©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Assembly and Cell Surface Expression of Heteromeric and Homomeric -Aminobutyric Acid Type A Receptors (*)

(Received for publication, August 16, 1995; and in revised form, October 16, 1995)

Christopher N. Connolly Belinda J. Krishek (1) Bernard J. McDonald Trevor G. Smart (1) Stephen J. Moss (§)

From the Medical Research Council Laboratory of Molecular Cell Biology and the Department of Pharmacology, University College London, Gordon Street, London WC1E 6BT Department of Pharmacology, The School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The ability of differing subunit combinations of -aminobutyric acid type A (GABA(A)) receptors produced from murine alpha1, beta2, and 2L subunits to form functional cell surface receptors was analyzed in both A293 cells and Xenopus oocytes using a combination of molecular, electrophysiological, biochemical, and morphological approaches. The results revealed that GABA(A) receptor assembly occurred within the endoplasmic reticulum and involved the interaction with the chaperone molecules immunoglobulin heavy chain binding protein and calnexin. Despite all three subunits possessing the ability to oligomerize with each other, only alpha1beta2 and alpha1beta22L subunit combinations could produce functional surface expression in a process that was not dependent on N-linked glycosylation. Single subunits and the alpha12L and beta22L combinations were retained within the endoplasmic reticulum. These results suggest that receptor assembly occurs by defined pathways, which may serve to limit the diversity of GABA(A) receptors that exist on the surface of neurons.


INTRODUCTION

-Aminobutyric acid type A (GABA(A)) (^1)receptors are believed to be the major sites of fast synaptic inhibition in the brain and are also the sites of action for many psychoactive drugs including the benzodiazepines and barbiturates (Olsen and Tobin, 1990). Molecular cloning has revealed a number of GABA(A) receptor subunits that can be divided by sequence homology into subunit classes with multiple members: alpha (1, 2, 3, 4, 5, 6) , beta(1, 2, 3, 4) , (1, 2, 3) , and (1) , creating considerable potential for structural diversity. Additional diversity of receptor structure is generated by alternative splicing of some of these subunit mRNAs (Burt and Kamatchi, 1991). In situ hybridization and immunocytochemical methodologies suggest a large temporal and spatial diversity of receptor structure in the brain with many neuron types often expressing multiple receptor subunits (Wisden and Seeburg 1992; Fritschy et al., 1992). It is believed that GABA(A) receptors are pentameric in their final assembled plasma membrane form (Nayeem et al., 1994); however, the precise subunit composition and stoichiometry of a single population of native GABA(A) receptors remains unknown.

Accordingly, the expression of cDNA clones has been used to examine the minimal subunit composition required to produce functional GABA(A) receptors, determined by electrophysiological methodologies. Expression of unitary subunits has produced conflicting results; some subunits expressed alone appear to be able to produce GABA-gated ion channels (Blair et al., 1988; Pritchett et al., 1988) or channels that are sensitive to inhibition by picrotoxin, a GABA(A) receptor channel blocker (Sigel et al., 1989), whereas other studies demonstrate that some single subunits do not produce functional receptors (Sigel et al., 1990; Angelotti and Macdonald, 1993; Krishek et al., 1994). Expression of some binary subunit combinations have also produced conflicting data. For example, GABA-gated channels have been reported upon co-expression of either alpha12 or beta22 subunits (Verdoorn et al., 1990; Draguhn et al., 1990). In contrast, the failure of co-expressed beta12 and alpha12 subunits to produce functional GABA(A) receptors has also been reported (Sigel et al., 1990; Krishek et al., 1994; Angelotti and Macdonald, 1993). There is, however, general agreement that co-expression of alpha and beta subunits is sufficient for the production of GABA-gated chloride currents, and the co-expression of alpha and beta with either the 2 or 3 subunits produce GABA(A) receptors that are sensitive to modulation by benzodiazepines (Pritchett et al., 1988, 1989; Burt and Kamatchi, 1991)

To further investigate these observations and attempt to seek an explanation for the failure of certain subunit combinations to produce functional GABA(A) receptors, we have examined the assembly and surface expression of homomeric and heteromeric GABA(A) receptors using biochemical, immunological, and electrophysiological methodologies. We have studied the assembly of GABA(A) receptors of varying subunit composition produced from alpha1, beta2, and 2L subunits expressed in both Xenopus oocytes and transiently transfected A293 cells. From in situ hybridization and immunochemical analyses these subunits are co-localized in many adult brain regions and comprise up to 30% of all benzodiazepine-sensitive GABA(A) receptors in the adult brain (Benke et al., 1994; Fritschy et al., 1992).

In this study we demonstrate that GABA(A) receptor assembly occurs in the endoplasmic reticulum (ER), where interactions with the molecular chaperones immunoglobulin heavy chain binding protein (BiP) and calnexin were detected. Access to the cell surface was, however, limited to receptors composed of alpha1beta2 and alpha1beta22L subunits. Single subunits and the binary combinations of alpha12L and beta22L, although capable of oligomerization, were retained within the ER, presumably via interactions with BiP and calnexin. Receptor assembly and transport to the cell surface was not dependent on N-linked glycosylation, although its efficiency was enhanced. These results suggest that GABA(A) receptor assembly occurs by defined mechanisms, which may serve to regulate the diversity of GABA(A) receptors expressed on the surface of neurons.


