 6), or gamma 2(A-R)myc (lane 7). Each recording represents the mean ± S.E. of at least nine determinants in at least three independent experiments. Results are significantly different from alpha 1 control (p < 0.001, t test).

Receptors composed of beta 2gamma 2 do not access the cell surface but are retained in the ER (5) (Fig. 2). Therefore, we assessed the ability of alpha 1(gamma 2) to assemble with beta 2. Consistent with a role for alpha 1 residues 57-68 in assembly with beta 2, cell surface expression of alpha 1(gamma 2) is abolished.

Given the critical role of arginine at position 66 of alpha 1 in GABA binding (22) and the complete conservation of this position within all subunit classes (Fig. 1), we mutated this arginine (in alpha 1) to the corresponding residue (alanine) in gamma 2 creating alpha 1(R-A). This mutation completely abolished the ability of alpha 1 to assemble with beta 2.

To determine whether this putative alpha 1 assembly signal might be transplanted into the gamma 2 subunit, we generated a gamma 2L(alpha 1) construct containing the residues 57-68 from alpha 1 in place of the homologous gamma 2 sequence. When expressed with beta 2, gamma 2L(alpha 1) is still incapable of assembling with beta 2 (Fig. 2A). Thus, although the alpha 1 region 57-68 is essential for the assembly of alpha 1 and beta 2 and critically dependent upon an arginine at position 66, this region is insufficient to direct this assembly event. This is corroborated by the gamma 2L(A-R) construct, which cannot assemble with beta 2.

Quantification of these observations was performed by whole cell ELISA to determine receptor cell surface expression (no detergent) versus total (detergent) receptor expression. The values presented here do not reflect a true percentage of surface-expressed receptors for two reasons; first, the alpha 1beta 2 values have been normalized to 100% and, second, surface values greater than 100% have been detected for beta 3 homomers (results not shown), suggesting a possible inhibitory influence of prior detergent treatment on this assay. In these experiments (Fig. 2B, Table I) it can be seen clearly that the assembly of beta 2 with alpha 1 requires residues 57-68 of alpha 1 (alpha 1S = 0.9 ± 1.3%), most notably an arginine at position 66 (alpha 1(R-A) = 5.0 ± 4.3%). In agreement with the results observed by immunofluorescence, although this region is critically involved in assembly with beta 2, transplantation of this signal to gamma 2 does not confer the ability to assemble with beta 2 (alpha 1(gamma 2) = 3.7 ± 3.1%).

                              
View this table:
[in this window]
[in a new window]
 
Table I
Summary of cell surface expression for mutant GABAA receptors
Values are obtained from Fig. 2-6 and are expressed as the percentage cell surface expression relative to alpha 1 (normalized to 100%) ± S.E.

Cell Surface Expression of alpha 1/gamma 2 Subunits with beta 1-- COS7 cells were transfected with alpha /gamma mycbeta 1FLAG, and immunofluorescence was performed using 9E10 antibodies. As shown previously (26) alpha 1 and beta 1 subunits are able to assemble and form heteromeric receptors on the cell surface (Fig. 3). Consistent with the presence of an assembly signal existing between residues 57-68 of the alpha 1 subunit, alpha 1S cannot reach the cell surface despite the presence of the beta 1 subunit (2.7 ± 3.1%). Similar results were observed for the alpha 1(rho 1) (results not shown).


View larger version (57K):
[in this window]
[in a new window]
 
Fig. 3.   Surface expression of beta 1 recombinant GABAA receptors requires the presence of the alpha 1 subunit. COS 7 cells coexpressing beta 1FLAG with either alpha 1myc, alpha 1Smyc, gamma 2Lmyc, alpha 1(gamma 2)myc, alpha 1(R-A)myc, gamma 2L(alpha 1)myc, or gamma 2(A-R)myc were examined by immunofluorescence (A) for the presence of the Myc-tagged subunits at the surface and intracellularly. Quantification of surface expression levels (B) were performed by cell ELISA in the absence (Surface) or presence (total) of detergent and normalized to alpha 1 levels. COS 7 cells were coexpressing beta 1FLAG with either alpha 1myc (lane 1), alpha 1Smyc (lane 2), gamma 2Lmyc (lane 3), alpha 1(gamma 2)myc (lane 4), alpha 1(R-A)myc (lane 5), gamma 2L(alpha 1)myc (lane 6), or gamma 2(A-R)myc (lane 7). Each recording represents the mean ± S.E. of at least nine determinants in at least three independent experiments. *, denotes significant difference from alpha 1 control (p < 0.001, t test).

