Detection and Binding Properties of GABAA Receptor Assembly Intermediates*

Thomas Klausberger, Noosha Ehya, Karoline Fuchs, Thomas Fuchs, Veronika Ebert, Isabella Sarto, and Werner SieghartDagger

From the Section of Biochemical Psychiatry, University Clinic for Psychiatry, A-1090 Vienna, Austria and the Brain Research Institute, University of Vienna, Division of Biochemistry and Molecular Biology, A-1090 Vienna, Austria

Received for publication, October 18, 2000, and in revised form, February 1, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Density gradient centrifugation of native and recombinant gamma -aminobutyric acid, type A (GABAA) receptors was used to detect assembly intermediates. No such intermediates could be identified in extracts from adult rat brain or from human embryonic kidney (HEK) 293 cells transfected with alpha 1, beta 3, and gamma 2 subunits and cultured at 37 °C. However, subunit dimers, trimers, tetramers, and pentamers were found in extracts from the brain of 8-10-day-old rats and from alpha 1beta 3gamma 2 transfected HEK cells cultured at 25 °C. In both systems, alpha 1, beta 3, and gamma 2 subunits could be identified in subunit dimers, indicating that different subunit dimers are formed during GABAA receptor assembly. Co-transfection of HEK cells with various combinations of full-length and C-terminally truncated alpha 1 and beta 3 or alpha 1 and gamma 2 subunits and co-immunoprecipitation with subunit-specific antibodies indicated that even subunits containing no transmembrane domain can assemble with each other. Whereas alpha 1gamma 2, alpha 1Ngamma 2, alpha 1gamma 2N, and alpha 1Ngamma 2N, combinations exhibited specific [3H]Ro 15-1788 binding, specific [3H]muscimol binding could only be found in alpha 1beta 3 and alpha 1beta 3N, but not in alpha 1Nbeta 3 or alpha 1Nbeta 3N combinations. This seems to indicate that a full-length alpha 1 subunit is necessary for the formation of the muscimol-binding site and for the transduction of agonist binding into channel gating.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Members of the ligand-gated ion channel family, such as the nicotinic acetylcholine receptor (nAChR),1 the GABAA receptor, the glycine receptor, or the 5-hydroxytryptamine, type 3 receptor, are heteromeric proteins composed of five subunits (1). The subunits of these proteins are co-translationally inserted into the membrane, lumen, or both, of the endoplasmatic reticulum, after which the subunits fold and oligomerize (2-4). During these folding and oligomerization events, ligand-binding sites of the receptors are formed. Proteins, once properly folded and oligomerized, are transported to their proper destination, whereas misfolded or improperly oligomerized subunits are retained in the endoplasmatic reticulum and degraded (2, 3, 5). Little is known about the molecular events involved in subunit oligomerization and formation of ligand-binding sites. In the present study the first steps of these events are investigated for GABAA receptors.

GABAA receptors are chloride channels that can be opened by GABA (6) 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 flux by interacting with separate and distinct allosteric binding sites (7). So far, at least 19 GABAA receptor subunits belonging to several subunit classes (six alpha , three beta , three gamma , one delta , one epsilon , one pi , one theta , and three rho ) have been identified in the mammalian brain (8, 9). Expression studies indicated that alpha , beta , and gamma  subunits have to combine to form GABAA receptors with a pharmacology resembling that of the majority of native receptors (7). Most reports agree that these receptors are composed of two alpha , two beta , and one gamma  subunit (10-13).

Density gradient centrifugation studies indicated that recombinant GABAA receptors composed of alpha 1beta 3gamma 2 subunits almost exclusively sediment as subunit pentamers. alpha 1beta 3 subunit combinations sediment as tetramers and pentamers, whereas combinations of alpha 1gamma 2 or beta 3gamma 2 subunits predominantly form heterodimers (12). These results suggested a subunit arrangement in GABAA receptors in which four alternating alpha  and beta  subunits are connected by a gamma  subunit (12).

Presently, however, nothing is known about the processes that lead from single subunits to completely assembled and pharmacologically functional receptors. Because no assembly intermediates could be identified in HEK cells transfected with alpha 1beta 3gamma 2 subunits under the conditions used and because not all of the subunit dimers that can be formed in HEK cells transfected with two different subunits might be formed when all three subunits are co-expressed, it is not clear whether alpha beta , alpha gamma , or beta gamma subunit dimers or some or all of these dimers are the starting point for GABAA receptor synthesis.

The pentameric receptor possesses binding sites for the endogenous neurotransmitter GABA, presumably located at the interface between alpha 1 and beta 3 subunits (14), for benzodiazepines, located between the alpha 1 and gamma 2 subunit (15), as well as for TPBS, presumably located either within or close to the channel formed by these subunits (16-18). Presently, nothing is known about the events leading to the formation of the various binding sites on GABAA receptors.

By lowering the temperature during culture of HEK cells transfected with alpha 1, beta 3, and gamma 2 subunits, in the present study we were able to detect assembly intermediates of GABAA receptors using sucrose density gradient centrifugation. Results indicated that different subunit dimers are formed during GABAA receptor assembly. Studies investigating the dimerization of complete and truncated subunits additionally suggested that the binding sites for [3H]muscimol or the benzodiazepine [3H]Ro 15-1788 can already be formed by the proper subunit dimers or trimers.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Antibodies-- The antibodies anti-peptide alpha 1(1-9) (19), anti-peptide beta 3(1-13) (12), anti-peptide beta 3(345-408) (20), anti-peptide gamma 2(319-366) (12), and anti-peptide gamma 2(1-33) (21) were generated and affinity purified as described previously. The monoclonal antibody bd17, recognizing beta 2/3 subunits (22), were purchased from Roche Molecular Biochemicals.

Generation of cDNA Constructs-- For the generation of recombinant receptors, alpha 1, beta 3, and gamma 2 subunits of GABAA receptors from rat brain were cloned and subcloned into pCDM8 expression vectors (Invitrogen, San Diego, CA) as described previously (12, 23).

Truncated subunits were constructed by polymerase chain reaction amplification using the full-length subunit as template. The polymerase chain reaction primers contained EcoRI and HindIII restriction sites, which were used to clone the fragments into pCDNAIAmp vectors (Invitrogen). The truncated subunits were confirmed by sequencing.