EXPERIMENTAL PROCEDURES

Cell Culture and Transfection

Human embryonic kidney 293 cells (ATCC CRL 1573) were maintained in Dulbecco's modified Eagle's medium/Ham's F-12 medium (Life Technologies Ltd.) supplemented with 10% fetal bovine serum. Exponentially growing cells were seeded at 2 times 10^6 cells/10-cm dish and transfected by calcium phosphate precipitation as described previously (Moss et al., 1992). 20 µg of DNA was used per 10-cm plate of A293 cells using equimolar ratios of expression constructs. Cells were analyzed 12-18 h (immunofluorescence and immunoprecipitation) or up to 24 h (pharmacology and electrophysiology) after transfection. Nuclear injection of Xenopus oocytes with murine GABA(A) receptor subunit constructs was performed as described by Krishek et al.(1994).

DNA Constructions

Murine alpha1, 2L (Wang et al., 1992; Kofuji et al., 1991), and beta2 (Kamatchi et al., 1995) subunit cDNAs were cloned as EcoRI fragments into the mammalian expression vector pGW1 (Moss et al., 1990). Expression of heterologous cDNAs in this vector is under the control of the cytomegalovirus promoter. The subunits were tagged with the 10-amino acid 9E10 epitope (EQKLISEEDL) from c-myc (Evan et al., 1985) using the oligonucleotides alpha1(GTCTTTAAGTTCATCTAGGTCTTCTTCTGATATTAGCTTTTGTTCTTGGGAGGGCTGTCC), beta2 (CATATTACTAGGGTCTAGGTCCTCTTCTGATATTAGCTTTTGTTCATTGACACTCTGAGC), and 2L(ATCTTCATAGTCATCTAGGTCTTCTTCTGATATTAGTTTTTGTTCATCTGACTTTTGGCT) or with the 8-amino acid FLAG epitope (DYKDDDDK) using the oligonucleotides alpha1(GTCTTTAAGTTCATCCTTGTCATCGTCGTCCTTGTAGTCTTGGGAGGGCTGTCC), beta2(CATATTACTAGGGTCCTTGTCATCGTCGTCCTTGTAGTCATTGACACTCTGAGC), and 2L(ATCTTCATAGTCATCCTTGTCATCGTCGTCCTTGTAGTCATCTGACTTTTGGCT). All tags were positioned between the fourth and fifth amino acid of the mature protein by site-directed mutagenesis (Kunkel, 1985). The fidelity of the final expression constructs were verified by DNA sequencing.

Antibodies

The 9E10 antibody was obtained from 9E10 hybridoma cells (Evan et al., 1985) and used directly as supernatant without purification. Anti-FLAG M2 mouse monoclonal antibody was purchase from IBI Ltd. The anti-BiP antibody was purchased from Cambridge Bioscience (clone 5A5), and the anti-calnexin antibody (AF8) was a kind gift from Michael Brenner (Harvard Medical School) (Hochstenbach et al., 1992). Rabbit anti-horseradish peroxidase was purchased from DAKO (Denmark). The secondary antibodies, goat anti-rabbit rhodamine, and goat anti-mouse fluorescein isothiocyanate were purchased from Pierce.

Immunofluorescence

A293 cells on poly-L-lysine (10 µg/ml)-coated coverslips were fixed in 3% paraformaldehyde (in phosphate-buffered saline) and washed twice in 50 mM NH(4)Cl (in phosphate-buffered saline). Subsequent washes and antibody dilutions were performed in phosphate-buffered saline containing 10% fetal bovine serum and 0.5% bovine serum albumin. When cells were permeabilized, 0.05% Triton X-100 was added to all solutions after fixation. 9E10 supernatant was diluted 1:5, and rabbit anti-horseradish peroxidase used at 0.5 µg/ml. Both secondary antibodies were used at 1 µg/ml. Cells were examined using a confocal microscope (MRC1000, Bio-Rad).

DAB Quenching

After fixation, cells were washed in 50 mM TrisbulletHCl (pH 7.5) and incubated in the same buffer containing 0.15% diaminobenzidine, 0.02% hydrogen peroxide, and 0.1 M imidazole for 1 h at 37 °C in the dark. Cells were then processed for immunofluorescence.

Immunoprecipitation

Cells were L-methionine starved for 30 min prior to labeling with [S]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). Postnuclear supernatants were preabsorbed with protein G-agarose and immunoprecipitated with 200 µl of 9E10 supernatant, 2 µg of anti-FLAG M2 (IBI Ltd), 2 µl of AF8, or 2 µl of anti-BiP, in the presence of protein G-agarose. Pellets were washed in lysis buffer (containing 0.5% deoxycholate and 0.2% SDS) and centrifuged through a 30% sucrose cushion, followed by three further 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.