In this study, we could detect only very low levels of gamma 2Lbeta 1 surface receptors by immunofluorescence (Fig. 3A) that could not be resolved from background (mock-transfected cells) when analyzed quantitatively (10.2 ± 11.8%, Fig. 3B). To determine whether the robust surface expression observed upon alpha 1beta 1 expression results from the use of the alpha 1 57-68 assembly signal, we assessed the ability of alpha 1(gamma 2) to assemble with beta 1. In contrast to the results observed with the beta 2 subunit, cell surface expression of alpha 1(gamma 2) is not significantly different (108.6 ± 23.4%) from that observed for wild-type alpha 1 (100 ± 7.6%) but higher than that observed for the wild-type gamma 2L (10.2 ± 11.8%). These findings suggest that, in contrast to the beta 2 subunit, the beta 1 subunit does not exhibit the same requirements for this region of the alpha 1 for the assembly of alpha 1beta 1 heteromeric receptors. This possibility is corroborated by the efficient assembly of beta 1 with alpha 1(R-A) (83.9 ± 16.1%, Fig. 3). However, this region is still essential for alpha 1beta 1 receptor assembly, as evidenced by the inability of alpha 1S (Fig. 3) and alpha 1(rho 1) (results not shown) to assemble with beta 1. Both of the gamma 2 mutants (gamma 2(alpha 1) and gamma 2(A-R)) were indistinguishable from the wild-type gamma 2L with respect to cell surface expression with beta 1, i.e. weak surface immunofluorescence and non-detectable surface levels by ELISA (Fig. 3).

Cell Surface Expression of alpha 1/gamma 2 Subunits with beta 3-- COS7 cells were transfected with alpha /gamma mycbeta 3FLAG, and immunofluorescence was performed using 9E10 antibodies. As shown previously (26) alpha 1 and beta 3 subunits are able to assemble and form heteromeric receptors on the cell surface (Fig. 3). Consistent with the presence of an assembly signal existing between residues 57 and 68 of the alpha 1 subunit, alpha 1S cannot reach the cell surface (3.5 ± 3.6%) despite the presence of the beta 3 subunit. Similar results were observed for the alpha 1(rho 1) (results not shown).

Receptors composed of beta 3gamma 2 have been reported to be capable of forming functional cell surface receptors (18). In this study, we observed robust surface expression by immunofluorescence (Fig. 4A) and ELISA (54.7 ± 19.7%, Fig. 4B). We assessed the ability of alpha 1(gamma 2) to assemble with beta 3. As observed with the beta 1 subunit, alpha 1(gamma 2) is capable of assembling with beta 3 and even exhibits enhanced levels of surface expression (163 ± 21.9%, p < 0.02, t test) compared with alpha 1 (100 ± 10.5%) or gamma 2L (54.7 ± 19.7%). These findings suggest that, like the beta 1 subunit but in contrast to the beta 2 subunit, beta 3 does not require this region of the alpha 1 for the assembly of alpha 1beta 3 heteromeric receptors. This possibility is corroborated by the efficient assembly of beta 3 with alpha 1(R-A) (81.2 ± 9.2%, Fig. 4). Both of the gamma 2 mutants (gamma 2(alpha 1) and gamma 2(A-R)) were indistinguishable from the wild-type gamma 2L with respect to cell surface expression with beta 3, i.e. strong surface immunofluorescence and detectable surface levels by ELISA (Fig. 4, gamma 2L(alpha 1) = 50.9 ± 16.2% and gamma 2L(A-R) = 85 ± 23.3%).


View larger version (62K):
[in this window]
[in a new window]
 
Fig. 4.   Surface expression of beta 3 recombinant GABAA receptors requires the presence of the alpha 1 subunit. COS 7 cells coexpressing beta 3FLAG with either alpha 1myc, alpha 1Smyc, gamma 2Lmyc, alpha 1(gamma 2)myc, alpha 1(R-A)myc, gamma 2L(alpha 1)myc, or gamma 2(A-R)myc were examined by immunofluorescence (A) for the presence of the Myc-tagged subunits at the surface and intracellularly. Quantification of surface expression levels (B) were performed by cell ELISA in the absence (Surface) or presence (total) of detergent and normalized to alpha 1 levels. COS 7 cells coexpress beta 3FLAG with either alpha 1myc (lane 1), alpha 1Smyc (lane 2), gamma 2Lmyc (lane 3), alpha 1(gamma 2)myc (lane 4), alpha 1(R-A)myc (lane 5), gamma 2L(alpha 1)myc (lane 6) or gamma 2(A-R)myc (lane 7). Each recording represents the mean ± S.E. of at least nine determinants in at least three independent experiments. *, denotes significant difference from alpha 1 control (*, p < 0.001; **, p < 0.05 t test).

