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Volume 17, Number 8,
Issue of April 15, 1997
pp. 2728-2737
Copyright ©1997 Society for Neuroscience
Stoichiometry and Assembly of a Recombinant GABAA
Receptor Subtype
Verena Tretter,
Noosha Ehya,
Karoline Fuchs, and
Werner Sieghart
Section of Biochemical Psychiatry, University Clinic for
Psychiatry, A-1090 Vienna, Austria
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
GABAA receptors are ligand-gated chloride ion channels
that are presumed to be pentamers composed of  ,  , and  subunits. The subunit stoichiometry, however, is controversial, and the subunit arrangement presently is not known. In this study the ratio of
subunits in recombinant 132 receptors was determined in
Western blots from the relative signal intensities of antibodies directed against the N terminus or the cytoplasmic loop of different subunits after the relative reactivity of these antibodies had been
determined with GABAA receptor subunit chimeras composed of
the N-terminal domain of one and the remaining part of the other
subunit. Via this method a subunit stoichiometry of two subunits,
two subunits, and one subunit was derived. Similar experiments
investigating the composition of 13 receptors expressed on the
surface of human embryonic kidney (HEK) 293 cells cotransfected with
1 and 3 subunits resulted in a stoichiometry of two and three
subunits. Density gradient centrifugation studies indicated that
combinations of 132 or 13 subunits expressed in HEK 293 cells are able to form pentamers, whereas combinations of 12 or
32 subunits predominantly form heterodimers. These results provide valuable information on the mechanism of GABAA
receptor assembly and support the conclusion that GABAA
receptors are pentamers in which a total of four alternating and
subunits are connected by a subunit.
Key words:
GABAA receptor;
stoichiometry;
assembly;
subunit arrangement;
human embryonic kidney 293 cells;
chimeric
subunits;
density gradient centrifugation;
Western blot
INTRODUCTION
GABA, the major inhibitory transmitter in the CNS,
mediates fast synaptic inhibition by opening the chloride ion channel
intrinsic to the GABAA receptor. This receptor is a
hetero-oligomeric protein and the site of action of a variety of
pharmacologically and clinically important drugs, such as
benzodiazepines, barbiturates, steroids, anesthetics, and convulsants
(Sieghart, 1995 ). So far, six , three , three , one  , and
two  subunits of these receptors, as well as several alternatively
spliced isoforms of some of these subunits, have been identified in
mammalian brain (Macdonald and Olsen, 1994; Sieghart, 1995). Expression
studies have indicated that an , a , and a subunit have to
combine to produce GABAA receptors with a pharmacology
resembling that of receptors found in the brain and that, depending on
the subunits used for transfection of cells, receptors with distinct
pharmacological and electrophysiological properties do arise (Sieghart,
1995). Overall it is assumed, however, that a total of five subunits
have to combine to form functional GABAA receptors (Nayeem
et al., 1994).
A variety of subunit-specific antibodies has been raised to investigate
the subunit composition of GABAA receptors.
Immunocytochemical studies demonstrating the colocalization of subunits
in GABAA receptor clusters on neuronal membranes (Fritschy
et al., 1992; Caruncho and Costa, 1994; Fritschy and Möhler,
1995; Somogyi et al., 1996), as well as studies investigating the
subunit composition of isolated receptors (Benke et al., 1991), have
indicated that receptors consisting of 1, 2/3, and 2 subunits
presumably are the major GABAA receptors in the brain.
Other studies have demonstrated that two different subunits are
present in at least some GABAA receptors (Duggan et al.,
1991; Lüddens et al., 1991; Zezula and Sieghart, 1991; Mertens et
al., 1993). Discrepant results were obtained on a possible
colocalization of different subunits in the same GABAA
receptor. Whereas in one study (Mossier et al., 1994) it was
demonstrated that GABAA receptors seem to contain only a
single type of subunit, other studies have indicated a significant
colocalization of the alternatively spliced short and long form of the
2 subunit (Khan et al., 1994) or of the 2 and 3 subunits
(Quirk et al., 1994) in the same receptor.
To resolve this discrepancy and to determine directly the subunit
stoichiometry of GABAA receptors, we developed a method in
the present study that allows the determination of subunit ratios in
multimeric proteins. Via this method the subunit stoichiometry of
recombinant 132 GABAA receptors expressed in human
embryonic kidney (HEK) 293 cells was determined. In addition, density
gradient centrifugation experiments investigating assembly
intermediates of recombinant GABAA receptors in HEK 293 cells transfected with 13, 12, 32, or 132
subunits provided important information on the subunit arrangement
within recombinant 132 receptors as well as on the mechanism
of GABAA receptor assembly.