Culture and Transfection of HEK 293 Cells-- Transformed HEK 293 cells (CRL 1573; American Type Culture Collection, Manassas, VA) were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum (JRH Biosciences, Lenexa, KS), 2 mM glutamine, 50 µM beta -mercaptoethanol, 100 units/ml penicillin G, and 100 µg/ml streptomycin in 75-cm2 culture dishes using standard cell culture techniques. HEK 293 cells (3 × 106) were transfected with a total amount of 20 µg of subunit cDNAs via the calcium phosphate precipitation method (24).

Density Gradient Centrifugation-- Transfected HEK cells were incubated 44 h at 37 °C or 8 h at 37 °C followed by 16 h at 25 °C. Cells from eight culture dishes were harvested and extracted in 1.6 ml of 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 phenylmethylsulfonyl fluoride, 1 mM benzamidine, and 100 mg/liter bacitracin) for 8 h at 4 °C. This buffer was used rather than a Triton X-100 or a deoxycholate buffer. Because of its low solubilizing ability it did not dissociate assembly intermediates and, thus, allowed their identification (3). Membranes from adult rat brains (preparation described in Ref. 25) were extracted in 3.5 ml of Lubrol extraction buffer/brain. The extracts were centrifuged for 40 min at 150,000 × g at 4 °C, and 200 µl of the extracts was layered onto the top of a density gradient 5-20% sucrose in Lubrol extraction buffer). For the determination of sedimentation coefficients, 2 µg of digoxygenated catalase (sedimentation coefficient, 11 s), 1.2 µg of digoxygenated alkaline phosphatase (sedimentation coefficient, 6.1 s), and 1 µg of digoxygenated carbonic anhydrase (sedimentation coefficient 3.3 s) were included in the overlays. The gradients were centrifuged at 120,000 × g at 4 °C for 23 h and were then fractionated by piercing at the tube bottom (12). Protein in individual fractions was precipitated (26) and dissolved in sample buffer (108 mM Tris-sulfate, pH 8.2, 10 mM EDTA, 25% (w/v) glycerol, 2% SDS, and 3% dithiothreitol) for SDS-PAGE. Proteins were identified by Western blot analysis, and signal intensity per fraction was quantified as described below. In a previous study (12) it has been demonstrated that after co-transfection of HEK cells with alpha 1, beta 3, and gamma 2 subunits all three subunits sedimented at a single peak of 8.7 s, representing the pentameric receptor. After co-transfection of HEK cells with alpha 1 and beta 3 subunits, both subunits again sedimented at a peak of 8.7 s. The beta 3 subunit, however, additionally formed subunit complexes that sedimented at 3.3, 5.5, 6.7, and 7.4 s (12). Assembly intermediates with similar sedimentation properties have been observed previously for the homologous nAChR using a similar procedure (3). Thus, the monomeric subunits of these receptors exhibited a sedimentation between 3 and 4 s, sedimentation of subunit dimers was observed at 6 s, trimers sedimented at 7 s, tetramers sedimented at 8 s, and pentamers sedimented at 9 s (3, 12). From this it was concluded that the 3.3-, 5.5-, 6.7-, and 7.4-s peaks of GABAA receptor assembly intermediates represent subunit mono-, di-, tri-, and tetramers (3).

SDS-PAGE, Western Blot, and Chemiluminescence Detection-- SDS-PAGE was performed as described (12, 27) using gels containing 12% acrylamide and 0.324% bisacrylamide. Proteins separated on the gels were tank-blotted onto prewetted polyvinylidene fluoride membranes. After blocking with 1.5% nonfat dry milk powder in PBS (2.7 mM KCl, 1.5 mM KH2PO4, 140 mM NaCl, and 4.3 mM Na2HPO4, pH 7.3) and 0.1% Tween 20 for 1 h at room temperature, the membranes were incubated overnight with alpha 1(1-9), beta 3(345-408), gamma 2(319-366), or digoxigenized alpha 1(1-9) antibodies (2 µg/ml) at 4 °C. The membranes were extensively washed (1.5% (w/v) dry milk powder and 0.1% Tween 20 in PBS) and were incubated with alkaline phophatase-coupled anti-digoxigenin F(ab)2 fragments (Roche Molecular Biochemicals) for 45 min at room temperature. Membranes were again washed extensively as described above, were equilibrated in assay buffer (0.1 M diethanolamine and 1 mM MgCl2, pH 10.0) for 10 min and were then incubated with 1 ml of 0.24 mM CSPD or CPD-star reagent (Tropix, Bedford, MA) diluted in assay buffer. After 5 min the fluid was removed, and the membranes were sealed in a foil and exposed to x-ray films (X-Omat S, Eastman Kodak Co.) for various time periods. Signals were quantified by a gel documentation system (Docu Gel 2000i; software: RFLP-Scan; MWG Biotech, Ebersberg, Germany).

Purification and Co-immunoprecipitation of GABAA Receptor Subunits-- Transfected HEK cells were incubated 44 h at 37 °C. Cells from four culture dishes were extracted with 800 µl of Lubrol extraction buffer for 8 h 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 beta 3(345-408) or gamma 2(319-366) antibodies. After addition of Immunoprecipitin (preparation described in Ref. 12 and 0.5% nonfat dry milk powder and shaking for additional 3 h at 4 °C, the precipitate was washed three times with IP low buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8.0) containing 1% Triton X-100. The precipitated proteins were dissolved in sample buffer and subjected to SDS-PAGE and Western blot analysis.

