Alternate Use of Distinct Intersubunit Contacts Controls GABA_A Receptor Assembly and Stoichiometry

Abstract

GABA_A receptors are the major inhibitory transmitter receptors in the CNS. Recombinant GABA_A receptors composed of α₁β₃γ₂ subunits have been demonstrated to assemble as pentamers consisting of two α₁, two β₃, and one γ₂ subunit. Using truncated and chimeric α₁subunits, we identified the α₁(80–100) sequence as a major binding site for γ₂ subunits. In addition, we demonstrated its direct interaction with γ₂(91–104), a sequence that previously has been identified to form the contact to α₁ subunits. The observation that the amino acid residues α₁P96 and α₁H101, which can be photolabeled by [³H]flunitrazepam, are located within or adjacent to the α₁(80–100) sequence, indicates that the benzodiazepine binding site of GABA_A receptors is located close to this intersubunit contact. The observation that α₁(80–100) interacts with γ₂ but not with β₃ subunits indicates the existence of an additional β₃ binding site on α₁ subunits. The preferred alternate use of the γ₂ and β₃binding sites in two different α₁ subunits of the same receptor ensures the incorporation of only a single γ₂subunit and thus, determines subunit stoichiometry of α₁β₃γ₂ receptors. Distinct binding sites and their alternate use can therefore explain how subunits of hetero-oligomeric transmembrane proteins assemble into a defined protein complex.

Members of the ligand-gated ion channel family, such as the nicotinic acetylcholine receptor (nAChR), the GABA_A receptor, the glycine receptor, or the 5-HT₃ receptor, are heteromeric proteins composed of five subunits (Bertrand and Changeux, 1995). The subunits of these proteins are cotranslationally inserted into the membrane, lumen, or both, of the endoplasmic reticulum, after which the subunits fold and oligomerize (Verrall and Hall, 1992; Green and Claudio, 1993; Connolly et al., 1996a; Griffon et al., 1999). During these folding and oligomerization events, each subunit must recognize its neighbors by specific high-affinity interactions. To achieve the correct order of subunits around the pore, in addition selective discriminations must be made between different subunits. So far, little is known about the molecular structures involved in these mechanisms.

GABA_A receptors are the major inhibitory neurotransmitter receptors in the CNS. These receptors are chloride ion channels that can be opened by GABA (Macdonald and Olsen, 1994) and are the site of action of various pharmacologically and clinically important drugs, such as benzodiazepines, barbiturates, steroids, anesthetics, and convulsants. These drugs modulate GABA-induced chloride ion flux by interacting with separate and distinct allosteric binding sites (Sieghart, 1995).

GABA_A receptors are hetero-oligomeric proteins consisting of five subunits (Nayeem et al., 1994; Tretter et al., 1997). So far at least 19 GABA_A receptor subunits belonging to several subunit classes (six α, three β, three γ, one δ, one ε, one π, one θ, and three ρ) have been identified in mammalian brain (Barnard et al., 1998; Sieghart et al., 1999). In situ hybridization and immunocytochemical studies indicate a distinct but overlapping temporal and regional expression of these subunits. The finding that multiple receptor subunits are expressed within single neurons (Fritschy et al., 1992; Pirker et al., 2000) raises the possibility for the formation of an extremely large variety of GABA_A receptor subtypes. However, not all receptors that can be formed theoretically are formed in the cells (Sieghart et al., 1999). Thus, GABA_A receptor heterogeneity is limited by the temporal and spatial pattern of subunit expression and by the selective oligomerization mediated by receptor assembly.

Recently, the subunit stoichiometry and arrangement of recombinant α₁β₃γ₂GABA_A receptors have been determined (Tretter et al., 1997). In this receptor only a single γ₂subunit is present and is situated between an α₁ and a β₃ subunit. In addition, the amino acid sequence γ₂(91–104) was identified to form the binding site to α₁subunits (Klausberger et al., 2000).

In the present study truncated and chimeric α₁ subunits were used to identify the α₁ sequence mediating assembly with γ₂ subunits. It was demonstrated that the sequence α₁(80–100) directly interacts with γ₂(91–104) and forms part of the α₁–γ₂ interface. The observation that α₁(80–100) mediates assembly with γ₂ but not with β₃subunits suggests the existence of an additional binding site for β₃ subunits. The preferred alternate use of the γ₂ and β₃ binding sites in different α₁ subunits of the same receptor indicates that the α₁–γ₂ intersubunit contact controls assembly and subunit stoichiometry of GABA_A receptors.

MATERIALS AND METHODS

Antibodies. The antibodies anti-peptide α₁(1–9), anti-peptide β₃(1–13), anti-peptide γ₂(319–366), and anti-peptide γ₂(1–33) were generated and affinity purified as described previously (Tretter et al., 1997; Jechlinger et al., 1998;Klausberger et al., 2000).

Generation of cDNA constructs. For the generation of recombinant receptors, α₁, β₃, and γ₂ subunits of GABA_A receptors from rat brain were cloned and subcloned into pCDM8 expression vectors (Invitrogen, San Diego, CA) as described previously (Tretter et al., 1997). Truncated subunits were constructed by PCR amplification using the full-length subunit as a template. The PCR primers contained EcoRI andHindIII restriction sites, which were used to clone the fragments into pCDNAIAmp vectors (Invitrogen). The truncated subunits were confirmed by sequencing. Chimeras were constructed using the “gene splicing by overlap extension” technique (Horton, 1993) and were cloned into pCDNAIAmp vectors using theEcoRI and HindIII restriction sites of the primers.