Interestingly, the beta 3 subunit (20, 31) and to a lesser extent the beta 1 subunit (32, 33) can form functional homomeric ion channels (not GABA-gated), whereas the beta 2 subunit is incapable of exiting the ER (5). A four-amino acid signal (GKER) that controls beta 3 homooligomerization has been identified (20). When transferred to beta 2 (beta 2GKER), this subunit is capable of cell surface expression as functional ion channels. The reciprocal construct (beta 3DNTK) can no longer assemble into homomeric receptors but is retained in the ER (20). To determine whether the assembly signal identified in the alpha 1 may also recognize the homomeric assembly signal present in the beta 3 subunit, we analyzed the assembly of alpha 1/gamma 2 subunits with the beta 2GKER and beta 3DNTK subunits.

Cell Surface Expression of alpha 1/gamma 2 Subunits with beta 2-- COS7 cells were transfected with alpha /gamma mycbeta 2GKER(FLAG), and immunofluorescence was performed using 9E10 antibodies. As expected, alpha 1 and beta 2GKER subunits are able to assemble and form heteromeric receptors on the cell surface (Fig. 5). Again, alpha 1S cannot reach the cell surface (9.7 ± 6.9%) despite the presence of the beta 2GKER subunit. Similar results were observed for the alpha 1(rho 1) (results not shown). In contrast to the ER retention of beta 2gamma 2L complexes (5) (3.0 ± 3.0% surface expression, Fig. 2), when beta 2GKER and gamma 2L are coexpressed, cell surface (74.3 ± 19.9%) receptors are produced (Fig. 5). Furthermore, surface expression with alpha 1(gamma 2) was increased for beta 2GKER (118.6 ± 21.9%) compared with beta 2 (10.2 ± 10%). More striking is the ability of the alpha 1(R-A) to assemble with beta 2GKER and reach the cell surface (90.1 ± 24.3%) compared with beta 2 (5.0 ± 4.3%). Analysis of the gamma 2 mutants reveals that the gamma 2L(alpha 1) and the gamma 2L(A-R) are also expressed on the cell surface at significant levels (55.8 ± 26.0% and 31.3 ± 9.9%) comparable with those observed for beta 3 (50.9 ± 16.2% and 85 ± 23.3%, respectively), not beta 2 (3.7 ± 3.1% and 7.5 ± 6.0%, respectively). The results with the beta 2GKER construct identifies the beta 3 homomeric assembly signal as capable of conferring beta 3-like assembly characteristics to the beta 2 subunit and suggests that, although beta 3 requires the presence of alpha 1 signal, the homomeric assembly signal in beta 3 does not depend critically upon the identity of the sequence between 57-68 of the alpha 1 and is capable of assembling with both alpha 1 or gamma 2L.


View larger version (48K):
[in this window]
[in a new window]
 
Fig. 5.   Surface expression of beta 2GKER recombinant GABAA receptors requires the presence of the alpha 1 subunit. COS 7 cells coexpressing beta 2GKER/FLAG with either alpha 1myc, alpha 1Smyc, gamma 2Lmyc, alpha 1(gamma 2)myc, alpha 1(R-A)myc, gamma 2L(alpha 1)myc, or gamma 2(A-R)myc were examined by immunofluorescence (A) for the presence of the Myc-tagged subunits at the surface and intracellularly. Quantification of surface expression levels (B) was performed by cell ELISA in the absence (Surface) or presence (total) of detergent and normalized to alpha 1 levels. COS 7 cells coexpressing beta 2GKER/FLAG with either alpha 1myc (lane 1), alpha 1Smyc (lane 2), gamma 2Lmyc (lane 3), alpha 1(gamma 2)myc (lane 4), alpha 1(R-A)myc (lane 5), gamma 2L(alpha 1)myc (lane 6), or gamma 2(A-R)myc (lane 7). Each recording represents the mean ± S.E. of at least nine determinants in at least three independent experiments. *, denotes significant difference from alpha 1 control (p < 0.001, t test).