Radioligand Binding Studies-- For binding studies frozen membranes from untransfected or transfected HEK cells were thawed, and cells were homogenized in 50 mM Tris/citrate buffer, pH 7.4, by using an Ultraturax, followed by three centrifugation (200 000 × g for 20 min at 4 °C) resuspension cycles. Cell pellets were resuspended in 50 mM Tris/citrate buffer, pH 7.4, at a protein concentration in the range of 0.5-1 mg/ml as measured with the BCA protein assay kit (Pierce) with bovine serum albumin as standard. Membranes were then incubated for 90 min at 4 °C in a total of 1 ml of a solution containing 50 mM Tris/citrate buffer, pH 7.4, 150 mM NaCl, and various concentrations (range, 0.1-1000 nM) of [3H]Ro 15-1788 (87.0 Ci/mmol; Amersham Pharmacia Biotech) in the absence or presence of 100 µM diazepam (Hoffmann La Roche, Basle, Switzerland). For muscimol binding assays, the membranes were incubated for 60 min at 4 °C in a total of 1 ml of a solution containing 50 mM Tris/citrate buffer, pH 7.4, and various concentrations (range, 1-300 nM) of [3H]muscimol (20.0 Ci/mmol; PerkinElmer Life Sciences) in the absence or presence of 10 µM GABA. For TPBS binding, membranes were incubated in a total of 1 ml of a solution containing 50 mM Tris/citrate buffer, pH 7.4, 200 mM NaBr and 2 nM [35S]TPBS (104.0 Ci/mmol; PerkinElmer Life Sciences) in the absence or presence of 40 µM IPTBO (from J. S. Collins, City of London Polytechnic, London, UK) for 180 min at room temperature.

Membranes were then filtered through Whatman GF/B filters, and the filters were rinsed twice with 3.5 ml of ice-cold 50 mM Tris/citrate buffer and were then subjected to scintillation counting. Unspecific binding in the presence of 100 µM diazepam, 10 µM GABA, or 40 µM IPTBO was subtracted from total [3H]Ro 15-1788, [3H]muscimol, or [35S]TBPS binding, respectively, to result in specific binding (23).

Immunofluorescence-- HEK cells were fixed with 2% paraformaldehyde in PBS 30-35 h 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 in PBS for 10 min, followed by an incubation with primary antibody in 1% bovine serum albumin in PBS. Primary antibodies were detected with goat anti-rabbit IgG(H+L) Bodipy FL (Molecular Probes, Eugene, OR) or donkey anti-mouse IgG(H+L)Cy3 (Amersham Pharmacia Biotech) in 1% bovine serum albumin in PBS. Labeling was visualized using a Zeiss Axiovert 135 M microscope attached to a confocal laser system (Carl Zeiss LSM 410), 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 GABAA receptor subunits (experiments not shown). These antibodies detected GABAA receptor subunits only after permeabilization of transfected cells. Results obtained from double labeling experiments were compared with single labeling experiments to demonstrate that the labeling pattern in double labeling experiments was not caused by cross-bleeding artifacts (experiments not shown).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Detection of Assembly Intermediates-- In an attempt to identify GABAA receptor assembly intermediates, extracts from adult rat brain or from HEK 293 cells transfected with alpha 1, beta 3, and gamma 2 subunits were subjected to sucrose density gradient centrifugation. Under these conditions, depending on their molecular mass, monomeric and multimeric proteins migrate into the gradient with different sedimentation coefficients. Gradients were fractionated, and the proteins in individual fractions were precipitated and subjected to SDS-PAGE and Western blot analysis with subunit specific antibodies. s values of receptors and receptor intermediates were determined by analyzing the sedimentation of standard proteins with known s values added to each gradient.

As shown in Fig. 1A for brain extracts and Fig. 1B for transfected HEK cells, the alpha 1, beta 3, as well as the gamma 2 subunit proteins sedimented at a single peak at 8.7 s. This sedimentation coefficient has been reported to represent the completely assembled pentameric GABAA receptor, and the protein shoulder above 8.7 s presumably is caused by an aggregation of pentameric receptors (3, 12). The absence of alpha 1, beta 3, and gamma 2 protein peaks with lower s values (Fig. 1) indicated that most of the GABAA receptors formed in adult brain as well as in transfected cells are pentamers and that receptor synthesis in these tissues was low and/or assembly of receptors was too fast to allow an identification of assembly intermediates under these conditions.


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Fig. 1.   Sucrose density gradient centrifugation of GABAA receptors. Receptors from adult rat brain (A) or from HEK cells transfected with alpha 1, beta 3, and gamma 2 subunits (B) were extracted and centrifuged on 5-20% linear sucrose density gradients. Gradients were fractionated, and proteins in individual fractions were precipitated and subjected to SDS-PAGE and Western blot analysis using alpha 1(1-9), beta 3(345-408), and gamma 2(319-366) antibodies. s values were measured by including digoxigenized standard proteins with known s values in each gradient. OD, optical density (arbitrary units); s, sedimentation value. The experiments were performed four times with comparable results.

In other experiments, the culture temperature was first kept at 37 °C for the first 8 h after transfection of HEK cells with alpha 1, beta 3, and gamma 2 subunits to normally initiate transcription and translation and was then reduced to 25 °C for the following 16 h to slow down the assembly of recombinant GABAA receptors. Receptors formed were extracted from the cells and were subjected to density gradient centrifugation. As shown in Fig. 2, under these conditions assembly intermediates could be identified. alpha 1 and gamma 2 subunits sedimented at 3.3, 4.5, 5.5, 6.8, 7.4, and 8.7 s (Fig. 2). The sedimentation pattern of the beta 3 subunit was similar to that of the alpha 1 and gamma 2 subunits, but the protein peak at 6.8 s could not be identified and presumably was present in the shoulder of the 7.4-s peak. It has been reported previously (12) that the peaks at 3.3, 5.5, 6.7, 7.4, and 8.7 s represent mono-, di-, tri-, tetra-, and pentamers of GABAA receptor subunits, respectively. The additional peak at 4.5 s could represent a monomer bound to a chaperone, because recently it has been suggested that chaperones might stabilize subunit monomers (4). The identification of all these protein peaks by all three antibodies was not due to a cross-reactivity of the antibodies, because none of the antibodies used for these experiments exhibited any cross-reactivity with other subunits as demonstrated by Western blot analysis of various recombinant receptors (27, 28).


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Fig. 2.   Sucrose density gradient centrifugation of GABAA receptors after culturing of transfected HEK cells at 25 °C. HEK cells were transfected with alpha 1, beta 3, and gamma 2 subunits and were incubated for 8 h at 37 °C and afterward for 16 h at 25 °C. Extracts of these cells were subjected to sucrose density gradient centrifugation as described in the legend to Fig. 1. Individual fractions of the gradients were analyzed in Western blots using alpha 1(1-9) (A), beta 3(345-408) (B), and gamma 2(319-366) (C) antibodies. OD, optical density (arbitrary units); s, sedimentation value. The experiments were performed eight times with comparable results.