Reimmunoprecipitation was performed on immunoprecipitates in reducing sample buffer. These were diluted 20-fold in lysis buffer containing 2% Triton X-100 prior to the second immunoprecipitation with either 9E10 supernatant, anti-calnexin, or anti-BiP as described above.

Electrophysiological Analysis

Whole cell recordings from transfected A293 cells and analysis of membrane currents from Xenopus oocytes were performed as described previously (Krishek et al., 1994). Currents from transfected cells were analyzed up to 24 h after transfection, wherase Xenopus oocytes were examined at 48 h after nuclear injection.


RESULTS

GABA(A)Receptor Subunit Combinations: Capability of Surface Membrane Expression

To study the assembly of murine GABA(A) receptors, heterologous expression of GABA(A) receptor alpha1, beta2, and 2L (a differentially spliced variant of 2 containing an 8-amino acid insert in the predicted major intracellular domain, which is lacking from the other splice variant, 2S) (Kofuji et al., 1991; Whiting et al., 1990) subunit cDNAs in both transiently transfected A293 cells (CRL 1573) and Xenopus oocytes was utilized.

To aid biochemical and morphological analyses, GABA(A) receptor subunits were tagged using the epitopes of 9E10 or FLAG. These epitopes were added to the alpha1, beta2, and 2L subunits by site-directed mutagenesis between amino acids 4 and 5 of the mature subunits to create alpha1, beta2, and 2L. The functional effects of these additions were tested by electrophysiological analysis in A293 cells and Xenopus oocytes. Receptors incorporating 9E10-tagged subunits produced GABA-activated responses, which were indistinguishable from receptors comprised of wild-type subunits (Draguhn et al., 1990; Smart et al., 1991; Angelotti and Macdonald, 1993) with regard to zinc insensitivity and benzodiazepine modulation (Fig. 1). Similar results were obtained with subunits containing the FLAG epitope. (^2)Therefore, the addition of these small epitopes to the extreme N-terminal domains of GABA(A) receptor subunits appears to be ``functionally silent.''


Figure 1: Functional properties of recombinant GABA(A) receptors incorporating the 9E10 epitope tag expressed in A293 cells. A, GABA equilibrium concentration response curves for alpha1beta2 (circle) and alpha1beta2 (bullet) constructs (n = 3-5 cells). The curves were fitted using a nonlinear least squares Marquadt routine with the following logistic state function: I/I(max) = 1/{1 + (EC/[A]^n)}, where I represents the GABA-activated current at a given concentration of GABA ([A]), and I(max) is the maximum current induced by a saturating concentration of GABA. EC describes the concentration of GABA inducing a half-maximal response, and n is the Hill coefficient. The determined EC values and Hill coefficients for the alpha1beta2 are 0.77 ± 0.07 µM and 1.45 ± 0.22, and for the alpha1beta2, the values are 0.82 ± 0.03 µM and 1.66 ± 0.12. B, whole cell recordings of GABA-activated currents after rapid application of GABA to A293 cells at -50 mV expressing alpha1beta2 constructs. The solid lines indicate the duration of the GABA application. W represents the recovery times (min) for the GABA-induced responses following exposure to 1 µM flurazepam or 10 µM Zn. C, concentration response curves for the recombinant GABA(A) receptors alpha1beta22L (circle) and alpha1beta22L (bullet) expressed in A293 cells. The EC values and Hill coefficients were 4.61 ± 0.83 µM and 1.38 ± 0.32 and 4.8 ± 0.63 µM and 0.98 ± 0.1, respectively. D, examples of GABA-induced membrane currents recorded from alpha1beta2 2L receptors after application of 1 µM flurazepam or 50 µM Zn. Note the enhanced response to GABA after flurazepam and the reduced inhibition by Zn in the 2L subunit containing GABA(A) receptors. The calibration in D applies to the upper and lower series of responses.



The subcellular distribution of these tagged GABA(A) receptors expressed in A293 cells was determined by immunofluorescence of the FLAG epitope followed by confocal microscopy. Expression of alpha1, beta2, or 2L alone revealed an ER-like staining pattern similar to that for horseradish peroxidase containing the C-terminal ER retention signal Lys-Asp-Glu-Leu (horseradish peroxidase-KDEL) (Fig. 2A), which is almost exclusively localized to the ER (Connolly et al., 1994). To further investigate the localization of these GABA(A) receptor subunits, immunofluorescence was performed on cells in which the peroxidase reaction had been performed using diaminobenzidine as the substrate. This results in the production of a dark insoluble precipitate, which cross-links all proteins that co-localize with horseradish peroxidase (Courtoy et al., 1984; Ajioka and Kaplan, 1987). The reaction product should therefore prevent the detection of fluorescence (DAB quenching) if the candidate protein shares the same intracellular localization as horseradish peroxidase.