Cell Surface Expression of alpha 1/gamma 2 Subunits with beta 3DNTK-- COS7 cells were transfected with alpha /gamma mycbeta 3DNTK(FLAG), and immunofluorescence was performed using 9E10 antibodies. As expected, alpha 1 and beta 3DNTK subunits are able to assemble and form heteromeric receptors on the cell surface (Fig. 6). Again, alpha 1S cannot reach the cell surface (7.9 ± 5.6%) despite the presence of the beta 3DNTK subunit. Similar results were observed for the alpha 1(rho 1) (results not shown). As observed for the beta 3gamma 2L receptors (54.7 ± 19.7% surface expression, Fig. 6), when beta 3DNTK and gamma 2L are coexpressed, cell surface (26.8 ± 11.5%) receptors are produced (Fig. 6). Furthermore, surface expression of alpha 1(gamma 2) with beta 3DNTK occurred at significant levels (99.6 ± 5.8%). In addition, the ability of the alpha 1(R-A) to assemble with beta 3DNTK and reach the cell surface (61.6 ± 13.2%) compared with beta 3 (81.2 ± 9.2%) was unaffected. Analysis of the gamma 2 mutants reveals that the gamma 2L(alpha 1) and the gamma 2L(A-R) are expressed on the cell surface at significant levels (38.3 ± 20.5 and 24.7 ± 13.2%), comparable with those observed for beta 3 (50.9 ± 16.2 and 85 ± 23.3%, respectively) but not beta 2 (3.7 ± 3.1% and 7.5 ± 6.0, respectively). Although the values obtained for the assembly of beta 3DNTK are consistently lower than that observed for the wild-type beta 3, the differences are not statistically significant, with the exception of alpha 1(gamma 2) and gamma 2(A-R) (p < 0.005, t test). Paradoxically, results observed for beta 3DNTK suggests that the beta 3 homomeric assembly signal is not an essential requirement for the assembly of beta 3 with either alpha 1 or gamma 2.


View larger version (57K):
[in this window]
[in a new window]
 
Fig. 6.   Surface expression of beta 3DNTK recombinant GABAA receptors requires the presence of the alpha 1 subunit. COS 7 cells coexpressing beta 3DNTK/FLAG with either alpha 1myc, alpha 1Smyc, gamma 2Lmyc, alpha 1(gamma 2)myc, alpha 1(R-A)myc, gamma 2L(alpha 1)myc, or gamma 2(A-R)myc were examined by immunofluorescence (A) for the presence of the Myc-tagged subunits at the surface and intracellularly. Quantification of surface expression levels (B) were performed by cell ELISA in the absence (Surface) or presence (total) of detergent and normalized to alpha 1 levels. COS 7 cells coexpressing beta 3DNTK/FLAG with either alpha 1myc (lane 1), alpha 1Smyc (lane 2), gamma 2Lmyc (lane 3), alpha 1(gamma 2)myc (lane 4), alpha 1(R-A)myc (lane 5), gamma 2L(alpha 1)myc (lane 6), or gamma 2(A-R) (lane 7). Each recording represents the mean ± S.E. of at least nine determinants in at least three independent experiments. *, denotes significant difference from alpha 1 control (p < 0.001, t test).

To determine the ability of the alpha 1/gamma 2 polypeptides to oligomerize with beta 2, cDNAs were cotransfected into COS7 cells, [35S]methionine-labeled, and immunoprecipitated via the Myc epitope tag on the alpha 1/gamma 2 subunits. Only the extracellular domain of beta 2 was used to eliminate any contribution from other subunit interactions (34) and events at the cell surface such as receptor turnover (6). Bands corresponding to the alpha 1/gamma 2 and beta 2 extracellular fragments were excised and quantified using a scintillation counter. The ratio of beta 2 co-immunoprecipitated with the alpha 1/gamma 2 subunit was normalized to that of wild-type alpha 1 (39%) such that the ratio (beta 2:alpha 1) of beta 2 co-immunoprecipitated by alpha 1 represents 100%. No significant reduction in binding was evident for any of the alpha 1/gamma 2 subunits (Fig. 7). This is not surprising, because each subunit must possess at least two interfaces (and presumably assembly signals) with other subunits (Fig. 8).