In other experiments, the sedimentation properties of GABAA receptors extracted from the brain of 8-10-day-old rats were investigated. In this developing tissue, GABAA receptors are continuously synthesized in different neurons, and it was hoped that amounts of assembly intermediates sufficient to be detected would be present. As shown in Fig. 3, this actually was the case: alpha 1, beta 3, and gamma 2 subunits extracted from 8-10-day-old rats, in contrast to those from adult rat brain, sedimented in multiple, overlapping peaks. Whereas the sedimentation pattern of alpha 1 and gamma 2 subunits was again similar, showing overlapping peaks and shoulders at 5.5, 6.8, and 8.7 s, the sedimentation pattern of beta 3 subunits was slightly different, showing prominent peaks at 3.3 and 5.5 s and overlapping peaks and shoulders between 6.8 and 8.7 s (Fig. 3).


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Fig. 3.   Sucrose density gradient centrifugation of GABAA receptors from the brain of young rats. Extracts from the brain of 8-10-day-old rats were analyzed as described in Fig. 1. OD, optical density (arbitrary units); s, sedimentation value. The experiments were performed five times with comparable results.

Formation of the [3H]Muscimol-binding Site-- Because the [3H]muscimol-binding site on GABAA receptors is located at the interface of alpha  and beta  subunits (14), it was interesting to investigate whether this binding site could already be formed by GABAA receptor assembly intermediates containing alpha 1 and beta 3 subunits. Because co-transfection of HEK cells with alpha 1 and beta 3 subunits leads to the formation of alpha 1beta 3 tetramers and pentamers (12), truncated alpha 1 (alpha 1N) and beta 3 (beta 3N) subunits containing the complete extracellular N-terminal domain but no transmembrane domains were cloned to investigate whether they can assemble with full-length beta 3 and alpha 1 subunits, respectively, forming smaller assembly intermediates. alpha 1 and beta 3 subunits and alpha 1N and beta 3N fragments were then co-transfected into HEK cells in various combinations, and expressed subunits were extracted from these cells and were immunoprecipitated with beta 3(1-13) antibodies. The precipitate was subjected to SDS-PAGE and Western blot analysis using digoxigenized alpha 1(1-9) antibodies. As shown in Fig. 4A, in extracts from HEK cells co-transfected with full-length alpha 1 and beta 3 subunits or full-length alpha 1 and beta 3N subunits, a strongly labeled protein band with apparent molecular mass 51 kDa was detected in Western blots. A protein band with identical molecular mass could be precipitated by alpha 1(1-9) antibodies from these cells as well as from HEK cells transfected with alpha 1 subunits only, but not from untransfected HEK cells (experiments not shown), indicating that this protein band represents the alpha 1 subunit of GABAA receptors. The weakly labeled lower molecular weight bands varied in labeling intensity in different experiments and could not be detected in untransfected HEK cells. They thus seemed to represent degradation products of the alpha 1 subunit. The precipitation of alpha 1 subunits by beta 3(1-13) antibodies was not due to a cross-reactivity of these antibodies because it could not be observed in HEK cells transfected with alpha 1 subunits only (experiment not shown).


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Fig. 4.   Co-immunoprecipitation of full-length and truncated alpha 1 and beta 3 or alpha 1 and gamma 2 subunits. HEK cells were co-transfected with alpha 1 and beta 3, alpha 1N and beta 3, alpha 1 and beta 3N, alpha 1N and beta 3N (A) or with alpha 1 and gamma 2, alpha 1N and gamma 2, alpha 1 and gamma 2N, alpha 1N and gamma 2N (B). Cell extracts were immunoprecipitated with beta 3(1-13) (A) or gamma 2(1-33) (B) antibodies. The precipitate was subjected to SDS-PAGE and Western blot analysis using digoxygenized alpha 1(1-9) antibodies. The experiment was performed three times with comparable results.

When extracts from HEK cells co-transfected with alpha 1N and full-length beta 3 subunits or alpha 1N and beta 3N constructs were precipitated with beta 3(1-13) antibodies, three protein bands with apparent molecular masses 30, 33, and 36 kDa could be detected in Western blots using digoxygenized alpha 1(1-9) antibodies. The molecular mass of the smallest band (30 kDa) corresponds with that expected for the unglycosylated alpha 1N fragment. Because two glycosylation sites are present in the alpha 1 subunit (29), the 33- and 36-kDa bands presumably represent partially and fully glycosylated alpha 1N fragments. The co-precipitation by beta 3(1-13) antibodies of alpha 1N or alpha 1 subunits indicates that not only full-lenth alpha 1 and beta 3 subunits but also alpha 1N and beta 3, alpha 1 and beta 3N, and even alpha 1N and beta 3N constructs are able to form hetero-oligomers.

The subcellular distribution of these subunit combinations in HEK cells was investigated by immunofluorescence and confocal laser microscopy. Double staining with alpha 1(1-9) and the beta 2/beta 3 subunit-specific bd17 antibodies of intact HEK cells transfected with full-length alpha 1 and beta 3 subunits (Fig. 5, A and B) demonstrated that these subunits formed receptors expressed on the cell surface. Permeabilization of the cells indicated the additional presence of a large number of intracellular subunits with an identical subcellular distribution (Fig. 5, C and D). In HEK cells transfected with alpha 1N constructs and beta 3 subunits, only beta 3 subunits could be detected on the cell surface (Fig. 5, E and F). These results are in agreement with previous reports demonstrating that beta 3 subunits are able to form homo-oligomeric receptors that are expressed on the cell surface (21, 30). The observation that the truncated and the full-length subunit could be detected in the permeabilized cells in the same subcellular compartments (Fig. 5, G and H), indicates that beta 3 subunits that assembled with truncated alpha 1 subunits were retained within the cell. No cell surface labeling was observed when HEK cells were co-transfected with alpha 1 and beta 3N or alpha 1N and beta 3N constructs (Fig. 5, I, J, M, and N). However, alpha 1 and beta 3N (Fig. 5, K and L) or alpha 1N and beta 3N subunits could be localized in the same subcellular compartments (Fig. 5, O and P).