Culture and transfection of human embryonic kidney 293 cells. Transformed human embryonic kidney (HEK 293) cells (CRL 1573; American Type Culture Collection, Rockville, MD) were cultured as described in Tretter et al. (1997). We transfected 3 × 10⁶ cells with 20 μg of subunit cDNA for single subunit transfection using the calcium phosphate precipitation method (Chen and Okayama, 1988). After cotransfection with two different subunits, for each subunit 10 μg of cDNA was used. When cells were cotransfected with three different subunits, 7 μg of cDNA was used per subunit. A total of ∼20 μg of cDNA per transfection and a cDNA ratio of 1:1:1 seemed to be optimal for the expression of GABA_A receptors under the conditions used, as judged by receptor binding studies in cells transfected with α₁, β₃, and γ₂ subunits. Changing the subunit ratio by doubling the amount of a single subunit at the cost of other subunits did not significantly change the number of [³H]Ro 15–1788 binding sites detected.

The cells were then harvested 36 hr after transfection. At this time point the number of [³H]Ro 15–1788 binding sites formed per milligram of protein was at its maximum for cells transfected with α₁, β₃, and γ₂ subunits. Results obtained, however, did not change when cells were harvested 34–48 hr after transfection. In addition, judged by Western blot analysis, expression levels of full-length, truncated, or chimeric subunits were comparable (see Figs. 1, 6) at all harvesting times.

Fig. 1.

Coimmunoprecipitation of truncated α₁ with full-length γ₂ subunits.A, Schematic drawing of the α₁ subunit and of C-terminally truncated α₁ constructs. The α₁ subunit consists of the N-terminal extracellular domain with the typical cysteine loop, of four transmembrane domains (TM1–4), and the large cytoplasmic loop between TM3 and TM4. The sequences of the C-terminally truncated α₁ constructs are indicated by the amino acid numbers given inparentheses. A 1 represents the first amino acid of the mature subunit. B, HEK cells were transfected with truncated α₁ constructs together with full-length γ₂ subunits, as indicated. Cell extracts were immunoprecipitated with γ₂(319–366) antibodies. α₁ fragments coprecipitated were identified by SDS-PAGE and Western blot analysis using digoxygenized α₁(1–9) antibodies (lanes 1–6). In control experiments (lanes 7, 8), truncated α₁(1–117) and α₁(1–100) constructs were transfected separately into HEK cells, and the fragments formed were precipitated with α₁(1–9) antibodies and subjected to SDS-PAGE and Western blot analysis using digoxygenized α₁(1–9) antibodies. The protein fragments formed from these constructs [apparent molecular mass: 14, 16, and 18 kDa for α₁(1–117) and apparent molecular mass: 12 and 14 kDa for α₁(1–100)] were expressed to a similar extent. Interestingly, the relative abundance of the unglycosylated, monoglycosylated, and diglycosylated α₁(1–117) fragments differed when these fragments were expressed in the absence or presence of γ₂ subunits, possibly suggesting that γ₂ subunits preferentially assemble with fully glycosylated α₁(1–117) fragments. All experiments were performed three times with comparable results.

Fig. 6.

Amino acid sequence α₁(80–100) directly binds to γ₂(91–104). A, B, HEK cells were cotransfected with the constructs as indicated. Cell extracts were immunoprecipitated with β₃(1–13) antibodies, and the precipitate was subjected to SDS-PAGE and Western Blot analysis using digoxygenized α₁(1–9) antibodies.A, Schematic representation of the experiment. + indicates binding, and − indicates absence of binding between the cotransfected constructs. B, Western blots demonstrating binding between constructs under condition “4”. C, Western blots demonstrating the extent of expression of the indicated constructs on single transfection into HEK cells. All experiments were performed three times with similar results.

Purification and immunoprecipitation of complete, truncated, and chimeric subunits. The culture medium was removed from transfected HEK 293 cells, and cells from four culture dishes were extracted with 800 μl of a Lubrol extraction buffer (1% Lubrol PX, 0.18% phosphatidylcholine, 150 mm NaCl, 5 mm EDTA, 50 mm Tris-HCl, pH 7.4, containing 0.3 mm PMSF, 1 mm benzamidine, and 100 μg/ml bacitracin) for 8 hr at 4°C. The extract was centrifuged for 40 min at 150,000 ×g at 4°C, and the clear supernatant was incubated overnight at 4°C under gentle shaking with 15 μg antibodies directed against the full-length subunit. After addition of Immunoprecipitin (Life Technologies, Gaithersburg, MD; for preparation, see Tretter et al., 1997) and 0.5% nonfat dry milk powder and shaking for additional 3 hr at 4°C, the precipitate was washed three times with a low-salt buffer for immunoprecipitation (IP low buffer) (50 mm Tris-HCl, 0.5% Triton X-100, 150 mm NaCl, and 1 mm EDTA, pH 8.0). The precipitated proteins were dissolved in sample buffer [108 mm Tris-sulfate, pH 8.2, 10 mm EDTA, 25% (w/v) glycerol, 2% SDS, and 3% dithiothreitol]. SDS-PAGE and Western blot analysis with digoxygenized antibodies was performed as described in Tretter et al. (1997).

All truncated or chimeric constructs used in this study could be expressed to a comparable extent after single transfection into HEK cells. After cotransfection of different constructs, however, the stability of fragments that could not bind stably to each other was reduced. This might have been caused by proteolytic degradation because of an unstable or unproductive interaction of the fragments. In all control experiments the extent of expression of fragments was therefore determined in singly transfected HEK cells.

