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INTRODUCTION |
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
, three
, three
, one
, one
, one
, one
, and three
) have
been identified in the mammalian brain (8, 9). Expression studies
indicated that
,
, and
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
, two
, and one
subunit
(10-13).
Density gradient centrifugation studies indicated that recombinant
GABAA receptors composed of
1
3
2 subunits almost
exclusively sediment as subunit pentamers.
1
3 subunit combinations sediment as
tetramers and pentamers, whereas combinations of
1
2 or
3
2 subunits predominantly form heterodimers (12). These results suggested
a subunit arrangement in GABAA receptors in which four alternating
and
subunits are connected by a
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
1
3
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 
, 
, or 
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
1 and
3 subunits (14), for
benzodiazepines, located between the
1 and
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
1,
3, and
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.
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EXPERIMENTAL PROCEDURES |
Antibodies--
The antibodies anti-peptide
1(1-9) (19), anti-peptide
3(1-13) (12),
anti-peptide
3(345-408) (20), anti-peptide
2(319-366) (12), and anti-peptide
2(1-33) (21) were generated and affinity purified as
described previously. The monoclonal antibody bd17, recognizing
2/3 subunits (22), were purchased from Roche Molecular Biochemicals.
Generation of cDNA Constructs--
For the generation of
recombinant receptors,
1,
3, and
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
-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
1,
3, and
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
1 and
3 subunits, both subunits again sedimented at a peak of
8.7 s. The
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
1(1-9),
3(345-408),
2(319-366), or digoxigenized
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
3(345-408) or
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 |
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
1,
3, and
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
1,
3, as well as the
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
1,
3, and
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 1,
3, and 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
1(1-9), 3(345-408), and
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.
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In other experiments, the culture temperature was first kept at
37 °C for the first 8 h after transfection of HEK cells with
1,
3, and
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.
1 and
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
3 subunit was similar to that of the
1 and
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 1,
3, and 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
1(1-9) (A), 3(345-408)
(B), and 2(319-366) (C)
antibodies. OD, optical density (arbitrary units);
s, sedimentation value. The experiments were performed eight
times with comparable results.
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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:
1,
3, and
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
1 and
2 subunits was again similar, showing overlapping peaks and shoulders at 5.5, 6.8, and 8.7 s, the sedimentation pattern of
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.
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Formation of the [3H]Muscimol-binding
Site--
Because the [3H]muscimol-binding site on
GABAA receptors is located at the interface of
and
subunits (14), it was interesting to investigate whether this binding
site could already be formed by GABAA receptor assembly
intermediates containing
1 and
3 subunits. Because co-transfection of HEK cells with
1
and
3 subunits leads to the formation of
1
3 tetramers and pentamers (12),
truncated
1 (
1N) and
3
(
3N) subunits containing the complete extracellular
N-terminal domain but no transmembrane domains were cloned to
investigate whether they can assemble with full-length
3
and
1 subunits, respectively, forming smaller assembly
intermediates.
1 and
3 subunits and
1N and
3N fragments were then
co-transfected into HEK cells in various combinations, and expressed
subunits were extracted from these cells and were immunoprecipitated
with
3(1-13) antibodies. The precipitate was subjected
to SDS-PAGE and Western blot analysis using digoxigenized
1(1-9) antibodies. As shown in Fig.
4A, in extracts from HEK cells
co-transfected with full-length
1 and
3
subunits or full-length
1 and
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
1(1-9) antibodies from these cells as well as from HEK cells transfected with
1 subunits only, but not from untransfected HEK cells
(experiments not shown), indicating that this protein band represents
the
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
1 subunit. The precipitation of
1
subunits by
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
1 subunits only
(experiment not shown).