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 7.   Oligomerization of alpha /gamma subunits with beta 2. COS 7 cells coexpressing the entire N-terminal extracellular region of beta 2 (beta 2- extFLAG) with either alpha 1myc, alpha 1Smyc, gamma 2Lmyc, alpha 1(gamma 2)myc, alpha 1(R-A)myc, gamma 2L(alpha 1)myc, or gamma 2(A-R)myc were labeled with [35S]methionine, immunoprecipitated with antibodies against the Myc epitope (9E10), separated by SDS-PAGE, and examined by autoradiography. Bands representing the alpha /gamma and beta 2-extFLAG were excised and counted by scintillation. The ratio of alpha /gamma :beta 2 was determined and normalized to that of alpha 1.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 8.   Identification and location of putative assembly signals in GABAA receptor subunits. A, putative assembly signals are illustrated for Interface 1 and their opposite interface (Interface 2) shown. alpha 1B may form an interface with either a (a) gamma  or a (b) beta  subunit. beta 3B may possess alternative signals capable of interacting with alpha 1A/gamma 2A. Two different gamma 2A signals have been identified. <GABA or >GABA represents the low or high affinity GABA binding sites, respectively. 1, Ref. 18. 2, Ref. 39. 3, Ref. 20. 4, Ref. 19. 5, Ref. 21. *, this study. B, arrangement of subunits in an alpha beta gamma pentameric receptor indicating subunit interfaces and binding sites for GABA (G) and benzodiazepines (Bz). The mirror image of this structure is equally possible (39).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To date 16 different GABAA receptor cDNAs have been isolated from a variety of vertebrates (4). Many of these subunits exhibit differing patterns of both spatial and developmental expression in the CNS, with many neurons often expressing multiple numbers of receptor subunits (8, 35). A major challenge in trying to analyze the diversity of GABAA receptor structure in the brain is determining what processes control receptor assembly. The determination of receptor composition may arise from temporal and spatial regulation, subcellular subunit segregation, and/or differential assembly/stability. Although there is extensive evidence for a role of temporal (9) and spatial (8, 35) regulation of subunit expression in determining receptor composition, these mechanisms cannot explain how receptor diversity is limited in neurons co-expressing multiple GABAA receptor subunits simultaneously (8, 35). To date, there is no supporting evidence for discrete subcellular assembly sites for GABAA receptors, although emerging evidence for the localized translation of proteins at synapses (11) may be relevant.

The potential for hierarchical assembly signals is supported by the strict control of receptor stoichiometry (12-17) and the observations that alpha 1beta 3 (versus beta 3 homomers) and alpha 1beta 2gamma 2 (versus alpha 1beta 2) receptors form to the exclusion of the other possible combinations (3, 31, 36). In vivo evidence for hierarchical receptor assembly has been provided by the analysis of alpha 6 knockout mice, which determined that the delta  subunit was concomitantly "knocked-out" by a partial alpha 6 polypeptide product that remains able to associate with the delta  subunit but cannot produce functional receptors (37).

The discovery of putative GABAA receptor assembly signals began with the identification of a natural splice variant of alpha 6 that lacked 10 amino acids in the N-terminal extracellular domain (38). This splice variant (termed alpha 6Short) was determined to be incapable of assembling into functional receptors (18, 38). The high degree of homology between all GABAA receptor subunits within this region suggests a common role in receptor assembly, a possibility vindicated for the alpha 1 (18) (but see Ref. 19) and gamma 3 (21). Putative assembly signals have also been discovered adjacent to this region in alpha 1 (for binding to gamma 2) (39) and gamma 2 (for binding to alpha 1 and beta 3) (19). Furthermore, two invariant tryptophans within this region have been shown to be essential for GABAA receptor assembly and benzodiazepine, but not GABA, binding (28). Indeed these tryptophans are completely conserved in all members of the ligand-gated ion channel superfamily and may provide some common structural feature necessary for receptor assembly (28).

Given the similarity between the homologous regions between alpha  (MEYTIDVFFRQSW) and gamma  (MEYTIDIFFAQTW) subunits, we examined the possibility that the sequence differences between these subunit classes might be responsible for the differential ability of the gamma 2 subunit to assemble with beta 1 and beta 3 (20, 33), but not beta 2 (5, 20), whereas the alpha 1 can assemble with all three beta  subunits (26).

Consistent with previous findings, alpha 1S and alpha 1(rho 1) (18) are not able to assemble with beta 1-3. In addition, gamma 2L cannot assemble with beta 2 (20) and only weakly with beta 1 (Table I) (3). In keeping with the possibility that alpha 1-(57-68) constitutes an assembly signal determining oligomerization with beta  subunits (18), alpha 1(gamma 2) is also unable to assemble with beta 2. In fact, a single site (Arg-66) was found to be critical for the assembly of alpha 1beta 2 receptors. However, the failure of gamma 2L(alpha 1) to assemble with beta 2 suggests that this putative assembly signal is necessary but not sufficient to direct this oligomerization step.