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Fig. 5.   Immunofluorescence of HEK cells co-transfected with full-length and truncated alpha 1 and beta 3 subunits. HEK cells were co-transfected with alpha 1 and beta 3 (A-D), alpha 1N and beta 3 (E-H), alpha 1 and beta 3N (I-L), or alpha 1N and beta 3N (M-P). Co-immunofluorescence was performed using alpha 1(1-9) antibodies (A, C, E, G, I, K, M, and O) and bd17 antibodies (B, D, F, H, J, L, N, and P) on the cell surface (A, B, E, F, I, J, M, and N) or in permeabilized cells (C, D, G, H, K, L, O, and P). Rabbit antibodies were detected using anti-rabbit IgG Bodipy FL antibodies, and the monoclonal mouse antibody was detected with anti-mouse IgG Cy 3. Co-immunofluorescence was investigated by confocal laser microscopy (single sections). The experiments were performed three to five times with similar results.

To investigate whether assembly products from full-length and truncated subunits are able to form specific [3H]muscimol-binding sites, membranes from nontransfected HEK cells or from cells transfected with alpha 1 and beta 3, alpha 1N and beta 3, alpha 1 and beta 3N, or alpha 1N and beta 3N were incubated with 5 nM of [3H]muscimol in the absence or presence of 10 µM GABA. For HEK cells transfected with alpha 1 and beta 3 subunits, a specific [3H]muscimol binding of 328 ± 30 fmol/mg protein was found (Table I), whereas in cells co-transfected with alpha 1 and beta 3N constructs, a specific [3H]muscimol binding of 21 ± 4 fmol/mg protein was detected (Table I). In nontransfected HEK cells (not shown), however, and in cells co-transfected with alpha 1N and beta 3 or with alpha 1N and beta 3N, no specific [3H]muscimol binding could be identified. Scatchard analysis of equilibrium binding data indicated a high affinity [3H]muscimol binding to HEK cells co-transfected with alpha 1 and beta 3N constructs (KD of 12.1 ± 4.1 nM, Bmax of 78 ± 29 fmol/mg protein, mean ± S.E., n = 4), and to cells transfected with alpha 1 and beta 3 subunits (KD of 7.9 ± 3.2 nM, Bmax of 805 ± 53 fmol/mg protein, mean ± S.E., n = 4). Whereas the affinity for [3H]muscimol of cells transfected with alpha 1 and beta 3N constructs or with alpha 1 and beta 3 subunits was comparable (p = 0.45, unpaired Student's t test), the Bmax values were significantly different (p < 0.0001, unpaired Student's t test). These results indicate that even intracellular and incomplete assembly intermediates can form specific high affinity [3H]muscimol-binding sites. For the formation of this binding site, however, a full-length alpha 1 subunit is necessary.

                              
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Table I
Assembly, cell surface expression, and [3H]muscimol binding of full-length and truncated alpha 1 and beta 3 subunits
Assembly and cell surface expression was investigated as described in Figs. 4 and 5, respectively. For [3H]muscimol binding HEK cells were co-transfected with alpha 1 and beta 3, alpha 1N and beta 3, alpha 1 and beta 3N, or alpha 1N and beta 3N. Membranes were incubated with 5 nM [3H]muscimol in the presence or absence of 100 µM GABA, and specific [3H]muscimol binding was determined as described under "Experimental Procedures." Values are given as the means ± S.E. from three separate experiments performed in triplicate.

Density gradient centrifugation of constructs formed after transfection of HEK cells with alpha 1 and beta 3N combinations indicated broad peaks at 5.0 and 6.1 s. Because dimers composed of full-length subunits sediment at 5.5 s and trimers at 6.7 s, these data are compatible with the formation alpha 1beta 3N dimers and trimers (Fig. 6). The lower sedimentation coefficients might have been due to the lower molecular mass of the truncated beta 3N construct. The broad peak at 6.1 s might have been due to the formation of a mixture of intermediates composed of (alpha 1)2beta 3N and alpha 1(beta 3N)2 subunits.


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Fig. 6.   Sucrose density gradient centrifugation of extracts from HEK cells co-transfected with alpha 1 and beta 3N subunits. OD, optical density (arbitrary units); s, sedimentation value. The experiments were performed two times with comparable results.

Formation of the Benzodiazepine-binding Site-- Because the benzodiazepine-binding site on GABAA receptors is located at the interface of alpha 1 and gamma 2 subunits (15), it was interesting to investigate whether this site could already be formed by alpha 1gamma 2 dimers. Previous studies have indicated that HEK cells transfected with alpha 1 and gamma 2 subunits form high affinity [3H]flunitrazepam-binding sites (23, 31) although predominantly forming subunit dimers (12). But the formation of minor amounts of higher oligomers and even completely assembled subunit pentamers could not be excluded by these studies.

To eliminate the possibility of formation of completely assembled subunit pentamers, in addition to the truncated alpha 1N construct a truncated gamma 2 subunit (gamma 2N) was cloned that again contained the complete extracellular N-terminal domain but no transmembrane domains. HEK cells were then co-transfected either with alpha 1 and gamma 2 subunits, alpha 1 and gamma 2N, alpha 1N and gamma 2, or alpha 1N and gamma 2N subunits. Expressed subunits were extracted from these cells and were immunoprecipitated with gamma 2(1-33) antibodies. As shown in Fig. 4B, the full-length alpha 1 or the truncated alpha 1N construct could be co-precipitated by gamma 2(1-33) antibodies from extracts of the appropriately co-transfected HEK cells. This was not due to a cross-reactivity of the gamma 2(1-33) antibody because this antibody (in contrast to alpha 1(1-9) antibodies) could not precipitate alpha 1 subunits from HEK cells transfected with alpha 1 subunits only (experiments not shown). These results therefore indicate that not only full-lenth alpha 1 and gamma 2 subunits but also alpha 1N and gamma 2, alpha 1 and gamma 2N, and even alpha 1N and gamma 2N are able to form hetero-oligomers.