Immunoprecipitation of receptors expressed on the cell surface. The culture medium was removed from HEK 293 cells transfected with cDNA (21 μg per 3 × 10⁶ cells) of GABA_Areceptor subunits (cDNA ratio 1:1:1), and the cells were washed once with PBS (in mm: 2.7 KCl, 1.5 KH₂PO₄, 140 NaCl, and 4.3 Na₂HPO₄, pH 7.3). Cells were then detached from the culture dishes by incubating with 2.5 ml of 5 mm EDTA in PBS for 5 min at room temperature. The resulting cell suspension was diluted in 6.5 ml of cold DMEM and centrifuged for 5 min at 1000 × g. The pellet from two dishes was incubated with 30 μg of α₁(1–9) antibodies in 3 ml of the same medium for 30 min at 37°C. Cells were again pelleted, and free antibodies were removed by washing twice with 10 ml of PBS buffer. Then receptors were extracted with IP low buffer containing 1% Triton X-100 for 1 hr under gentle shaking. Cell debris was removed by centrifugation (30 min; 150,000 × g; 4°C). After addition of Immunoprecipitin and 0.5% nonfat dry milk powder and shaking for 3 hr at 4°C, the precipitate was centrifuged for 10 min at 10,000 × g and washed three times with IP low buffer. The precipitated proteins were dissolved in sample buffer and subjected to SDS-PAGE and Western blot analysis using digoxygenized antibodies. Secondary antibodies (anti-digoxygenin-AP, Fab fragments; Roche Diagnostics GmbH, Mannheim, Germany) were visualized by the reaction of alkaline phosphatase with CSPD (Tropix, Bedford, MA). Protein bands were quantified by densitometry of Kodak X-Omat S films with the Docu Gel 2000i gel documentation system using restriction fragment length polymorphism scan software (MWG Biotech, Ebersberg, Germany). The linear range of the detection system was established by determining the antibody response to a range of antigen concentrations after immunoblotting. The experimental conditions were designed such that immunoreactivities obtained in the assay were within this linear range, thus permitting a direct comparison of the amount of antigen applied per gel lane between samples. Different exposures of the same membrane were used to ensure that the measured signal was in the linear range of the x-ray film.

To verify that only receptors on the cell surface were labeled by the antibodies, parallel samples were incubated with antibodies directed against the intracellular loop of GABA_A receptor subunits (data not shown). These antibodies could not precipitate any GABA_A receptor subunits under the conditions used. A possible redistribution of the antibodies during the extraction procedure could be excluded by an experiment performed analogous to that described in Klausberger et al. (2000).

Immunofluorescence. HEK cells were fixed with 2% paraformaldehyde in PBS 30–35 hr after transfection, followed by a 10 min wash in 50 mm NH₄Cl in PBS. Washes between incubation steps were performed in PBS. For detection of intracellular receptors, cells were permeabilized with 0.1% Triton X-100 for 5 min. Blocking was performed in 5% bovine serum albumin (BSA) in PBS for 10 min, followed by an incubation with primary antibody in 1% BSA in PBS. Primary antibodies were detected with goat anti-rabbit IgG_(H+L) bodipy FL (Molecular Probes, Eugene, OR) in 1% BSA in PBS. Labeling was visualized using a Zeiss Axiovert 135 M microscope attached to a confocal laser system (Carl Zeiss LSM 410, BRD), equipped with an argon laser and a helium–neon laser and suitable filter sets. To verify that labeling of cells without permeabilization was restricted to the cell surface, parallel samples were stained with antibodies directed against the intracellular loop of GABA_A receptor subunits (data not shown). These antibodies detected GABA_Areceptor subunits only after permeabilization of transfected cells.

Electrophysiological investigations. HEK cells were cotransfected with GABA_A receptor subunits together with pEGFP-N1 (Clontech, Palo Alto, CA) as a transfection marker. Electrophysiologic recordings were performed at room temperature 1–2 d after transfection using the perforated patch technique (Rae et al., 1991). GABA and ZnCl₂ were applied using a DAD-12 superfusion system (Adams and List Associates Ltd., Westbury, NY). Extracellular solution contained (in mm): 140 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, 5 glucose, and 10 HEPES, pH 7.4. The pipette solution contained (in mm): 140 KCl, 11 EGTA, 1 CaCl₂, 1 MgCl₂, 10 HEPES, and 0.2 amphotericin B, pH 7.2. The cells were clamped at −60 mV, and currents were filtered at 1 kHz, recorded with an Axopatch 200A amplifier (Axon Instruments, Foster City, CA) and analyzed with Clampfit software (Axon Instruments).

RESULTS

Truncated α₁ constructs are able to assemble with full-length γ₂ subunits

In the present study C-terminally truncated α₁ subunits (Fig.1A) were cloned, and it was investigated which of these fragments could assemble with full-length γ₂ subunits. For this, HEK cells were cotransfected with γ₂ subunits and either full-length or truncated α₁ subunits. Expressed subunits were extracted from these cells and were immunoprecipitated with γ₂(319–366) antibodies. The precipitate was subjected to SDS-PAGE and Western blot analysis using digoxygenized α₁(1–9) antibodies. As shown in Figure1B, full-length α₁ subunits (protein band of 51 kDa), as well as the fragments α₁(1–249) (two bands of 39 and 41 kDa), α₁(1–221) (three bands of 26, 28, 30 kDa), α₁(1–134) (three bands of 17, 19, 21 kDa), and α₁(1–117) [three bands of 14, 16 (very weak), and 18 kDa] could be coimmunoprecipitated with full-length γ₂ subunits from appropriately transfected HEK cells. Because all α₁ fragments investigated contained two glycosylation sites, the three bands presumably represented unglycosylated, partially, and fully glycosylated fragments. The observation that only one or two protein bands could be observed for the full-length α₁ subunit or the α₁(1–249) construct might indicate that these subunits predominantly occur in the double-glycosylated or double- and mono-glycosylated state, respectively. This conclusion is supported by the apparent molecular mass of these proteins that amounted to 51 kDa or 41 kDa for the full-length α₁ subunit or the α₁(1–249) construct, respectively, although the ungycosylated mass of these proteins can be calculated to be 47 or 37 kDa. Alternatively, the differentially glycosylated protein bands with higher molecular mass might not have been resolved by the 15% polyacrylamide gel used in this investigation.