Figure 2: Surface expression and intracellular localization of GABA(A) receptor unitary, binary, and tertiary subunit combinations. A293 cells were analyzed for GABA(A) receptor/horseradish peroxidase-KDEL expression 1 day after transfection. A, co-immunofluorescence of individual GABA(A) receptor subunits (via FLAG epitope) and horseradish peroxidase-KDEL was performed on permeabilized cells (using anti-horseradish peroxidase antiserum). B, DAB quenching in cells co-expressing horseradish peroxidase-KDEL and alpha1beta2 (alphabeta), alpha12L (alpha), or beta22L (beta), followed by immunofluorescence for the FLAG tag. Areas were selected to illustrate immunofluorescence versus DAB reaction product. C, GABA(A) receptor detection, via FLAG tag, on the surface of nonpermeabilized cells expressing alpha1beta2 (alphabeta), beta2alpha1 (betaalpha), or alpha1beta22L (alphabeta). The scale bar represents 10 microns.



To confirm this, we examined the ability to detect endogenous markers in the presence of DAB reaction product produced from horseradish peroxidase-KDEL. After completion of the DAB reaction, under conditions identical to those shown in Fig. 2B, fluorescence was performed on horseradish peroxidase-KDEL-expressing cells using either antibodies against BiP and calnexin (localized to the ER; Fig. 3A) or fluorescent-labeled wheat germ agglutinin and lens culinaris lectins (markers for the Golgi apparatus; Fig. 3B). In cells expressing horseradish peroxidase-KDEL, no fluorescence was detected using antibodies to the ER markers, BiP, and calnexin, although fluorescence was detected in untransfected cells. In contrast, high levels of fluorescence for the Golgi markers, wheat germ agglutinin, and lens culinaris were observed in horseradish peroxidase-KDEL-transfected cells. These results confirm DAB quenching as a valid protocol for the detection of components within the same compartment.


Figure 3: Localization of ER and Golgi markers using DAB quenching from horseradish peroxidase-KDEL. A293 cells were transfected with the horseradish peroxidase-KDEL cDNA expression construct. 12-18 h after transfection, the cells were fixed and exposed to 0.02% H(2)O(2), 0.15% diaminobenzidine, and 0.1 M imidazole for 1 h at 37 °C. The cells were then processed for immunohistochemical analysis using antisera directed against either ER or Golgi markers. A, ER markers: BiP (Clone 5A5) and calnexin (AF8). B, Golgi markers detected by rhodamine-labeled wheat germ agglutinin (WGA) and fluorescein isothiocyanate-labeled lens culinaris lectin (LC). The left-hand panels are phase images, whereas the right-hand panels represent fluorescent images recorded from the same field. The scale bar represents 10 microns.



Using this methodology we analyzed all three binary subunit combinations alpha1beta2, alpha12L, and beta22L (Fig. 2B). Immunofluorescence was rare (approximately 0.01%) in alpha12L- or beta22L-transfected cells and was never coincident with cells exhibiting DAB staining, suggesting co-localization of these binary subunit combinations with horseradish peroxidase-KDEL. Similar results were also seen with single subunits utilizing DAB quenching.^2 These results are consistent with the restriction to an ER-like staining pattern observed for these combinations in the absence of horseradish peroxidase-KDEL (Fig. 2B; alpha and beta) and their failure to reach the cell surface, as determined in nonpermeabilized cells.^2 Together these results demonstrate that single subunits and the alpha12L and beta22L binary subunit combinations are restricted to the ER.

In contrast, the alpha1beta2 combination was capable of leaving the ER as determined by the co-existence of immunofluorescence and DAB staining within the same cells, consistent with the apparent surface staining (Fig. 2B, alphabeta) and confirmed in nonpermeabilized cells (Fig. 2C, alphabeta and betaalpha). Only when all three subunits were co-expressed could the 2L subunit be detected on the cell surface (Fig. 2C, alphabeta). These results correlated with electrophysiological recordings made from A293 cells expressing all subunit combinations. Consistent with the results from the immunofluorescence experiments, only cells expressing either alpha1beta2 or alpha1beta22L receptor subunits exhibited resolvable GABA-gated currents (Table 1). Similar results were obtained from expression studies performed using Xenopus oocytes up to 72 h after injection. Thus it appears that only the combinations alpha1beta2 and alpha1beta22L can access the cell surface. The accumulation of all nonsurface receptor combinations (alpha, beta, , alpha, and beta) in the ER strongly suggests that receptor assembly occurs in this intracellular compartment.



Immunoprecipitation of GABA(A)Receptor Subunits

Immunoprecipitation from detergent extracts of [S]methionine-labeled A293 cells revealed that the alpha1 subunit exists in three forms with molecular masses of 48, 50, and 52 kDa (Fig. 4, alpha, -). The predicted molecular mass for the polypeptide backbone is approximately 48 kDa (Schofield et al., 1987; Wang et al., 1992). The alpha1 subunit contains two consensus sequences (Asn-Xaa-Ser/Thr) for N-linked glycosylation at positions 10 and 110. Treatment of the alpha1-expressing cells with tunicamycin (a potent inhibitor of N-linked glycosylation) produced a single band coincident with the lowest form of alpha1 subunit of 48 kDa (Fig. 4, alpha, +). Thus, the three alpha1 forms differ in their extent of N-linked glycosylation, and their sizes are consistent with the presence of zero, one, and two sites of N-linked glycosylation.