MATERIALS AND METHODS
Generation and purification of antibodies. The
anti-1(1-9) antibody was generated and affinity-purified as
described previously (Zezula and Sieghart, 1991). Peptide 3(1-13)
was custom-synthesized with an additional C-terminal cysteine and was
coupled to keyhole limpet hemocyanin. Rabbits were immunized with this
adduct, and anti-3(1-13) antibodies were purified from the serum of
the rabbits by affinity chromatography on a column consisting of the
peptide 3(1-13) coupled to thiopropyl-Sepharose. The
anti-1(328-382) and the anti-2(319-366) antibodies were
generated by immunizing rabbits with a maltose binding protein
(MBP)-1(328-382)-7His or MBP-2(319-366)-7His fusion protein,
respectively (Mossier et al., 1994). Then the antibodies were purified
by using the corresponding glutathione S-transferase fusion
protein coupled to Affi-Gel 10 (Bio-Rad, Hercules, CA).
Cloning of chimeric receptor subunits. For the generation of
recombinant receptors, 1, 3, and 2 subunits of
GABAA receptors from rat brain were cloned and subcloned
into the pCDM8 expression vector (Invitrogen, San Diego, CA) as
described previously (Fuchs et al., 1995). To generate chimeric
receptor subunits, we cloned the cDNAs for the 1, 3, and 2
subunits into the vector pALTER-1 (Promega, Madison, WI), using
HindIII and NotI restriction sites of the
polylinker. Then oligonucleotide-directed mutagenesis was performed to
introduce a unique BglII site at the 5 -end of the first
transmembrane region of each subunit. Mutagenesis was performed with
the Altered Sites II in vitro Mutagenesis System (Promega) according to the instructions of the manufacturer. The following oligonucleotides were used: 1-mut, 5-TTG AAT AAC AAA GTA GCC GAT
AGA TCT CTT CAA GTG AAA GTG AGT-3; 3-mut, 5-CTG AAG TAT GAA GTA
CCC AAT AGA TCT CTT CAA CCG AAA ACT-3; 2-mut, 5-CTG GAT GGT AAA
GTA CCC CAT AGA TCT GCT CAG ATC AAA GTA-3. Mutations were confirmed by
dideoxy-DNA sequencing. Chimeras were generated by cutting with
restriction enzymes (HindIII and BglII for the N-terminal part or BglII and NotI for the
C-terminal part of the genes) and isolating and religating of the
desired fragments with the HindIII and NotI cut
expression vector pCDNA I Amp (Invitrogen). Chimeras were confirmed by
selective restriction enzyme cleavage and by sequencing across the
chimera boundaries. Large-scale DNA purification for transfections was
performed with the Qiagen plasmid purification procedure (Qiagen GmbH,
Hilden, Germany).
Expression of recombinant 132 receptors and chimeric
receptor proteins. HEK 293 cells (CRL 1573) from American Type
culture collection (Rockville, MD) were maintained in DMEM (Life
Technologies, Grand Island, NY) supplemented with 10% fetal calf serum
(HyClone Laboratories, Logan, UT), 2 mM glutamine, 50 µM -mercaptoethanol, 100 U/ml penicillin G, and 100 µg/ml streptomycin and nonessential amino acids (Life Technologies)
in 75 cm2 Petri dishes by the use of standard cell culture
techniques. HEK 293 cells (3 × 106) were transfected
with a total of 21 µg of subunit cDNA (cDNA ratio 1:3:2 = 1:1:1 or 1:1:4) or with 20 µg of individual chimera cDNA via the
calcium phosphate precipitation method (Chen and Okayama, 1988). The
cells were harvested 44 hr after transfection.
Purification of recombinant 132 receptor. The
transfected HEK cells from a total of 150 Petri dishes were harvested,
and the pelleted cells were extracted in 50 ml of deoxycholate buffer [0.5% deoxycholate, 0.05% phosphatidylcholine and (in
mM) 10 Tris-HCl, pH 8.0, 150 NaCl, 0.3 PMSF, and 1 benzamidine with 100 mg/l bacitracin] for 30 min at 4°C. The extract
was centrifuged for 40 min at 150,000 × g at 4°C and
was applied to a column consisting of the benzodiazepine Ro 7-1986 coupled to Affi-Gel 15 (Bio-Rad) (Fuchs and Sieghart, 1989). The column
was washed with 3 vol of deoxycholate buffer, 6 vol of
immunoprecipitation (IP) high buffer (50 mM Tris-HCl, 0.5%
Triton X-100, 600 mM NaCl, and 1 mM EDTA, pH
8.3), and 6 vol of IP low buffer (50 mM Tris-HCl, 0.2%
Triton X-100, 150 mM NaCl, and 1 mM EDTA, pH
8.0) and was eluted with 6 M guanidine-HCl, 0.2 M acetic acid, and 0.5% Triton X-100. Before
electrophoresis the receptor was precipitated with methanol/chloroform
(Wessel and Flügge, 1984), and the pellet was 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.