Interestingly, a striking overlap between the alpha A site and the GABA binding site (loop D) exists for alpha 1beta 2 receptors (22). Boileau et al. (22) showed that residues Phe-64, Arg-66, and Ser-68 were found to be part of (or close to) the GABA binding site, with Phe-64 and Arg-66 critical for modulation by GABA. In keeping with other studies (18, 28), these authors found that Gln-67 and Trp-69 were essential for the production of functional receptors. Moreover, this region is not only important in the alpha 1 subunit contribution to GABA binding, but the high affinity GABA binding site has been shown to be produced by the homologous region in beta 2 (beta 2A interface to alpha 1) (24). In addition, the benzodiazepine binding site on the gamma 2 subunit resides within the same homologous region, with A79 (homologous position to Arg-66 in alpha 1) lining the benzodiazepine binding pocket (23). A similar overlap between assembly signal and benzodiazepine binding is observed for the opposing interface on the alpha 1 subunit, with residues 74-123 providing the binding site (40) and residues 81-100 (MTVLRLNNLMASKIWTPDTFF, see Fig. 1) involved in oligomerization with gamma 2 (39).

In contrast to the tight correlation between receptor assembly and the formation of the GABA/benzodiazepine binding site in alpha 1beta 2gamma 2 receptors, little overlap exists for the opposing side of this interface, the beta B site. This side of the interface has been determined to be constructed from 3 distinct regions; loop A between residues 93 and 101, loop B between residues 157 and 160, and loop C between residues 202 and 209 to generate the GABA binding domain on the beta 2 subunit (41, 42). The residues required for assembly (GDKAVTGVER in beta 3) fall between loops B and C.

In contrast to the findings for the beta 2 subunit, although beta 1, beta 3, beta 2GKER, and beta 3DNTK all require the presence of residues 57-68 from either alpha 1 or gamma 2, they do not exhibit the same dependence upon this sequence as beta 2. The most likely explanation for these results is a general requirement for this region to either induce correct folding or subunit architecture but not to provide an assembly signal. This would be consistent with the role of these sequences in the formation of GABA and benzodiazepine binding sites. Alternatively, assembly signals for beta 1 and beta 3 may reside within this region, as for assembly with beta 2, but be interchangeable between alpha 1 and gamma 2. However, this seems unlikely given that beta 1 assembles only poorly with gamma 2L, yet robustly with alpha 1(gamma 2). Thus, it seems more likely that beta 1 and beta 3 utilize unique assembly signals in alpha 1 and gamma 2. This is consistent with previous findings (19) in which assembly signals just downstream from the alpha 1 57-68 region have been identified for gamma 2 binding to beta 3 (Figs. 1 and 8). However, unlike the gamma 2, gamma 3 does utilize the homologous assembly signal (MEYQIDIFFAQTW) used by alpha 1 (to assemble with beta 2) to assemble with beta 3 (21).

A more complex scenario is also possible. Perhaps beta 1 and beta 3, but not beta 2, may possess and utilize alternative assembly signals dependent on availability. In this way GABAA receptors may form their preferred receptor compositions depending on subunit availability. In other words, a process of hierarchical assembly may operate, as observed previously (3, 31, 36). Such a possibility is supported by the observation that although "GKER" in beta 3 is sufficient to drive assembly with alpha 1 and gamma 2 (see beta 2GKER), it is not necessary to do so (see beta 3DNTK). Thus, it seems possible that multiple, alternative assembly signals may exist for GABAA receptor formation, providing a flexible mechanism for the construction of GABAA receptors. In such a scenario, a beta  subunit would be able to select its most desirable companion available without committing itself to just one partner. This is observed in delta  knockout mice, in which alpha 4 and alpha 6 subunits, normally assembling with the delta  subunit, are now free to assemble with gamma 2 (35, 43, 44). Thus, the selection of a second choice would not be expected to be possible in the presence of a favorite. This is well illustrated by the fatal attraction suffered for the delta  subunit in cerebellar granule neurons of alpha 6 knockout mice (37) (that retain the alpha 6 assembly signal), implying a complete devotion of the delta  subunit for alpha 6 assembly signal within this environment.

In summary, we have identified a single amino acid within the proposed assembly signal of alpha 1 for beta  subunits that is absolutely required for alpha 1beta 2, but not alpha 1beta 1 or alpha 1beta 3, receptor formation. beta 1, beta 2, and beta 3 exhibit distinct assembly profiles with alpha 1 and gamma 2, utilizing distinct assembly signals in alpha 1/gamma 2. We have identified an assembly signal in beta 3 that is sufficient to drive assembly with both alpha 1 and gamma 2. However, it is not necessary, implicating the presence of multiple assembly signals capable of fulfilling the same function, selecting a subunit with which to assemble.