Figure 4: Immunoprecipitation and glycosylation of GABA(A) receptor alpha1, beta2, and 2L subunits from transfected A293 cells. A293 cells expressing alpha1 (alpha), beta2 (beta), 2L (), or untransfected (C) were [S]methionine-labeled in the absence(-) or presence (+) of 5 µg/ml tunicamycin (present 2 h prior to and during labeling). Receptor subunits were then immunoprecipitated using 9E10 antibody coupled to protein G-Sepharose. Immune complexes were then separated by SDS-polyacrylamide gel electrophoresis using 8% gels. The molecular masses of marker proteins (Bio-Rad) are indicated on the right. A co-immunoprecipitating band migrating at approximately 75 kDa (*) was consistently observed.



The beta2 subunit exhibits apparent molecular masses of 53 and 56 kDa plus a weak band at 50 kDa (Fig. 4, beta, -). Again, this subunit contains two consensus sequences for N-linked glycosylation at positions 8 and 80. Tunicamycin treatment produced a major band at 50 kDa (Fig. 4, beta, +), as predicted by cDNA cloning (Kamatchi et al., 1995). Thus, the beta2 forms are also consistent with the presence of zero, one, and two sites of N-linked glycosylation. The 2L subunit migrated as a broad band at approximately 42 kDa (Fig. 4, , -) and, following tunicamycin treatment, as a broad band of around 30 kDa (Fig. 4, , +); this shift is consistent with the predicted presence of three consensus sites for N-linked glycosylation at positions 13, 90, and 208. The apparent molecular masses of both glycosylated and unglycosylated forms of 2L are much smaller than its 48-kDa predicted molecular mass derived from cDNA cloning (Pritchett et al., 1989). This may be due to proteolysis, a common observation for this subunit (Moss et al., 1992; Haddingham et al., 1992) and may explain the appearance of a smear rather than discreet bands.

Oligomerization of GABA(A)Receptor Subunits

A possible explanation for the differential ability of GABA(A) subunit combinations to reach the cell surface as functional receptors may be found in their respective abilities to oligomerize with each other, as is often a prerequisite for exit from the ER (Hurtley and Helenius, 1989). The ability of GABA(A) receptor subunits to oligomerize was therefore analyzed by co-immunoprecipitation of binary combinations (alpha12L, beta22L, and alpha1beta2).

Consistent with the surface expression of the alpha1beta2 subunit combination, it was found that beta2 co-immunoprecipitates both the 48-kDa unglycosylated and the 50-kDa partially glycosylated but not the 52-kDa fully glycosylated forms of the alpha1 subunit (Fig. 5A, lane 3). The reciprocal co-immunoprecipitation was not so clear and may be complicated by the co-migration of both the alpha1 and beta2 bands at 50 kDa (Fig. 5A, lane 4). Despite being transport incompetent, the alpha1 and 2L subunits could also co-immunoprecipitate each other (Fig. 5B, lanes 3 and 4). As seen with the beta2 subunit, the 2L also binds exclusively to the two lower forms of alpha1 (Fig. 5B, lane 3). Thus, neither beta2 nor 2L show detectable oligomerization with the 52-kDa fully glycosylated form of the alpha1 subunit, even though it is the major species present. Similarly, the beta2 and 2L subunits are also capable of oligomerizing despite their transport incompetence. In this case, 2L binds predominantly to the nonglycosylated 50-kDa form of beta2 (Fig. 5C, lane 3). It also appears that both alpha1 and beta2 bind preferentially to lower molecular mass 2L (Fig. 5, B, lane 4, and C, lane 4, respectively) compared to the major species present when immunoprecipitated directly (Fig. 5, B, lane 2, and C, lane 2). These preferences for subunit binding occur in the presence of the nonbinding forms, which exist within 5 min of [S]methionine labeling, a period in which oligomerization has occurred.^2 Similar patterns of subunit oligomerization were seen under nonreducing conditions, suggesting that they did not result from intermolecular disulfide bridges, as well as after solubilization in a range of other detergents including CHAPS (1%) and digitonin (1%).^2 To test the oligomerization of the triple subunit combination alpha1beta22L immunoprecipitation utilizing 2L subunit was performed. These results demonstrated association of the beta2 and alpha1 subunits with the 2L subunit as expected.^2


Figure 5: Oligomerization of GABA(A) receptor subunits expressed in A293 cells. A293 cells expressing single or binary combinations of GABA(A) receptor subunits were labeled with [S]methionine, cells were lysed, and GABA(A) receptor subunits were purified using 9E10 antibody coupled to protein G-Sepharose. GABA(A) receptor subunits were then separated by SDS-polyacrylamide gel electrophoresis using 8% gels. Control cells show the presence of a contaminating band at approximately 40 kDa, which is sometimes observed regardless of the antibody used (arrowhead). A, oligomerization of alpha1 and beta2 subunits: lane 1, alpha1; lane 2, beta2; lane 3, beta2alpha1; lane 4, alpha1beta2. B, oligomerization of alpha1 and 2L subunits: lane 1, alpha1; lane 2, 2L; lane 3, 2Lalpha1; lane 4, alpha12L. C, oligomerization of beta2 and 2L subunits: lane 1, beta2; lane 2, 2L; lane 3, 2Lbeta2; lane 4, beta22L.