Immunopurification of chimeric receptor subunits.
Formaline-fixed Staphylococcus aureus cells (1 ml; Immunoprecipitin, Life Technologies, Gaithersburg, MD) were
pelleted and resuspended in 900 µl of buffer containing 3% SDS/10%
-mercaptoethanol in PBS. The suspension was heated at 95°C for 30 min, centrifuged at 8000 × g, and washed three times
with IP low buffer. Finally, the pellet was suspended in 900 µl of IP
low buffer containing 100 mg/l bacitracin, 1 mM
benzamidine, and 0.3 mM PMSF, and the suspension was used
for immunoprecipitation.
Transfected HEK cells were grown at 37°C for 44 hr, and receptor
chimeras were extracted with deoxycholate buffer (see above) for 30 min
at 4°C at a concentration of 1.3 mg of protein per milliliter of
buffer. The extract was centrifuged at 150,000 × g for
30 min at 4°C, and the clear supernatant was incubated overnight at
4°C under gentle shaking with 20 µg of antibody per milliliter. After addition of Immunoprecipitin and 0.5% nonfat dry milk powder and
shaking for an additional 3 hr at 4°C, the precipitate was washed
three times with IP low buffer. The precipitated protein was dissolved
in sample buffer for SDS-PAGE.
SDS-PAGE, Western blot, and chemiluminescence detection.
SDS-PAGE was performed according to Neville and Glossmann (1974) with 10% acrylamide/0.27% bisacrylamide. Various dilutions of receptor and chimeric subunit protein were applied to SDS-PAGE. Samples
that should be compared directly (chimeras and/or receptors detected
with two different antibodies) were run on the same gel. After
electrophoresis, gels containing the receptor samples or the chimeras
were tank-blotted onto prewetted polyvinylidene fluoride membranes in
the same sandwich under identical conditions. After blocking with 1.5%
nonfat dry milk powder in PBS and 0.1% Tween 20 for 1 hr at room
temperature, we incubated the membranes overnight with digoxigenated
primary antibody at the following concentrations: anti-1(1-9), 2 µg/ml; anti-3(1-13), 2 µg/ml; anti-2(319-366), 1 µg/ml;
anti-1(328-382), 1 µg/ml. After extensive washing [1.5% (w/v)
dry milk powder and 0.1% Tween 20 in PBS], the membranes were
incubated with anti-digoxigenin F(ab)2 fragments coupled to
alkaline phosphatase (Boehringer Mannheim, Mannheim, Germany) for 45 min at room temperature. Membranes were washed extensively and
equilibrated in assay buffer (0.1 M diethanolamine and 1 mM MgCl2, pH 10.0) for 10 min. Then membranes
were incubated with 1 ml of 0.25 mM CSPD or CDP-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, Kodak, Rochester, NY) for various time
periods. Signals were quantified by a gel documentation system (Docu
Gel 2000i; software: RFLP-Scan; MWG Biotech, Ebersberg, Germany).
Determination of surface expression of receptors. HEK 293 cells were transfected with various combinations of GABAA
receptor subunits. After 44 hr the culture medium was removed and the
cells were washed once with PBS containing (in mM) 2.7 KCl,
1.5 KH2PO4, 0.14 NaCl, and 4.3 Na2HPO4, pH 7.3. Cells then were detached from the Petri dishes by incubating with 2.5 ml of 5 mM EDTA in
PBS for 5 min at room temperature. The resulting cell suspension was diluted in 6.5 ml of cold DMEM and centrifuged for 5 min at 1000 × g. The pellet from two dishes was incubated with 30 µg
of antibody in 3 ml of the same medium for 30 min at 37°C. Cells
again were pelleted and washed twice with 10 ml of DMEM and twice with
10 ml of PBS buffer. Then receptors were extracted with IP low buffer containing 1% Triton X-100 for 1 hr under gentle shaking. Cell debris
was removed by centrifugation, and antibody-labeled receptors were
isolated by immunoprecipitation. The resulting protein pellet was
dissolved in sample buffer for SDS-PAGE.