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Fig. 4.
Co-immunoprecipitation of full-length and
truncated 1 and 3 or
1 and 2 subunits. HEK cells were
co-transfected with 1 and 3,
1N and 3, 1 and
3N, 1N and 3N
(A) or with 1 and 2,
1N and 2, 1 and
2N, 1N and 2N
(B). Cell extracts were immunoprecipitated with
3(1-13) (A) or 2(1-33)
(B) antibodies. The precipitate was subjected to SDS-PAGE
and Western blot analysis using digoxygenized 1(1-9)
antibodies. The experiment was performed three times with comparable
results.
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When extracts from HEK cells co-transfected with
1N and
full-length
3 subunits or
1N and
3N constructs were precipitated with
3(1-13) antibodies, three protein bands with apparent
molecular masses 30, 33, and 36 kDa could be detected in Western blots
using digoxygenized
1(1-9) antibodies. The molecular
mass of the smallest band (30 kDa) corresponds with that expected for
the unglycosylated
1N fragment. Because two
glycosylation sites are present in the
1 subunit (29),
the 33- and 36-kDa bands presumably represent partially and fully
glycosylated
1N fragments. The co-precipitation by
3(1-13) antibodies of
1N or
1 subunits indicates that not only full-lenth
1 and
3 subunits but also
1N and
3,
1 and
3N, and even
1N and
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
1(1-9) and the
2/
3 subunit-specific bd17 antibodies of
intact HEK cells transfected with full-length
1 and
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
1N constructs and
3 subunits, only
3 subunits could be detected on the cell surface (Fig.
5, E and F). These results are in agreement with previous reports demonstrating that
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
3 subunits that assembled with truncated
1 subunits were retained within the cell. No cell
surface labeling was observed when HEK cells were co-transfected with
1 and
3N or
1N and
3N constructs (Fig. 5, I, J,
M, and N). However,
1 and
3N (Fig. 5, K and L) or
1N and
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
1 and
3 subunits. HEK cells were
co-transfected with 1 and 3
(A-D), 1N and 3
(E-H), 1 and 3N
(I-L), or 1N and 3N
(M-P). Co-immunofluorescence was performed using
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.
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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
1 and
3,
1N and
3,
1 and
3N, or
1N and
3N were
incubated with 5 nM of [3H]muscimol in the
absence or presence of 10 µM GABA. For HEK cells transfected with
1 and
3 subunits, a
specific [3H]muscimol binding of 328 ± 30 fmol/mg
protein was found (Table I), whereas in
cells co-transfected with
1 and
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
1N and
3 or with
1N and
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
1 and
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
1 and
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
1 and
3N constructs or with
1 and
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
1 subunit is
necessary.
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Table I
Assembly, cell surface expression, and [3H]muscimol binding
of full-length and truncated 1 and 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 1 and 3,
1N and 3, 1 and 3N, or
1N and 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.
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Density gradient centrifugation of constructs formed after transfection
of HEK cells with
1 and
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
1
3N dimers and trimers (Fig.
6). The lower sedimentation coefficients
might have been due to the lower molecular mass of the truncated
3N construct. The broad peak at 6.1 s might have
been due to the formation of a mixture of intermediates composed of
(
1)2
3N and
1(
3N)2 subunits.

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Fig. 6.
Sucrose density gradient centrifugation of
extracts from HEK cells co-transfected with
1 and
3N subunits. OD,
optical density (arbitrary units); s, sedimentation value.
The experiments were performed two times with comparable results.
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Formation of the Benzodiazepine-binding Site--
Because the
benzodiazepine-binding site on GABAA receptors is located
at the interface of
1 and
2 subunits
(15), it was interesting to investigate whether this site could already
be formed by
1
2 dimers. Previous studies
have indicated that HEK cells transfected with
1 and
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
1N
construct a truncated
2 subunit (
2N) was
cloned that again contained the complete extracellular N-terminal
domain but no transmembrane domains. HEK cells were then co-transfected
either with
1 and
2 subunits,
1 and
2N,
1N and
2, or
1N and
2N subunits. Expressed subunits were extracted from these cells and were
immunoprecipitated with
2(1-33) antibodies. As shown in
Fig. 4B, the full-length
1 or the truncated
1N construct could be co-precipitated by
2(1-33) antibodies from extracts of the appropriately
co-transfected HEK cells. This was not due to a cross-reactivity of the
2(1-33) antibody because this antibody (in contrast to
1(1-9) antibodies) could not precipitate
1 subunits from HEK cells transfected with
1 subunits only (experiments not shown). These results
therefore indicate that not only full-lenth
1 and
2 subunits but also
1N and
2,
1 and
2N, and even
1N and
2N are able to form hetero-oligomers.
To investigate whether the structures formed from
1 and
2,
1N and
2,
1 and
2N, or
1N and
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
1(1-9) or
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
1N and
2,
1 and
2N, or
1N and
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
1 and
2
subunits (experiments not shown).