Taken together with the immunolocalization studies, these experiments detailing the oligomerization of receptor subunits demonstrate that GABA(A) receptor assembly is primarily localized to the ER.

GABA(A)Receptor Assembly Is Independent of N-Linked Glycosylation

To examine the significance of N-linked glycosylation in receptor assembly, subunit oligomerization in the presence of tunicamycin was analyzed. Transfected cells expressing receptor cDNAs were treated with tunicamycin to prevent N-glycosylation and labeled with [S]methionine. Subunit oligomerization was then examined by co-immunoprecipitation using 9E10 antibodies directed against the alpha1 subunit. Precipitation of this subunit from cells co-expressing the beta2 and 2L subunit (Fig. 6, lane 3) demonstrated that subunit oligomerization could occur in the absence of N-linked glycans, because the nonglycosylated alpha1 subunit (identified in Fig. 6, lane 1) co-precipitated the nonglycosylated beta2 subunit (identified in Fig. 6, lane 2) as well as the nonglycosylated 2L subunit (30 kDa). Furthermore the oligomerization of the binary subunit complex alpha12L was also apparently unaffected by tunicamycin treatment as determined by co-precipitation (Fig. 6, lane 4).


Figure 6: Effect of tunicamycin on GABA(A) receptor subunit oligomerization. A293 cells transfected with alpha1 (lane 1); beta2 (lane 2); alpha1, beta2, and 2L (lane 3); alpha1 and 2L (lane 4); or control untransfected cells were labeled with [S]methionine, immunoprecipitated using 9E10 antibody coupled to protein G-Sepharose, and resolved by SDS-polyacrylamide gel electrophoresis using 8% gels. Cells were treated with 5 µg/µl tunicamycin for 2 h prior to and during labeling with [S]methionine. C represents immune precipitations from control untreated cells. The presence of a contaminating band of 40 kDa, which is sometimes observed regardless of antibody used, is indicated (*).



Although the inhibition of N-linked glycosylation did not affect subunit oligomerization, N-linked glycosylation may be important for subsequent maturation. Therefore, we examined cell surface receptor expression in the presence of tunicamycin. This treatment caused other effects, most notably a reduction in cell number, reduced transfection efficiency, and changes in morphology. However, cell surface expression was not prevented, as determined by immunofluorescence for receptors consisting of alpha1beta22L subunits (Fig. 7A) but not for intracellular markers such as BiP, although the efficiency was significantly reduced from 95 ± 7.5% in the absence to 37 ± 15.52% in the presence of tunicamycin (Fig. 7B). This is consistent with the report of the alpha1 subunit (lacking N-linked glycosylation sites by site-directed mutagenesis) in the presence of beta1 and 2, which produced functional GABA(A) receptors with pharmacological properties similar to those observed for the wild-type receptor but with reduced levels (Buller et al., 1994).


Figure 7: Effect of tunicamycin treatment on cell surface expression. A, immunofluorescence for the alpha1 subunit in the presence (+) or the absence(-) of TX100 was performed on A293 cells transfected with alpha1beta22L (-tunicamycin). In a duplicate experiment, the cells were treated with tunicamycin constantly post-transfection, as well as 2 h pretransfection (+tunicamycin). B, quantitation of the frequency of surface (-TX100) fluorescence as a percentage of the frequency of total (+TX100) fluorescence. In the absence of tunicamycin 38.23 ± 3.03% (n = 519) were positive in the absence of TX100, with 40.3 ± 7.84% (n = 445) in the presence of TX100. In the presence of tunicamycin 6.05 ± 2.53% (n = 843) were positive in the absence of TX100, with 16.3 ± 7.2% (n = 336) in the presence of TX100 (where n = total number of cells counted).



GABA(A)Receptor Subunits Interact with ER Chaperone Proteins

The correct folding of many proteins have been shown to involve the interaction of BiP (Pelham, 1989). In addition, another ER chaperone, calnexin, appears to be involved exclusively with glycoproteins (Ou et al., 1993). As seen earlier, immunoprecipitation of either alpha1, beta2, or 2L GABA(A) receptor subunits routinely co-immunoprecipitated a protein of approximately 75 kDa (Fig. 4, asterisk), which may represent BiP. We therefore sought to determine if these two chaperone proteins participate in the assembly of GABA(A) receptors by retention of unassembled subunits within the ER, as observed for the alpha1, beta2, 2L, alpha12L, and beta22L combinations. Cells expressing the alpha1 subunit alone (which is transport-incompetent and therefore retained in the ER) were first immunoprecipitated with 9E10 antibody. This precipitate was then reprecipitated with either anti-BiP, anti-calnexin, or 9E10 antibodies, and bands migrating with expected molecular masses for both BiP and calnexin were evident (Fig. 8A). In addition, weak bands co-migrating with the nonprecipitated proteins (e.g. BiP and calnexin when performed via 9E10) were also present (weakly visible in Fig. 8A).