To investigate whether the structures formed from alpha 1 and gamma 2, alpha 1N and gamma 2, alpha 1 and gamma 2N, or alpha 1N and gamma 2N subunits were transported to the cell surface, appropriately transfected HEK cells were again investigated by immunofluorescence and confocal laser microscopy. As shown in Fig. 7 (A and B) for intact cells and in agreement with previous reports (4, 21) no GABAA receptor subunits could be detected on the cell surface with alpha 1(1-9) or gamma 2(1-33) antibodies. After permeabilization of the cells, however, both subunits were detected in intracellular compartments (Fig. 7, C and D). For HEK cells co-transfected with alpha 1N and gamma 2, alpha 1 and gamma 2N, or alpha 1N and gamma 2N, again no subunits could be detected on the cell surface. In permeabilized cells, however, a similar subcellular distribution of subunits was observed as in cells transfected with full-length alpha 1 and gamma 2 subunits (experiments not shown).


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Fig. 7.   Immunofluorescence of HEK cells co-transfected with full-length and truncated alpha 1 and gamma 2 subunits. HEK cells were transfected with alpha 1 and gamma 2 subunits. alpha 1 subunits were labeled on the cell surface (A) or in permeabilized cells (C) using alpha 1(1-9) antibodies. gamma 2 subunits were labeled on the cell surface (B) or in permeabilized cells (D) using gamma 2(1-33) antibodies. Rabbit antibodies were detected using anti-rabbit IgG Bodipy FL antibodies. Immunofluorescence was investigated by confocal laser microscopy (single sections). The experiment was performed five times with similar results.

To investigate whether assembly products composed of full-length and truncated or of two truncated subunits are able to form benzodiazepine-binding sites, membranes from nontransfected HEK cells or from cells co-transfected with alpha 1 and gamma 2, alpha 1N and gamma 2, alpha 1 and gamma 2N, or alpha 1N and gamma 2N were incubated with 5 nM [3H]Ro 15-1788 in the absence or presence of 100 µM diazepam. In nontransfected HEK cells or in cells transfected with gamma 2 subunits only, no specific [3H]Ro 15-1788 binding was observed. A specific [3H]Ro 15-1788 binding was observed, however, in HEK cells co-transfected with alpha 1 and gamma 2, alpha 1N and gamma 2, alpha 1 and gamma 2N, or alpha 1N and gamma 2N, and the number of [3H]Ro 15-1788-binding sites/mg protein was comparable (Table II). These results indicate that not only full-length alpha 1 and gamma 2 subunits (31) but also truncated alpha 1 and gamma 2 subunits lacking transmembrane domains are capable of forming a benzodiazepine-binding site. Interestingly, however, the total number of binding sites observed in the four preparations was small compared with that observed in alpha 1beta 3gamma 2 transfected HEK cells in parallel experiments (874 ± 19 fmol/mg protein). To investigate whether this was due to a low affinity or a low number of binding sites formed, Scatchard analysis was performed. Cells transfected with alpha 1 and gamma 2 subunits exhibited a KD of 135 ± 38 nM and a Bmax of 301 ± 23 fmol/mg protein (mean ± S.E., n = 4). Similar values were obtained when cells were transfected with alpha 1N and gamma 2 (KD of 124 ± 44 nM, Bmax of 322 ± 49 fmol/mg protein, mean ± S.E., n = 4), alpha 1 and gamma 2N, or alpha 1N and gamma 2N (data not shown). KD and Bmax values of cells transfected with alpha 1N and gamma 2 subunits were comparable with those of cells transfected with alpha 1 and gamma 2 subunits (p = 0.82 and p = 0.91, respectively) but were significantly different (p = 0.02 and p = 0.0003, respectively) from cells transfected with alpha 1beta 3gamma 2 subunits (KD of 0.96 ± 0.25 nM, Bmax of 1050 ± 86 fmol/mg protein, mean ± S.E., n = 4).

                              
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Table II
Assembly, cell surface expression, and [3H]Ro 15-1788 binding of full length and truncated alpha 1 and gamma 2 subunits
Assembly and cell surface expression was investigated as described in Figs. 4 and 7, respectively. For [3H]Ro 15-1788 binding, HEK cells were co-transfected with alpha 1 and gamma 2, alpha 1N and gamma 2, alpha 1 and gamma 2N, or alpha 1N and gamma 2N. Membranes were incubated with 5 nM [3H]Ro 15-1788 in the presence or absence of 100 µM diazepam, and specific [3H]Ro 15-1788 binding was determined as described under "Experimental Procedures." Values are given as the means ± S.E. from three separate experiments performed in triplicate.

Formation of the TPBS-binding Site-- The TPBS-binding site of GABAA receptors can be identified in receptors composed of homo-oligomeric beta 3 subunits, alpha 1beta 3 and alpha 1beta 3gamma 2 subunits (23) and for the formation of this site the presence of the second transmembrane domain of the beta 3 subunit in a receptor is essential (18). It therefore was no surprise that only HEK cells transfected with alpha 1 and beta 3 subunits but not those transfected with alpha 1 and beta 3N, or alpha 1N and beta 3N subunits exhibited a specific [35S]TBPS binding (experiments not shown). HEK cells transfected with alpha 1N and beta 3 subunits were not investigated for [35S]TBPS binding because in these cells homo-oligomeric beta 3 receptors are formed (see above) that in any case exhibit high affinity [35S]TBPS binding (23).

To investigate whether the TPBS-binding site can be formed by assembly intermediates containing alpha 1 and beta 3 subunits, a beta 3 fragment was cloned (beta 3TM3) that not only contained the extracellular N-terminal domain but also the first three transmembrane domains of the beta 3 subunit. The beta 3TM3 fragment could be co-precipitated with alpha 1 subunits from HEK cells co-transfected with alpha 1 and beta 3TM3 (experiments not shown). Double staining of intact HEK cells transfected with full-length alpha 1 and beta 3TM3 constructs (Fig. 8, A and B) demonstrated that none of these subunits were expressed on the cell surface, but both subunits could be detected in the permeabilized cells in the same subcellular compartments (Fig. 8, C and D). However, no specific [35S]TPBS binding could be identified in these cells. This failure to detect specific [35S]TPBS binding was not due to an improper folding of the beta 3TM3 fragment, because in the same cells a specific [3H]muscimol binding of 50 ± 4 fmol/mg protein could be observed. The quantitative difference between [3H]muscimol binding in HEK cells transfected with alpha 1beta 3N (Table I) and alpha 1beta 3TM3 was significant (p = 0.007, unpaired Student's t test) and reproducible. Because the KD for [3H]muscimol binding to cells transfected with alpha 1 and beta 3N (12.1 ± 4.1 nM) was not significantly different from that of cells transfected with alpha 1 and beta 3 subunits (7.9 ± 3.2 nM), no change in the KD was to be expected in cells transfected with alpha 1beta 3TM3. The increase in [3H]muscimol binding, thus, presumably was due to an increase in the number of binding sites. This could have been caused by an increased stabilization of the [3H]muscimol-binding site because of the presence of the three beta 3 transmembrane domains in the assembly product of alpha 1 and beta 3TM3 or by the formation of a second muscimol-binding site in a possible assembly product composed of two alpha 1 and two beta 3TM3 subunits.