Binding between γ₂ subunits and these fragments seemed to be the result of a specific assembly process because after cotransfection of HEK cells with full-length γ₂subunits and the fragment α₁(1–221), high-affinity binding sites for the benzodiazepine [³H]Ro15–1788 were formed. These sites are assumed to be located at the interface of α₁ and γ₂ subunits in GABA_A receptors (Sigel and Buhr, 1997). The number of [³H]Ro15–1788 binding sites formed (16.8 ± 2.3 fmol/mg protein) was comparable with that observed after transfection of HEK cells with full-length α₁ and γ₂ subunits (17.6 ± 1.2 fmol/mg protein) but was significantly smaller (p < 0.001; unpaired Student's ttest) than that of HEK cells transfected with α₁, β₃, and γ₂ subunits in parallel experiments (874 ± 19 fmol/mg of protein). Data given are mean values ± SEM from three different experiments performed in triplicate.

In contrast to the protein fragments formed from α₁(1–117), the fragments formed from α₁(1–100) could not be coprecipitated with γ₂ subunits from appropriately cotransfected HEK cells (Fig. 1B), although the extent of expression of these fragments (12 and 14 kDa) was similar to that of the fragments derived from the α₁(1–117) construct (Fig. 1B, lanes 7, 8). The inability of γ₂(319–366) antibodies to coprecipitate the fragment α₁(1–100) confirmed previous conclusions (Jechlinger et al., 1998) that these antibodies did not cross-react with α₁ subunits. These results indicate that the amino acid sequence of the α₁ subunit that is responsible for binding to γ₂ subunits is located in the N-terminal 117 amino acids of the α₁ subunit.

Amino acid sequence α₁(80–100) mediates binding to γ₂ subunits

To identify this contact site, it was investigated which α₁ amino acid sequence could induce binding to γ₂ subunits after incorporation into a fragment that originally could not bind to these subunits. The fragment β₃(1–115) seemed to be suitable for this purpose because it is homologous to α₁(1–117) but could not be coprecipitated with γ₂subunits (or β3 subunits) after coexpression in HEK cells (Fig.2). To incorporate binding sites of the α₁ subunit, several chimeras were constructed by replacing the C-terminal part of the β₃(1–115) fragment with the corresponding α₁ sequences (Fig. 2). These chimeras were transfected into HEK cells together with full-length γ₂ subunits. Expressed subunits were precipitated from cell extracts with γ₂(319–366) antibodies. The precipitate was subjected to SDS-PAGE, and the proteins were detected with digoxygenized β₃(1–13) antibodies in Western blots. The actual expression of the chimeras was confirmed by precipitation and detection with β₃(1–13) antibodies (data not shown).

Fig. 2.

α₁(80–100) forms the contact site to γ₂ subunits. C-terminal sequences of the fragments α₁(1–117), β₃(1–115), and of different chimeras are shown. Amino acid sequences of the α₁subunit are boxed. HEK cells were cotransfected with these constructs together with γ₂ subunits, and a possible coimmunoprecipitation was investigated, as described in Results. + indicates binding, and − indicates absence of binding between these constructs and full-length γ₂subunits. The experiments were performed three times with similar results.

In β₃(1–115)chimα₁(101–117) the 17 C-terminal amino acids of the β₃(1–115) fragment were replaced by amino acids 101–117 of the α₁ subunit. As indicated in Figure 2, this chimera could not be coprecipitated with full-length γ₂ subunits from appropriately cotransfected HEK cells, demonstrating the specificity of the γ₂(319–366) antibodies used and indicating that amino acids α₁(101–117) are not able to induce binding to γ₂ subunits. In β₃(1–115)chimα₁(80–117), the amino acid sequence β₃(78–115) was replaced by α₁(80–117). This construct was able to bind to full-length γ₂ subunits (Fig.2), but not to full-length β₃ subunits (data not shown). Because amino acids α₁(101–117) were not sufficient to induce binding to γ₂subunits as discussed above, this indicated that amino acids 80–100 of the α₁ subunit are important for binding to γ₂ subunits. To directly confirm this conclusion, the construct β₃(1–115)chimα₁(80–100) was generated (Fig. 2), in which amino acids α₁(80–100) were incorporated into β₃(1–115), replacing amino acids β₃(78–98). As expected, this chimera was able to bind to γ₂ subunits.

To investigate which part of the α₁(80–100) sequence is responsible for binding to γ₂subunits, four additional chimeras were constructed. In β₃(1–115)chimα₁(80–86), amino acids β₃(78–84) were replaced by the amino acids α₁(80–86), in β₃(1–115)chimα₁(87–93) the sequence β₃(85–91) was replaced by α₁(87–93), in β₃(1–115)chimα₁(94–100) the sequence β₃(92–98) was replaced by amino acids α₁(94–100), and in β₃(1–115)chimα₁(80–93) the sequence β₃(78–91) was replaced by α₁(80–93) in the β₃(1–115) fragment. None of these chimeras was able to bind to γ₂ subunits. These results indicate that the whole α₁(80–100) sequence is necessary for binding to γ₂ subunits.