Density gradient centrifugation. Transfected HEK 293 cells
from eight Petri dishes were harvested and extracted in 1.6 ml of
Lubrol extraction buffer [1% Lubrol PX, and (in mM) 150 NaCl, 5 EDTA, 50 Tris-HCl, pH 7.4, 0.3 PMSF, 1 benzamidine with 0.18% phosphatidylcholine, and 100 mg/l bacitracin] overnight at 4°C. The
clear extract (250 µl) 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 digoxigenated catalase
(sedimentation coefficient 11 s), 1.2 µg of digoxigenated alkaline phosphatase (sedimentation coefficient 6.1 s) and 1 µg of digoxigenated 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 hr and then
were fractionated by piercing at the bottom. Protein in individual
fractions was precipitated (Wessel and Flügge, 1984) and
dissolved in sample buffer for SDS-PAGE. Then the samples were analyzed
by Western blotting and densitometry.
RESULTS
Identification of individual receptor subunits in recombinant
GABAA receptors
HEK 293 cells were transfected with a mixture of cDNAs encoding
1, 3, and 2 subunits of GABAA receptors. To
eliminate incompletely assembled receptors, we extracted recombinant
receptors that formed by using a deoxycholate-containing buffer and
subjected them to affinity chromatography on a column containing the
benzodiazepine Ro 7-1986 (Fuchs and Sieghart, 1989). Affinity-purified
GABAA receptors then were subjected to SDS-PAGE and Western
blot analysis by the use of polyclonal antibodies specific for the
1, 3, or 2 subunits. In agreement with previous results
(Zezula and Sieghart, 1991), anti-1(1-9), antibodies identified a
protein with apparent molecular mass of 51 kDa (Fig. 1).
A protein with identical apparent molecular mass was identified by
anti-1(328-382) antibodies. Anti-3(1-13) antibodies strongly
identified a protein with apparent molecular mass of 54 kDa and weakly
recognized additional proteins with a slightly higher or lower
molecular mass, as was shown previously with anti-3(345-408)
antibodies (Slany et al., 1995) or the monoclonal antibody bd 17 (Fuchs
et al., 1989). Anti-2(319-366) antibodies identified a protein with
apparent molecular mass of 49 kDa (Mossier et al., 1994). As shown in
Figure 1 (lane labeled Ab-Mix), the similarity of the
apparent molecular masses of the individual proteins and the
microheterogeneity of the labeled protein bands precluded a direct
quantification of recombinant receptor subunits after pulse labeling
with radiolabeled methionine, a method that has been applied
successfully for the determination of the subunit stoichiometry of the
nicotinic ACh receptor (Anand et al., 1991).
Fig. 1.
Identification of GABAA receptor
subunits in affinity-purified recombinant 132 receptors. HEK
293 cells were transfected with 132 subunits. Receptors were
extracted, purified by affinity chromatography, and subjected to
SDS-PAGE and Western blot analysis by using the digoxigenated
subunit-specific antibodies and chemiluminescence detection, as
indicated. Chemiluminescence exposure time was varied to result in
comparable signal intensities for the individual antibodies. Antibody
mix (Ab-Mix): a mixture of anti-peptide
1(1-9), anti-peptide 3(1-13), and
anti-peptide 2(319-366) antibodies was used for the
detection of GABAA receptor subunits. The experiment was
performed three times with similar results.
[View Larger Version of this Image (29K GIF file)]
Determination of subunit ratios in GABAA receptors
Determination of subunit ratios using 3-1 chimeras
Immunological signals as shown, for instance, in Figure 1, because
of differences in the reactivity of the antibodies used (avidity
differences, different number of epitopes identified), do not reflect
directly the amount of the respective protein present in the purified
receptor preparation. To determine the relative reactivity of the
anti-3(1-13) and the anti-1(328-382) antibodies used, we
constructed a chimeric GABAA receptor subunit consisting of
the extracellular domain of the 3 subunit and the four transmembrane domains and the cytoplasmic loop of the 1 subunit (Fig.
2). Because the epitopes identified by the anti-3 and
anti-1 antibodies now were present in the same protein, the
reactivity of the individual antibodies against their epitopes could be
compared directly.
Fig. 2.
Determination of the 1:3 subunit ratio using
a 3-1 chimera. A chimeric protein consisting of the N terminus
of the 3 subunit and the four transmembrane domains plus the
cytoplasmic loop of the 1 subunit was expressed in HEK 293 cells.