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Fig. 8.   Immunofluorescence of HEK cells co-transfected with full-length alpha 1 and beta 3TM3 constructs. Co-immunofluorescence was performed as described in the legend to Fig. 5. The experiments were performed three times with similar results.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Different Subunit Dimers Are Formed during GABAA Receptor Assembly-- The present study aimed to detect subunits or subunit combinations that could form the starting point of the GABAA receptor assembly process. However, neither in the adult rat brain nor in HEK cells transfected with alpha 1beta 3gamma 2 subunits and kept under standard tissue culture conditions could assembly intermediates be identified by density gradient centrifugation. This indicated that receptor synthesis in these tissues is either low and/or assembly of receptors is too fast to allow intermediates to be identified. When protein folding and subunit oligomerization of recombinant GABAA receptors was slowed down by reducing the culture temperature to 25 °C, however, subunit monomers, dimers, trimers, tetramers, and pentamers could be detected by sucrose density gradient centrifugation. Interestingly, alpha 1, beta 3, as well as gamma 2 subunits could be identified in subunit dimers and all other oligomers. A similar result was obtained from brains of young rats, where a high expression of GABAA receptor subunits caused by ongoing development of the tissue leads to a constant high concentration of assembly intermediates. An identification of the subunit composition of the dimers was not possible, because the peaks for dimers and trimers were overlapping and could not be completely separated by density gradient centrifugation. A possible co-immunoprecipitation of two subunits in the dimer peak thus could have been caused by the respective subunit dimer or by a contamination with subunit trimers. In addition, the similarity of the apparent molecular masses of the alpha 1, beta 3, and gamma 2 subunits and the microheterogeneity of the labeled protein bands (12) prevented an identification of the exact dimers formed after radiolabeling of subunits by culturing with [35S]methionin.

Although the formation of alpha 1beta 3, alpha 1gamma 2, and beta 3gamma 2 heterodimers has been demonstrated previously in cells co-transfected with these subunit combinations (12), the presence of all three subunits in the dimer peak of brains from young rats or of cells transfected with alpha 1, beta 3, and gamma 2 subunits does not necessarily mean that all possible heterodimers are formed in these tissues. The data could also be explained by the formation of two different heterodimers or by the formation of heterodimers and/or homodimers. In addition, some of the dimers could be dead end products for the assembly of GABAA receptors and be subsequently degraded (5). The present data therefore cannot clarify the question of whether assembly of GABAA receptors can start from more than one possible dimer.

This question so far has also not been unequivocally answered for the nAChR. Thus, it has been reported that alpha  subunits of the nAChR first form heterodimers with gamma  and delta , but not with beta  subunits. The alpha gamma and alpha delta heterodimers then were proposed to assemble with the beta  subunit and with each other to form the complete alpha 2beta gamma delta receptor (32). In another study, alpha beta gamma trimers were the first stable assembly intermediates identified (3), and it was proposed that the complete receptor is then formed by a stepwise addition of the delta  and the second alpha  subunit. In any case, the complete assembly of the nAChR is a complex, slow, and inefficient process (33), and its mechanism is still not entirely clarified.

[3H]Muscimol but No [35S]TBPS-binding Sites Are Formed by alpha 1beta 3 Dimers and/or Trimers-- Already during the early steps of assembly of the nAChR, subunit oligomerization and folding events lead to the formation of ligand-binding sites. Thus, a monomeric but properly folded alpha  subunit is sufficient for binding of the competitive antagonist alpha -bungarotoxin, whereas the formation of binding sites for agonists and low molecular weight antagonists occurs in alpha gamma and alpha delta dimers (34). In the present study we therefore investigated the formation of ligand-binding sites on GABAA receptor intermediates.

In agreement with previous studies it was demonstrated that full-length alpha 1 and beta 3 subunits on co-transfection into HEK cells form [3H]muscimol as well as [35S]TBPS-binding sites (23). These subunits are able to form pentameric receptors (12) and are expressed on the cell surface (4, 21, 30). C-terminally truncated alpha 1 or beta 3 subunits, containing only the extracellular N-terminal domain also could assemble with each other or with full-length beta 3 or alpha 1 subunits, respectively, but the assembly products remained in intracellular compartments and could not be detected on the cell surface. Specific [3H]muscimol but no [35S]TBPS-binding sites could be observed in HEK cells co-transfected with full-length alpha 1 subunits and beta 3N constructs. The absence of [35S]TBPS-binding sites in these cells as well as in cells co-transfected with alpha 1N and beta 3N subunits is not surprising, because recently it has been demonstrated that the presence of a TM2 region of the beta 3 subunit is essential for the formation of these sites (18).

Although similar amounts of subunits were expressed in alpha 1beta 3N- or alpha 1beta 3-transfected cells and although the affinity of [3H]muscimol for the sites formed was comparable, the number of [3H]muscimol-binding sites in alpha 1beta 3N-transfected cells was small compared with that in alpha 1beta 3-transfected cells. Immunoprecipitation studies and density gradient centrifugation indicated that most of the alpha 1 subunits and beta 3N fragments formed in the cells were assembled into heterodimers and heterotrimers, and only small amounts of these subunits remained unassembled. The comparatively small number of high affinity [3H]muscimol-binding sites, thus, indicates that only a small part of the alpha 1beta 3N heterodimers or heterotrimers formed contained these sites. This could have been due to a partially incorrect assembly of subunits or a low probability of formation of high affinity [3H]muscimol-binding sites caused by the lacking transmembrane regions of the beta 3N construct or the incomplete assembly of the receptor. The latter suggestion is supported by the finding that only a small number of unassembled nAChR alpha  subunits exhibited alpha -bungarotoxin binding but that the number of binding sites increased with additional assembly steps (3).