Figure 8: Interaction of GABA(A) receptor subunits with the ER chaperones: calnexin and BiP. A, cells transfected with the GABA(A) alpha1 subunit were labeled with [S]methionine and then immunoprecipitated using 9E10 antibody coupled to protein G-Sepharose. The precipitated material was then reprecipitated with antibodies against BiP (lane 1), calnexin (AF8) (lane 2), or 9E10 (lane 3). B, A293 cells expressing alpha1 (lane 1), beta2 (lane 2), or 2L (lane 3) and untransfected cells (lane 4) were immunoprecipitated using either anti-calnexin antibody (AF8) or by 9E10 antibody (9E10). Migration of the 90-kDa calnexin (AF8) and the 75-kDa BiP bands as well as the mature GABA(A) receptor subunits are identified.



This interaction with BiP and calnexin is not unique to the alpha1 subunit, because the alpha1, beta2, and 2L subunits can also be co-immunoprecipitated by anti-calnexin antibody (Fig. 8B) with no apparent preference for different forms. In all three cases, a protein migrating at the same molecular mass as BiP is also co-immunoprecipitated, suggesting some overlap in the binding abilities of BiP and calnexin. This overlap appears to occur with some endogenous proteins in A293 cells as evidenced by the untransfected control lane (Fig. 8B), in which anti-calnexin antibody co-immunoprecipitated a band coincident with BiP. When these apparent complexes were immunoprecipitated by 9E10, calnexin is only weakly observed, consistent with its low turnover rate and subsequently low [S]methionine incorporation (Hammond et al., 1994). Upon expression of all three, subunits BiP is still immunoprecipitated (Moss et al., 1995). However, whether this is due to interactions occurring between unitary, binary, or tertiary subunit complexes under these conditions is difficult to ascertain.


DISCUSSION

To date, 15 different GABA(A) receptor cDNAs have been isolated from a variety of vertebrates (Burt and Kamatchi, 1991). 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 (Wisden and Seeburg, 1992). A major challenge in trying to analyze the diversity of GABA(A) receptor structure in the brain is determining what processes control receptor assembly. Regulation of receptor assembly could occur at numerous stages, including subunit oligomerization or export to the cell surface. Unfortunately, to date there is little experimental data on these important questions. To address this, we have examined the assembly of GABA(A) receptors of differing subunit compositions constructed from those of alpha1, beta2 and 2L subunits expressed in both A293 cells and Xenopus oocytes using immunological, biochemical, and electrophysiological methodologies. These subunits are thought to co-localize in many brain regions and comprise up to 30% of all benzodiazepine-sensitive GABA(A) receptors in the adult brain (Fritschy et al., 1992; Benke et al., 1994). For immunological and biochemical analyses the 9E10 (Evan et al., 1985) or the FLAG epitopes were added between amino acids 4 and 5 of each of these subunits. As demonstrated by electrophysiological analyses, these additions appeared to be functionally silent.

Using immunolocalization, epitope tagged alpha1, beta2, or 2L subunits expressed alone are incapable of leaving the ER, as are the binary subunit combinations of alpha12L and beta22L, demonstrated by co-localization with horseradish peroxidase-KDEL (Connolly et al., 1994). The only combinations of receptors produced from these subunits that are capable of exiting the ER and accessing the cell surface are alpha1beta2 and alpha1beta22L. In agreement with this only the latter two subunit combinations exhibited resolvable GABA-gated currents when expressed in either A293 cells or Xenopus oocytes.

Previous studies have produced conflicting data on the expression of single subunits and the combinations of alpha12L and beta22L, as determined by electrophysiological analysis. Blair et al. (1988) reported the production of GABA-gated channels on the expression of single alpha1 or beta1 subunits in Xenopus oocytes, and Sigel et al.(1989) reported finding chloride currents that could be blocked by picrotoxin on the expression of the rat beta1 subunit. Moreover, Verdoorn et al.(1990) and Draguhn et al.(1990) found robust receptor expression from alpha12 subunits and smaller currents from beta22L subunits in A293 cells. In contrast to these results Angelotti and Macdonald(1993) and Krishek et al.(1994) found no GABA-gated currents when expressing alpha12 or beta12 subunits in L929 or A293 cells. These discrepancies could be due to differences in the expression systems used or differences in the type of beta subunit used in some of these experiments (beta1 versus beta2). Expression of murine beta1 subunits alone in both A293 cells and Xenopus oocytes can produce low levels of surface expression, (^3)in common with the observations of Blair et al.(1988) and Sigel et al.(1989). Therefore some of this variability in expression may be subunit-specific. A second reason for such discrepancies may result from over-expression. It is possible that the longer expression periods used in many previous studies (48-72 h in A293 cells and 2-6 days in oocytes (Blair et al., 1988; Verdoorn et al., 1990) compared with 12-24 h and 2-3 days for 293 cells and Xenopus oocytes, respectively, used in this study) may have resulted in the escape of normally transport-incompetent receptor complexes through saturation of the ER retention system, resulting in low levels of surface expression.