The sequence α₁(80–100) is important for the assembly of GABA_A receptors composed of α₁β₃γ₂ subunits

To investigate the importance of the α₁(80–100) sequence not only for the assembly of truncated subunits and dimers, but also for assembly of full-length subunits and pentameric receptors, a full-length α₁ chimera (α₁*) was constructed in which the sequence α₁(79–100) was replaced by the sequence β₃(77–98). The additional exchange of the amino acid 79 of the α₁ subunit in α₁* was necessary to avoid the generation of two adjacent prolines that could have destroyed the conformation of the resulting chimera (Fig. 2). In control experiments, it was demonstrated that the extent of expression of the α₁* chimera was similar to that of the α₁ subunit in HEK cells (data not shown).

HEK cells were then cotransfected with α1*, β₃, and γ₂ subunits and subunits expressed were investigated by immunofluorescence and confocal laser microscopy. As shown in Figure 3, α₁* (Fig. 3A) and β₃ subunits (Fig. 3B) could be easily detected on the surface of intact cells, but for the γ₂ subunit only a weak labeling was observed (Fig. 3C), although the labeling of the γ₂ subunit in permeabilized cells (Fig.3F) was comparable with that of α₁ (Fig. 3D) and β₃ (Fig. 3E) subunits. In HEK cells cotransfected with α₁, β₃, and γ₂ subunits, all three subunits could be detected on the cell surface (Fig.3G–I). Because previous results have indicated that α₁ subunits alone in contrast to α₁β₃ subunit combinations do not form receptors that are incorporated into the plasma membrane to a significant extent, these results suggested that α₁* predominantly formed receptors with β₃ subunits that are expressed on the cell surface, but the ability to form receptors containing γ₂ subunits was significantly reduced.

Fig. 3.

Immunofluorescence of HEK cells cotransfected with GABA_A receptor subunits. HEK cells were cotransfected with α_1*, β₃, and γ₂subunits (A–F) or with α₁, β₃, and γ₂ subunits(G–I). Immunofluorescence was performed using α₁(1–9) antibodies (A, D, G), β₃(1–13) antibodies (B, E, H), or γ₂(1–33) antibodies (C, F, I) on the cell surface (A–C, G–I) or in permeabilized cells(D–F) by confocal laser microscopy (single sections). Scale bar, 10 μm. The experiment was performed four times with similar results.

To quantify this phenomenon, HEK cells were cotransfected with α₁, β₃, and γ₂ subunits or with α₁*, β₃, and γ₂ subunits. GABA_Areceptors expressed on the surface of the cells were labeled by an incubation of intact cells with α₁(1–9) antibodies. Antibody labeled receptors were extracted and precipitated by addition of Immunoprecipitin. The precipitate was subjected to SDS-PAGE and Western blot analysis using digoxygenized α₁(1–9) antibodies (Fig.4). In contrast to α₁ subunits (51 kDa, the weak 46 kDa band presumably represents a degradation product), the protein band of α₁* exhibited an apparent molecular mass of 53 kDa because of an additional glycosylated asparagine at position 80 of the newly introduced β₃ subunit insert. The protein bands were quantified, and results obtained indicated that α₁* and α₁ subunits were expressed to a similar extent on the surface of transfected cells. Then, the Western blot was stripped and analyzed using digoxygenized β₃(1–13) antibodies (Fig. 4). Finally, blots were again stripped and were probed with γ₂(1–33) antibodies. Whereas similar amounts of β₃ subunits (54 kDa) were coprecipitated with α₁ subunits from α₁β₃γ₂or α₁*β₃γ₂transfected cells, the amount of γ₂ subunits (49 kDa) coprecipitated with α₁* subunits was only 32 ± 3% (mean ± SEM, n = 3; from three different transfections) of that coprecipitated with α₁ subunits. Similar results were obtained when the order of detection of subunits was changed and Western blots were first probed with γ₂(1–33) antibodies and after stripping were re-analyzed with α₁(1–9) or β₃(1–13) antibodies. These results indicate that α₁* was able to form receptors with β₃ subunits, but that the ability to form receptors containing γ₂ subunits was reduced by 68%.

Fig. 4.

Western blot analysis of GABA_Areceptors labeled on the surface of HEK cells. HEK cells were cotransfected with α₁, β₃, and γ₂ subunits or with α₁*, β₃, and γ₂ subunits. GABA_A receptors expressed on the cell surface were immunolabeled by adding α₁(1–9) antibodies to intact cells, and were then extracted, immunoprecipitated, and analyzed by SDS-PAGE and Western blots using digoxygenized α₁(1–9), β₃(1–13), or γ₂(1–33) antibodies.

Properties of GABA_A receptors composed of α₁*β₃γ₂ or α₁*β₃ subunits

To investigate the properties of the receptors formed, HEK cells cotransfected with α₁*, β₃, and γ₂ subunits were subjected to patch-clamp analysis, and whole-cell recordings were compared with those from cells transfected with α₁, β₃, and γ₂ subunits. GABA exhibited an apparent EC₅₀ of 68 ± 10 μm (mean ± SEM; n = 11 cells from different plates; total of four transfections) (Fig. 5E) in HEK cells transfected with α₁*, β₃, and γ₂ subunits and elicited a maximal current of 713 ± 170 pA at a GABA concentration of 1000 μm (Fig. 5A). In contrast, GABA exhibited an EC₅₀ of 7.7 ± 2.3 μm (mean ± SEM; n= 8 cells from different plates; total of four transfections) (Fig.5E) in HEK cells transfected with α₁, β₃, and γ₂ subunits and elicited a maximal current of 2988 ± 469 pA at a concentration of 300 μm (Fig. 5B). These data not only indicated that GABA exhibited a 10-fold reduced potency for activating α₁*β₃γ₂receptors, but also that the maximal current of cells transfected with α₁*β₃γ₂subunits was only ∼24% of that transfected with α₁β₃γ₂subunits.