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Fig. 7.
Immunofluorescence of HEK cells
co-transfected with full-length and truncated
1 and
2 subunits. HEK cells were
transfected with 1 and 2 subunits.
1 subunits were labeled on the cell surface
(A) or in permeabilized cells (C) using
1(1-9) antibodies. 2 subunits were
labeled on the cell surface (B) or in permeabilized cells
(D) using 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
1 and
2,
1N and
2,
1 and
2N, or
1N and
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
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
1 and
2,
1N and
2,
1 and
2N, or
1N and
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
1 and
2 subunits (31) but also truncated
1 and
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
1
3
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
1 and
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
1N and
2 (KD of 124 ± 44 nM, Bmax of 322 ± 49 fmol/mg
protein, mean ± S.E., n = 4),
1
and
2N, or
1N and
2N (data
not shown). KD and Bmax values of cells transfected with
1N and
2
subunits were comparable with those of cells transfected with
1 and
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
1
3
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 1 and 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 1 and 2,
1N and 2, 1 and 2N, or
1N and 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.
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|
Formation of the TPBS-binding Site--
The TPBS-binding site of
GABAA receptors can be identified in receptors composed of
homo-oligomeric
3 subunits,
1
3 and
1
3
2 subunits (23) and for
the formation of this site the presence of the second transmembrane
domain of the
3 subunit in a receptor is essential (18).
It therefore was no surprise that only HEK cells transfected with
1 and
3 subunits but not those
transfected with
1 and
3N, or
1N and
3N subunits exhibited a specific
[35S]TBPS binding (experiments not shown). HEK cells
transfected with
1N and
3 subunits were
not investigated for [35S]TBPS binding because in these
cells homo-oligomeric
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
1 and
3
subunits, a
3 fragment was cloned (
3TM3)
that not only contained the extracellular N-terminal domain but also
the first three transmembrane domains of the
3 subunit.
The
3TM3 fragment could be co-precipitated with
1 subunits from HEK cells co-transfected with
1 and
3TM3 (experiments not shown).
Double staining of intact HEK cells transfected with full-length
1 and
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
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
1
3N (Table I) and
1
3TM3 was significant (p = 0.007, unpaired Student's t test) and reproducible.
Because the KD for [3H]muscimol
binding to cells transfected with
1 and
3N (12.1 ± 4.1 nM) was not
significantly different from that of cells transfected with
1 and
3 subunits (7.9 ± 3.2 nM), no change in the KD was to be
expected in cells transfected with
1
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
3 transmembrane domains in the assembly product of
1 and
3TM3 or by the formation of a
second muscimol-binding site in a possible assembly product composed of
two
1 and two
3TM3 subunits.

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Fig. 8.
Immunofluorescence of HEK cells
co-transfected with full-length 1
and 3TM3 constructs.
Co-immunofluorescence was performed as described in the legend to Fig.
5. The experiments were performed three times with similar
results.
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 |
DISCUSSION |
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
1
3
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,
1,
3, as
well as
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
1,
3,
and
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
1
3,
1
2, and
3
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
1,
3, and
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
subunits of the nAChR first
form heterodimers with
and
, but not with
subunits. The

and 
heterodimers then were proposed to assemble with the
subunit and with each other to form the complete
2

receptor (32). In another study, 

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
and the second
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
1
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
subunit
is sufficient for binding of the competitive antagonist
-bungarotoxin, whereas the formation of binding sites for agonists and low molecular weight antagonists occurs in 
and 
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
1 and
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
1 or
3 subunits, containing only the extracellular N-terminal
domain also could assemble with each other or with full-length
3 or
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
1 subunits and
3N constructs. The absence of [35S]TBPS-binding sites in these cells as well as in
cells co-transfected with
1N and
3N
subunits is not surprising, because recently it has been demonstrated
that the presence of a TM2 region of the
3 subunit is
essential for the formation of these sites (18).
Although similar amounts of subunits were expressed in
1
3N- or
1
3-transfected cells and although the
affinity of [3H]muscimol for the sites formed was
comparable, the number of [3H]muscimol-binding sites in
1
3N-transfected cells was small compared
with that in
1
3-transfected cells.
Immunoprecipitation studies and density gradient centrifugation
indicated that most of the
1 subunits and
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
1
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
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
subunits exhibited
-bungarotoxin binding but that the number of binding sites increased with additional assembly steps (3).
Because
3 subunits alone can form subunit pentamers
exhibiting high affinity [35S]TBPS-binding sites, a
co-transfection with
1N and
3 subunits could not be used to investigate whether [35S]TBPS
binding can be formed by assembly intermediates. Therefore, a
3TM3 construct containing the N-terminal domain and
three of the four transmembrane domains of the
3 subunit
was transfected into HEK cells together with full-length
1 subunits. The observed absence of
1
subunits and
3TM3 fragments on the cell surface suggests
that neither
1
3TM3 hetero- nor
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
1
3TM3-transfected cells then either
indicates that this site cannot be formed by an incompletely assembled
receptor or that a complete
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
1
2 Dimers--
In the present work we
demonstrated that
1 and
2,
1N and
2,
1 and
2N, as well as
1N and
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
1 and
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
1 and
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
and
subunits (14), transmembrane domains seem also to support its
formation. This is indicated by the observation that in HEK cells
co-transfected with
1N and
3N constructs,
in contrast to those transfected with
1 and
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,
1 transmembrane domains seem to
be more important than the corresponding
3 domains for
the formation of a [3H]muscimol-binding site. This is
indicated by the observation that HEK cells transfected with
full-length
1 subunits and
3N constructs
but not those transfected with full-length
3 subunits and
1N constructs exhibit specific
[3H]muscimol-binding sites. Because
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
subunit.
In contrast to the [3H]muscimol-binding site that is
expressed in cells transfected with
1
3 or
1
3N combinations only, comparable amounts
of [3H]Ro 15-1788-binding sites are expressed in
1
2,
1N
2,
1
2N, or
1N
2N transfected cells. Binding affinity
was similar for all these combinations but was more than 100-fold lower
than that of
1
3
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
1 and
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 
interface strongly influences the conformation of the GABA-binding site
located at the other side (
interface) of the same
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.