    FOOTNOTES

* This work was supported by Wellcome Trust Grant 059321 (to C. N. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 1382-632527; Fax: 1382-667120; E-mail: c.n.connolly@dundee.ac.uk.

Published, JBC Papers in Press, December 5, 2002, DOI 10.1074/jbc.M210229200

    ABBREVIATIONS

The abbreviations used are: GABAA, gamma -aminobutyric acid, type A; ER, endoplasmic reticulum; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. McKernan, R. M., and Whiting, P. J. (1996) Trends Neurosci. 19, 139-143[CrossRef][Medline] [Order article via Infotrieve]
2. Wohlfarth, K. M., Bianchi, M. T., and Macdonald, R. L. (2002) J. Neurosci. 22, 1541-1549[Abstract/Free Full Text]
3. Angelotti, T. M., and Macdonald, R. L. (1993) J. Neurosci. 13, 1418-1428[Abstract]
4. Moss, S. J., and Smart, T. G. (2001) Nat. Rev. Neurosci. 2, 240-250[CrossRef][Medline] [Order article via Infotrieve]
5. Connolly, C. N., Wooltorton, J. R., Smart, T. G., and Moss, S. J. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9899-9904[Abstract/Free Full Text]
6. Connolly, C. N., Kittler, J. T., Thomas, P., Uren, J. M., Brandon, N. J., Smart, T. G., and Moss, S. J. (1999) J. Biol. Chem. 274, 36565-36572[Abstract/Free Full Text]
7. Wan, Q., Xiong, Z. G., Man, H. Y., Ackerley, C. A., Braunton, J., Lu, W. Y., Becker, L. E., MacDonald, J. F., and Wang, Y. T. (1997) Nature 388, 686-690[CrossRef][Medline] [Order article via Infotrieve]
8. Wisden, W., and Seeburg, P. H. (1992) Curr. Opin. Neurobiol. 2, 263-269[Medline] [Order article via Infotrieve]
9. Smith, S. S., Gong, Q. H., Li, X., Moran, M. H., Bitran, D., Frye, C. A., and Hsu, F. C. (1998) J. Neurosci. 18, 5275-5284[Abstract/Free Full Text]
10. Gardiol, A., Racca, C., and Triller, A. (1999) J. Neurosci. 19, 168-179[Abstract/Free Full Text]
11. Job, C., and Eberwine, J. (2001) Nat. Rev. Neurosci. 2, 889-898[CrossRef]
12. Im, W. B., Pregenzer, J. F., Binder, J. A., Dillon, G. H., and Alberts, G. L. (1995) J. Biol. Chem. 270, 26063-26066[Abstract/Free Full Text]
13. Chang, Y., Wang, R., Barot, S., and Weiss, D. S. (1996) J. Neurosci. 16, 5415-5424[Abstract/Free Full Text]
14. Tretter, V., Ehya, N., Fuchs, K., and Sieghart, W. (1997) J. Neurosci. 17, 2728-2737[Abstract/Free Full Text]
15. Farrar, S. J., Whiting, P. J., Bonnert, T. P., and McKernan, R. M. (1999) J. Biol. Chem. 274, 10100-10104[Abstract/Free Full Text]
16. Sieghart, W., Fuchs, K., Tretter, V., Ebert, V., Jechlinger, M., Hoger, H., and Adamiker, D. (1999) Neurochem. Int. 34, 379-385[CrossRef][Medline] [Order article via Infotrieve]
17. Baumann, S. W., Baur, R., and Sigel, E. (2001) J. Biol. Chem. 276, 36275-36280[Abstract/Free Full Text]
18. Taylor, P. M., Connolly, C. N., Kittler, J. T., Gorrie, G. H., Hosie, A., Smart, T. G., and Moss, S. J. (2000) J. Neurosci. 20, 1297-1306[Abstract/Free Full Text]
19. Klausberger, T., Fuchs, K., Mayer, B., Ehya, N., and Sieghart, W. (2000) J. Biol. Chem. 275, 8921-8928[Abstract/Free Full Text]
20. Taylor, P. M., Thomas, P., Gorrie, G. H., Connolly, C. N., Smart, T. G., and Moss, S. J. (1999) J. Neurosci. 19, 6360-6371[Abstract/Free Full Text]