Fig. 5.

Functional properties of GABA_Areceptors containing the α₁* subunit.A—D, Whole-cell recordings from HEK cells cotransfected with α₁*, β₃, and γ₂, or α₁, β₃, and γ₂, α₁* and β₃, or α₁ and β₃subunits after application of GABA concentrations producing maximal currents. Shown are single experiments that were reproduced with similar results 8–11 times in different cells. E,Relative currents induced by various GABA concentrations in cells transfected with subunit combinations as indicated. Data shown are mean values ± SEM of relative currents from 8–11 individual dose–response curves obtained from different cells derived from a total of four transfections. F, Bar graph showing the effect of 100 μm Zn²⁺ on currents activated by 100 μm GABA. HEK cells were transfected, as indicated. Height of bars indicates fraction of control current remaining in the presence of Zn²⁺, measured 1.5 sec after application was started; error bars indicate ± SEM (n = 6–8).

Because surface expression studies indicated a significant formation of α₁*β₃ receptors in α₁*β₃γ₂transfected cells, the properties of receptors in α₁*β₃ transfected cells were also investigated. Although homo-oligomeric receptors composed of β₃ subunits could also have been formed under the conditions used, they would not have contributed to the GABA evoked current because these receptors apparently are not gated by GABA (Connolly et al., 1996b). Using various GABA concentrations, it was demonstrated that GABA exhibited an EC₅₀ of 10.5 ± 2.1 μm (mean ± SEM; n= 8 cells from different plates; total of four transfections) (Fig.5E) in HEK cells transfected with α₁* and β₃ subunits and elicited a maximal current of 270 ± 63 pA at a concentration of 300 μm (Fig. 5C). In contrast, GABA exhibited an EC₅₀ of 3.0 ± 1.2 μm (mean ± SEM; n = 9 cells from different plates; total of three transfections) (Fig.5E) in cells transfected with α₁ and β₃ subunits and elicited a maximal current of 426 ± 146 pA at a concentration of 100 μm(Fig. 5D). These data supported the conclusion that the α₁* construct was able to form functional receptors with β₃ subunits. The potency of GABA for activating α₁*β₃receptors, however, was significantly (p < 0.05; Student's t test) reduced compared with receptors composed of α₁β₃subunits. Similarly, the maximal currents elicited by GABA in α₁*β₃ transfected cells were significantly smaller than those in α₁β₃ transfected cells (p < 0.05).

Although α₁*β₃receptors significantly contribute to receptors formed in α₁*β₃γ₂transfected HEK cells, as indicated by surface expression studies, because of the low maximum currents observed in α₁*β₃ receptors (Fig.5C), these receptors overall have a comparatively small contribution to currents elicited in α₁*β₃γ₂transfected cells (Fig. 5A) that is apparent only as a slightly increased range of GABA concentrations that are able to elicit currents in these cells (Fig. 5E). Thus, most of the current elicited in the cells investigated was produced by α₁*β₃γ₂receptors. The low apparent potency of GABA to activate currents in these cells as well as the increased dose range of GABA for stimulation of currents clearly indicated the formation of α₁*β₃γ₂receptors in addition to α₁*β₃ receptors.

This conclusion was supported by investigating the effects of 100 μm Zn²⁺ on whole-cell currents stimulated by 100 μm GABA. In agreement with previous results (Draguhn et al., 1990; Gingrich and Burkat, 1998), currents mediated by the wild-type α₁β₃γ₂receptors were only weakly reduced (86 ± 4% of control;n = 6; total of four transfections), whereas currents mediated by α₁β₃receptor were reduced to 7 ± 3% (n = 6; total of three transfections) (Fig. 5F) in the presence of Zn²⁺. For HEK cells transfected with α₁*, β₃, and γ₂ subunits, currents mediated by 100 μm GABA were reduced to 50 ± 4% (n = 7; total of four transfections), and for cells transfected with α₁* and β₃ subunits, GABA-mediated currents were reduced to 8 ± 2% (n = 8; total of four transfections) in the presence of 100 μmZn²⁺ (Fig. 5F). Because α₁β₃ and α₁*β₃ receptors exhibit a comparable Zn²⁺ sensitivity, it is reasonable to assume that the Zn²⁺sensitivity of α₁*β₃γ₂and α₁β₃γ₂receptors was also comparable. The 36% increase in Zn²⁺ sensitivity of α₁*β₃γ₂-transfected cells therefore indicated that ∼40% of the α₁*β₃γ₂current was mediated by the additionally formed α₁*β₃ receptors. Combined with the observation that the main conductance level of αβ receptors (15–18 pS) is only half of that of αβγ receptors (∼30 pS; Hevers and Lüddens, 1998), and assuming that the same holds true for α₁*β₃and α₁*β₃γ₂receptors, a ratio of α₁*β₃:α₁*β₃γ₂receptors of 80:60 can be calculated, indicating that α₁*β₃γ₂receptors represented 43% of receptors formed in these cells. Given the many assumptions that had to be made in the course of this calculation, this percentage is in good agreement with the data from the immunoprecipitation experiments shown in Figure 4.