Because beta 3 subunits alone can form subunit pentamers exhibiting high affinity [35S]TBPS-binding sites, a co-transfection with alpha 1N and beta 3 subunits could not be used to investigate whether [35S]TBPS binding can be formed by assembly intermediates. Therefore, a beta 3TM3 construct containing the N-terminal domain and three of the four transmembrane domains of the beta 3 subunit was transfected into HEK cells together with full-length alpha 1 subunits. The observed absence of alpha 1 subunits and beta 3TM3 fragments on the cell surface suggests that neither alpha 1beta 3TM3 hetero- nor beta 3TM3 homo-pentamers were formed in these cells or that pentamers formed were retained within the cells. The finding that no [35S]TBPS sites could be detected in alpha 1beta 3TM3-transfected cells then either indicates that this site cannot be formed by an incompletely assembled receptor or that a complete beta 3 subunit in any case is essential for the correct formation of the [35S]TBPS site.

[3H]Ro 15-1788-binding Sites Are Formed on alpha 1gamma 2 Dimers-- In the present work we demonstrated that alpha 1 and gamma 2, alpha 1N and gamma 2, alpha 1 and gamma 2N, as well as alpha 1N and gamma 2N subunits are able to form hetero-oligomers that are not expressed on the cell surface but form specific [3H]Ro 15-1788-binding sites. It has been reported previously that assembly of GABAA receptor alpha 1 and gamma 2 subunits on co-transfection into HEK cells predominantly stops at the stage of dimers (12). Because it is unprobable that higher oligomers are formed in the presence of truncated subunits, these results indicate that the formation of the benzodiazepine-binding site already occurs at the stage of heterodimers and that even intracellular and truncated alpha 1 and gamma 2 subunits lacking transmembrane domains are capable of binding benzodiazepines. The comparatively low affinity and number of the binding sites formed, however, indicates that [3H]Ro 15-1788-binding sites formed by heterodimers do not significantly contribute to the total number of these binding sites formed in the brain.

Implications for the Function of GABAA Receptors-- Although the [3H]muscimol-binding site is formed by the N-terminal domain of the GABAA receptor alpha  and beta  subunits (14), transmembrane domains seem also to support its formation. This is indicated by the observation that in HEK cells co-transfected with alpha 1N and beta 3N constructs, in contrast to those transfected with alpha 1 and beta 3N constructs, no [3H]muscimol-binding sites could be identified. Because binding of GABA to the [3H]muscimol-binding site in intact GABAA receptors causes a conformational change in the transmembrane domains leading to the opening of the chloride ion channel (7), a close conformational interaction of the two subunit domains is to be expected. In addition, studies have indicated that point mutations within the second transmembrane domain (35) or the first extracellular loop between TM2 and TM3 (36) of subunits strongly influence gating of the channel.

Interestingly, however, alpha 1 transmembrane domains seem to be more important than the corresponding beta 3 domains for the formation of a [3H]muscimol-binding site. This is indicated by the observation that HEK cells transfected with full-length alpha 1 subunits and beta 3N constructs but not those transfected with full-length beta 3 subunits and alpha 1N constructs exhibit specific [3H]muscimol-binding sites. Because alpha  subunits not only contribute to the formation of the [3H]muscimol site (14) but also to the formation of the benzodiazepine-binding site (15), it is tempting to speculate that conformational changes in the chloride channel induced by GABA as well as the modulation of the GABA-induced current by benzodiazepines might predominantly be mediated by the alpha  subunit.

In contrast to the [3H]muscimol-binding site that is expressed in cells transfected with alpha 1beta 3 or alpha 1beta 3N combinations only, comparable amounts of [3H]Ro 15-1788-binding sites are expressed in alpha 1gamma 2, alpha 1Ngamma 2, alpha 1gamma 2N, or alpha 1Ngamma 2N transfected cells. Binding affinity was similar for all these combinations but was more than 100-fold lower than that of alpha 1beta 3gamma 2 receptors, suggesting that the affinity of the [3H]Ro 15-1788-binding site is influenced by the presence of additional subunits in a completely assembled receptor. Interestingly, however, the affinity of the benzodiazepine-binding site formed on subunit dimers is not influenced by the absence of alpha 1 and gamma 2 transmembrane domains, possibly reflecting an absence of a direct interaction between the benzodiazepine-binding site and the channel forming transmembrane domains. This conclusion is supported by the observation that binding of benzodiazepines does not cause direct opening of the chloride channel in the absence of GABA but enhances the frequency of GABA-induced channel opening (6). It thus can be speculated that binding of benzodiazepines to its site at the alpha gamma interface strongly influences the conformation of the GABA-binding site located at the other side (alpha beta interface) of the same alpha  subunit. This could either enhance the affinity of GABA for its binding site (6, 7) or produce a conformational change similar to that produced by binding of GABA and thus reduce the number of GABA molecules necessary for opening the channel, as indicated by a previous study (37). Each of these mechanisms would enhance the frequency of channel opening by GABA. Further studies will have to decide between these possibilities.

    FOOTNOTES

* This work was supported by Grant 7835 of the Jubiläumsfonds of the Austrian National Bank and by Grant P12637-Med of the Austrian Science Foundation.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.

Dagger To whom correspondence should be addressed: Brain Research Inst., University of Vienna, Div. of Biochemistry and Molecular Biology, Spitalgasse 4, A-1090 Vienna, Austria. Tel.: 43-1-4277-62950; Fax: 43-1-4277-62959; E-mail: Werner.Sieghart@univie.ac.at.

Published, JBC Papers in Press, February 23, 2001, DOI 10.1074/jbc.M009508200

    ABBREVIATIONS

The abbreviations used are: nAChR, nicotinic acetylcholine receptor; TPBS, t-butylbicyclophosphorothionate; HEK, human embryonic kidney; PAGE, polyacrylamide gel electrophoresis; IPTBO, 4-(isopropyl)-1-phospho-2,6,7-trioxabicyclo-(2, 2, 2)octane-1-oxide; PBS, phosphate buffered saline; GABAA, gamma -aminobutyric acid, type A.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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