21. Sarto, I., Klausberger, T., Ehya, N., Mayer, B., Fuchs, K., and Sieghart, W. (2002) J. Biol. Chem. 277, 30656-30664[Abstract/Free Full Text]
22. Boileau, A. J., Evers, A. R., Davis, A. F., and Czajkowski, C. (1999) J. Neurosci. 19, 4847-4854[Abstract/Free Full Text]
23. Teissere, J. A., and Czajkowski, C. (2001) J. Neurosci. 2, 4977-4986
24. Newell, J. G., Davis, M., Bateson, A. N., and Dunn, S. M. (2000) J. Biol. Chem. 275, 14198-14204[Abstract/Free Full Text]
25. Torres, V. I., and Weiss, D. S. (2002) J. Biol. Chem. 277, 43741-43748[Abstract/Free Full Text]
26. Connolly, C. N., Krishek, B. J., McDonald, B. J., Smart, T. G., and Moss, S. J. (1996b) J. Biol. Chem. 271, 89-96[Abstract/Free Full Text]
27. Evan, G. I., Lewis, G., Ramsay, G., and Bishop, J. M. (1985) Mol. Cell. Biol. 5, 3610-3616[Medline] [Order article via Infotrieve]
28. Srinivasan, S., Nichols, C. J., Lawless, G. M., Olsen, R. W., and Tobin, A. J. (1999) J. Biol. Chem. 274, 26633-26638[Abstract/Free Full Text]
29. Hackam, A. S., Wang, T. L., Guggino, W. B., and Cutting, G. R. (1998) J. Neurochem. 70, 40-46[Medline] [Order article via Infotrieve]
30. Koulen, P., Brandstatter, J. H., Enz, R., Bormann, J., and Wassle, H. (1998) Eur. J. Neurosci. 10, 115-127[CrossRef][Medline] [Order article via Infotrieve]
31. Wooltorton, J. R., Moss, S. J., and Smart, T. G. (1997) Eur. J. Neurosci. 9, 2225-2235[Medline] [Order article via Infotrieve]
32. Krishek, B. J., Moss, S. J., and Smart, T. G. (1996) Mol. Pharmacol. 49, 494-504[Abstract]
33. Sigel, E., Baur, R., Malherbe, P., and Mohler, H. (1989) FEBS Lett. 257, 377-379[CrossRef][Medline] [Order article via Infotrieve]
34. Eertmoed, A. L., and Green, W. N. (1999) J. Neurosci. 19, 6298-6308[Abstract/Free Full Text]
35. Burt, D. R., and Kamatchi, G. L. (1991) FASEB J. 5, 2916-2923[Abstract/Free Full Text]
36. Rabow, L. E., Russek, S. J., and Farb, D. H. (1995) Synapse 21, 189-274[Medline] [Order article via Infotrieve]
37. Jones, A., Korpi, E. R., McKernan, R. M., Pelz, R., Nusser, Z., Makela, R., Mellor, J. R., Khan, Z. U., Gutierrez, G., and DeBlas, A. L. (1994) J. Neurochem. 63, 371-374[Medline] [Order article via Infotrieve]
38. Korpi, E. R., Kuner, T., Kristo, P., Kohler, M., Herb, A., Luddens, H., and Seeburg, P. H. (1994) J. Neurochem. 63, 1167-1170[Medline] [Order article via Infotrieve]
39. Klausberger, T., Sarto, I., Ehya, N., Fuchs, K., Furtmuller, R., Mayer, B., Huck, S., and Sieghart, W. (2001) J. Neurosci. 21, 9124-9133[Abstract/Free Full Text]
40. Smith, G. B., and Olsen, R. W. (2000) Neuropharmacology 39, 55-64[CrossRef][Medline] [Order article via Infotrieve]
41. Boileau, A. J., Newell, J. G., and Czajkowski, C. (2002) J. Biol. Chem. 277, 2931-2937[Abstract/Free Full Text]
42. Wagner, D. A., and Czajkowski, C. (2001) J. Neurosci. 21, 67-74[Abstract/Free Full Text]
43. Tretter, V., Hauer, B., Nusser, Z., Mihalek, R. M., Hoger, H., Homanics, G. E., Somogyi, P., and Sieghart, W. (2001) J. Biol. Chem. 276, 10532-10538[Abstract/Free Full Text]
44. Korpi, E. R., Mihalek, R. M., Sinkkonen, S. T., Hauer, B., Hevers, W., Homanics, G. E., Sieghart, W., and Luddens, H. (2002) Neuroscience 109, 733-743[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.