Amino acid sequence α₁(80–100) binds to the γ₂(91–104) sequence

Recently it has been demonstrated that incorporation of the amino acid sequence γ₂(91–104) into the fragment α₁(1–100), that per se could not bind to α₁ subunits, resulted in the chimera α₁(1–100)chimγ₂(91–104) that was able to bind to α₁ subunits. From this it was concluded that the amino acid sequence γ₂(91–104) forms the contact site to α₁ subunits (Klausberger et al., 2000). It therefore seemed interesting to investigate whether the γ₂(91–104) sequence directly interacts with the α₁(80–100) sequence.

To clarify this question, it first was investigated whether the α₁(1–100) fragment and the fragment β₃(1–115), which was used to identify the α₁(80–100) contact site (Fig. 2), could bind to each other. For this, fragments β₃(1–115) and α₁(1–100) were cotransfected into HEK cells. The extract of HEK cells expressing β₃(1–115) and α₁(1–100) fragments was then immunoprecipitated with β₃(1–13) antibodies, and the precipitate was subjected to SDS-PAGE and Western blot analysis using digoxygenized α₁(1–9) antibodies. As shown in Figure 6, A andB, α₁(1–100) fragments were not coprecipitated by β₃(1–13) antibodies, confirming the absence of cross-reactivity of these antibodies with the α₁(1–100) fragments and indicating that β₃(1–115) could not bind to α₁(1–100) fragments. Similarly, the construct β₃(1–115)chimα₁(80–100), which contains the putative binding site for γ₂subunits, was unable to bind to α₁(1–100) fragments after cotransfection into HEK cells (Fig.6A,B). In the reverse experiment, the construct α₁(1–100)chimγ₂(91–104), containing the binding site for α₁ subunits, could also not bind to the β₃(1–115) fragment. Only when the α₁(80–100) sequence was incorporated into the β₃(1–115) fragment and the γ₂(91–104) sequence was incorporated into the α₁(1–100) fragment, the resulting chimeras could bind to each other (Fig. 6A,B). These results indicate that the α₁(80–100) and the γ₂(91–104) sequences can directly bind to each other.

In control experiments (Fig. 6C) it was demonstrated that each of the constructs used in this experiment was expressed to a similar extent after single transfection into HEK cells. Constructs α₁(1–100) and α₁(1–100)chimγ₂(91–104) each contained a single glycosylation site and thus, gave rise to two fragments: a weakly labeled of 12 kDa and a strongly labeled of 14 kDa. Construct β₃(1–115) contained two glycosylation sites and thus, formed three fragments, two strongly labeled of 16 and 18 kDa and a weakly labeled fragment of 14 kDa. Construct β₃(1–115)chimα₁(80–100) contained only one glycosylation site and formed a strongly labeled protein band of 14 and weakly labeled band of 16 kDa.

Interestingly, predominantly the unglycosylated α₁(1–100)chimγ₂(91–104) fragment of 12 kDa seemed to assemble with β₃(1–115)chimα₁(80–100) on cotransfection of these fragments into HEK cells, although the glycosylated fragment of 14 kDa was the predominant one expressed after single transfection of HEK cells (Figs. 1B,6C). This suggests that assembly of subunit fragments already starts when subunits are not fully glycosylated. This conclusion is supported by previous observations (Klausberger et al., 2000, 2001) as well as by observations with other constructs (Fig.1B).

DISCUSSION

Amino acid sequence α₁(80–100) forms the binding site to γ₂ but not to β₃ subunits

The present study demonstrated that the N-terminal extracellular domain of the α₁ subunit [α₁(1–221)] could bind to full-length γ₂ subunits after coexpression in HEK cells, as indicated by coimmunoprecipitation with subunit-specific antibodies. Binding between α₁(1–221) and γ₂ subunits represented a specific assembly process, as indicated by the formation of specific [³H]Ro15–1788 binding sites that are assumed to be formed on the interface of α₁ and γ₂ subunits of GABA_Areceptors. These results are consistent with previous studies indicating that N-terminal sequences of GABA_Areceptor (Hackam et al., 1997; Klausberger et al., 2000) or K⁺ channel (Shen et al., 1993) subunits can assemble with full-length subunits.

A subsequent reduction in the size of the truncated subunit indicated that the α₁(1–117), but not the α₁(1–100) construct was still able to bind to γ₂ subunits. The respective binding site was then identified by incorporating various α₁sequences into the β₃(1–115) fragment. This fragment is homologous to α₁(1–117) but in contrast to the latter construct could not bind to γ₂ subunits after coexpression in HEK cells. The incorporation of the sequence α₁(80–100) into the β₃(1–115) fragment was sufficient to induce binding to γ₂ but not to β₃ subunits, suggesting that the α₁ binding sites for γ₂and β₃ subunits are different.

The observation that the α₁(1–100) fragment was unable to bind to γ₂ subunits although it contained the α₁(80–100) sequence is consistent with previous results indicating that γ₂(1–113) was the smallest fragment that could bind to α₁ subunits, although the respective binding site was identified to be formed by the γ₂(91–104) sequence (Klausberger et al., 2000). The additional length of the fragments presumably is required for stabilizing the conformation of the actual binding sites located in a more N-terminal position.

In other experiments a chimeric α₁ subunit (α₁*) was constructed in which the α₁(79–100) sequence was replaced by the homologous β₃(77–98) sequence. Chimera α₁* was then coexpressed with β₃ and γ₂ subunits in HEK cells. Confocal immunofluorescence microscopy, whole-cell patch-clamp experiments, as well as immunolabeling and quantification of receptors on the cell surface indicated a 60–70% reduction in receptors containing α₁*, β_3, and γ₂ subunits, although the level of expression of α₁* subunits and its extent of assembly with β₃subunits was unimpaired. These results confirmed the importance of the α₁(80–100) sequence for assembly with γ₂ but not with β₃subunits. The remaining formation of α₁*β₃γ₂receptors can be explained by the existence of additional binding sites between α₁ and γ₂subunits that partially can compensate for the absence of the α₁(80–100) sequence in α₁* subunits.