In spite of the apparent inability of the alpha12L and beta22L subunit combinations to leave the ER, they were still capable of rapid oligomerization. In fact, all oligomerization events were not dependent on N-linked glycosylation and could occur with unglycosylated subunits. Furthermore, N-linked glycosylation was not required for the transport of receptor heteroligomers to the cell surface. Interestingly, the beta2 subunit appeared to oligomerize more efficiently to lower molecular mass forms of the alpha1 (and possibly the 2L subunits), and the 2L subunit appeared to oligomerize more efficiently to lower molecular mass forms of both the alpha1 and the beta2 subunits. The significance of these interactions is uncertain; they may represent preferred patterns of subunit oligomerization if these incompletely processed forms are represented in cell surface receptors in the presence of glycosylated forms. Whether these interactions are important in controlling the final assemblies of GABA(A) receptors produced warrants further investigation.

GABA(A) receptor subunits interacted with at least two ER chaperone proteins, BiP and calnexin, whose function is to retain misfolded and unassembled proteins. Interactions with BiP are thought to result from the exposure of hydrophobic domains in incorrectly folded proteins (Pelham, 1989), whereas calnexin appears to show specificity for glycoproteins containing partially ``glucose-trimmed'' carbohydrate side chains (Hammond et al., 1994). Calnexin is thought to hold glycoproteins in the ER until folding/assembly is complete, thus preventing their aggregation and/or premature exit from the ER (Hochstenbach et al., 1992; Ou et al., 1993; Hammond et al., 1994). Presumably single GABA(A) receptor subunits and the binary combinations of alpha12L and beta22L are retained in the ER via the interaction with chaperone proteins such as calnexin and BiP.

The demonstration of the intracellular localization of GABA(A) receptor heteroligomers has important consequences for our understanding of GABA(A) receptor structure. To date, the method of choice for examining the complexity of GABA(A) receptor structure in the brain has been the use of subunit-specific antisera to immunoprecipitate receptor complexes from solubilized brain tissue (e.g. Duggan and Stephenson, 1990; Mertens et al., 1993; Mckernan et al., 1991; Mossier, 1994). These procedures are complex and often yield contradictory results (cf. Pollard et al.(1993), Quirk et al.(1994), and Khan et al.(1994)). It is possible that some of the interactions seen following immunoprecipitation in these experiments and thereby proposed to represent cell surface receptor subunit combinations may in fact represent ER-retained forms of GABA(A) receptors. Such complexes as demonstrated in our study may have differing subunit combinations from GABA(A) receptors expressed on the surface of neurons.

Finally, the inability of GABA(A) receptor heteroligomers consisting of alpha12L and beta22L subunits to reach the cell surface suggests that assembly of GABA(A) receptors might share similar mechanisms to those employed by muscle acetylcholine receptors. Assembly of these receptors is believed to utilize intracellular dimer or trimer intermediates (Green and Miller, 1995). Further studies will elucidate whether the ER-retained subunit heteroligomers described in our work are important intermediates in the production of fully assembled pentameric GABA(A) receptors. This may be of significance because the consensus of opinion (Burt and Kamatchi, 1991; Pritchett et al., 1988, 1989), derived from a combination of molecular, pharmacological, and electrophysiological methodologies, suggests that in vivo benzodiazepine-responsive GABA(A) receptors are composed of alphabeta in unknown stoichiometries. The relevance of these alpha12L and beta22L intracellular subunit complexes to the final functional GABA(A) receptors produced is currently under investigation.


FOOTNOTES

*
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.

§
To whom correspondence should be addressed: The MRC LMCB, University College London, Gordon Street, London WC1E 6BT, UK. Tel.: 44-71-380-7912; Fax: 44-71-380-7805.

(^1)
The abbreviations used are: GABA(A), -aminobutyric acid type A; ER, endoplasmic reticulum; BiP, immunoglobulin heavy chain binding protein; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DAB, diaminobenzidine.

(^2)
C. N. Connolly and S. J. Moss, unpublished results.

(^3)
Krishek, B. J., Moss, S. J., and Smart, T. G.(1996) Mol. Pharmacol., in press.


ACKNOWLEDGEMENTS

We thank Dan Cutler (Medical Research Council, Laboratory of Molecular Cell Biology) for the generous gift of the horseradish peroxidase-KDEL construct and for comments on the manuscript. Michael Brenner (Harvard Medical School) is also thanked for supplying the anti-calnexin antibody (AF8), and Neil Hotchin is thanked for advice.


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