Amino acid sequences α₁(80–100) and γ₂(91–104) form part of the α–γ interface and are located close to the benzodiazepine binding site of GABA_Areceptors

Recently it was demonstrated that the sequence γ₂(91–104) forms the contact site to α₁ subunits (Klausberger et al., 2000). To investigate whether the sequences α₁(80–100) and γ₂(91–104) directly interact with each other, these sequences were incorporated into GABA_A receptor fragments β₃(1–115) and α₁(1–100), respectively, which could not bind to each other. The observation that α₁(80–100) had to be incorporated into β₃(1–115) and γ₂(91–104) into α₁(1–100) to induce coprecipitation of the fragments indicated that the α₁(80–100) and the γ₂(91–104) sequences can directly bind to each other and thus, form part of the α₁–γ₂ subunit interface. This is the first time that interacting sequences from two different subunits could be identified in this receptor superfamily. It is possible, however that subunits rearrange during assembly (Mitra et al., 2001). Whether the identified sequences also form part of the final intersubunit contact, thus, will have to be determined by further studies. Interestingly, two amino acid residues (L90 and S92) identified previously as important for homo-oligomeric assembly of glycine receptor α₁ subunits (Griffon et al., 1999) are located in a region homologous to α₁(80–100), but their identity is different in GABA_A receptors. In addition, L90 as well as a further amino acid residue (P79) that also is important for binding between glycine receptor subunits, are located in a region homologous to γ₂(91–104). This indicates that GABA_A receptors, glycine receptors, and possibly also nAChRs (Kreienkamp et al., 1995) use homologous regions with receptor-specific assembly signals for forming intersubunit contacts.

Interestingly, the amino acid H101 of the α₁subunit that can be photolabeled by [³H]flunitrazepam (Smith and Olsen, 2000) and seems to be involved in the formation of the benzodiazepine binding pocket (Sigel and Buhr, 1997) is located immediately adjacent to the α₁(80–100) sequence forming the intersubunit contacts to γ₂ subunits. In addition, P96 of the α₁ subunit, which also can be photolabeled by [³H]flunitrazepam (Smith and Olsen, 2000), contributes to the α_1–γ₂ subunit interface. This indicates that the benzodiazepine binding site of GABA_A receptors is located close to the intersubunit contact between α₁ and γ₂ subunits.

Other amino acid residues possibly contributing to the benzodiazepine binding site are α₁Y159, α₁G200, α₁S204, α₁T206, α₁Y209, γ₂M130, and γ₂F77 (Sigel and Buhr, 1997). Although located outside the α₁(80–100) and γ₂(91–104) sequences, all these residues are embedded in domains with a hydrophobicity comparable with that of these sequences (data not shown). These residues might thus be involved in the formation of other parts of this interface. In any case, all residues forming the benzodiazepine binding site must be in spatial proximity to α₁H101.

Implications for GABA_A receptor assembly and subunit stoichiometry

The present observation that the α₁(80–100) sequence binds to γ₂ but not to β₃subunits suggests the existence of at least three distinct subunit binding sites on α₁ subunits: a (+) and a (−) site for binding of β₃ subunits and an additional (✣) site for binding of γ₂subunits (Fig. 7). Whereas the (+) and the (✣) sites are located at the same side and are possibly situated closely together, the (−) site is located at the other side of the subunit. The recently identified sequence α₁(58–67), which mediates binding to β₃ subunits (Taylor et al., 2000), seems to form part of the (−) site because one of its residues (α₁F64; Smith and Olsen, 1994) contributes to the GABA binding site assumed to be located at the α₁–β₃ interface of GABA_A receptors (Fig. 7).

Fig. 7.

Stoichiometry and subunit arrangement of the recombinant α₁β₃γ₂GABA_A receptor (Tretter et al., 1997). A mirror image arrangement is equally possible. +, ✣, and − indicate the clockwise and counterclockwise binding sites of a subunit, respectively. BZ or GABA indicate site of interaction of benzodiazepines or GABA with GABA_A receptors, respectively.

Interestingly, one α₁ subunit of GABA_A receptors uses the (✣) site for binding to a γ₂ subunit, whereas the other one uses the (+) site for a β₃ subunit (Fig. 7). Previous studies have indicated that cells transfected with α₁, β₃, and γ₂ subunits predominantly form pentameric receptors composed of 2α₁, 2β_3, and one γ₂subunit, whereas cells transfected with α₁ and β₃ subunits form tetramers and pentamers (Tretter et al., 1997). This delayed formation of subunit pentamers indicates that a γ₂ subunit can be more easily accommodated into an α₁β₃ tetramer than an additional β₃ subunit. A preferential use of the α₁(✣) site under these conditions ensures the incorporation of a γ₂ subunit into the receptor. Because the alternate use of the α₁(+) and the α₁(✣) site in the two α₁ subunits of GABA_A receptors seems to be sterically or energetically favored, binding to the α₁(+) site of the second α₁ subunit should be preferred when a γ₂ subunit is already present in an assembly intermediate. In this case the preferred use of the α₁(+) site prevents the incorporation of a second γ₂ subunit into the receptor. This conclusion is supported by most of the experimental data available, suggesting that there is only one γ₂ subunit in GABA_A receptors (for review, see Sieghart et al., 1999) and indicates that the α₁–γ₂ intersubunit contact controls subunit assembly and stoichiometry of GABA_A receptors.