The chimera was extracted and precipitated using anti-3(1-13)
antibodies; increasing amounts of this protein were subjected to
SDS-PAGE and Western blot analysis with digoxigenated anti-3(1-13)
or anti-1(328-382) antibodies, anti-digoxigenin F(ab)2
fragments coupled to alkaline phosphatase, and the chemiluminescence
substrate CDP-star. In the same experiment affinity-purified
recombinant 132 receptors were subjected to SDS-PAGE and
Western blot analysis by using the same primary and secondary
antibodies and substrate. Chemiluminescence signals were detected by
exposure to x-ray film and were quantified with a gel documentation
system. Signals obtained were subjected to linear regression analysis.
Shown is a typical experiment that was repeated a total of five times
with comparable results.
[View Larger Version of this Image (30K GIF file)]
The chimeric 3-1 protein was expressed in HEK 293 cells and
purified by immunoprecipitation with anti-3(1-13) antibodies. Increasing amounts of the chimera then were subjected to SDS-PAGE and
Western blot analysis with digoxigenated anti-3(1-13) and anti-1(328-382) antibodies. As shown in Figure 2, the digoxigenated antibodies identified the same protein with the expected molecular mass, and the signal intensity of the antibodies increased linearly with increasing amounts of chimeric protein applied to the gel. The
ratio of the slopes (signal intensity per protein unit) of the standard
curves indicated that the anti-1(328-382) antibodies reacted 4.04 times stronger with the chimeric protein than the anti-3(1-13)
antibodies (Fig. 2). This was not much of a surprise, because the
number of epitopes recognized by the anti-1(328-382) antibodies
(directed against 45 amino acids) probably was larger than that
recognized by the anti-3(1-13) antibodies (directed against 13 amino acids).
In the same experiment increasing amounts of affinity-purified
132 receptor were subjected to SDS-PAGE and Western blot analysis with the same digoxigenated antibodies. Each of the antibodies now identified the respective GABAA receptor subunits, and
the signals obtained again increased linearly with increasing amounts of receptor protein applied to the gel. The slope ratio obtained indicated that the reaction of the anti-1(328-382) antibodies with
the 1 subunit was 4.82 times stronger than the reaction of the
anti-3(1-13) antibodies with the 3 subunit. Taking into account
the 4.04 times higher reactivity of the anti-1(328-382) antibodies,
we calculated the ratio of the 1 and 3 subunits in the
recombinant 132 receptors to be 1.19:1 (Fig. 2). This experiment was performed independently a total of five times, resulting
in an average subunit ratio of 1:3 = 0.95 ± 0.25 (mean ± SD).
Determination of subunit ratios using 3-2 chimeras
To compare the reactivity of the anti-3(1-13) and
anti-2(319-366) antibodies, we constructed a chimeric
GABAA receptor subunit consisting of the N terminus and
extracellular domain of the 3 and the four transmembrane domains and
the cytoplasmic loop of the 2 subunit. The chimeric protein was
identified by the anti-3(1-13) as well as by the
anti-2(319-366) antibodies, and increasing amounts of the purified
chimeric protein resulted in a linear increase of both signals (Fig.
3). The slope ratio indicated that the signal obtained
with the anti-2(319-366) antibodies in this experiment was 8.48 times stronger than that obtained with the anti-3(1-13) antibodies.
In the same experiment and under the same conditions, increasing
amounts of affinity-purified 132 receptor were subjected to
SDS-PAGE and Western blot analysis. The signals obtained with the
anti-2(319-366) and anti-3(1-13) antibodies again increased
linearly with the protein concentration applied to the gel (Fig. 3).
The reaction of the anti-2(319-366) antibodies with the 2
subunit was 4.55 times stronger than the reaction of the
anti-3(1-13) antibodies with the 3 subunit. After correction of
this result for the difference in the response of the two antibodies
toward the chimeric 3-2 protein, a 3:2 subunit ratio of
1:0.53 was obtained. This experiment was performed independently a
total of six times, resulting in an average subunit ratio of
3:2 = 2.19 ± 0.63 (mean ± SD).
Fig. 3.
Determination of the 3:2 subunit ratio by
using a 3-2 chimera. A chimeric protein consisting of the N
terminus of the 3 subunit and the four transmembrane domains plus
the cytoplasmic loop of the 2 subunit was expressed in HEK 293 cells. The chimera was extracted and precipitated using
anti-3(1-13) antibodies; increasing amounts of this protein were
subjected to SDS-PAGE and Western blot analysis with digoxigenated
anti-3(1-13) or anti-2(319-366) antibodies, anti-digoxigenin
F(ab)2 fragments coupled to alkaline phosphatase, and the
chemiluminescence substrate CDP-star. In the same experiment
affinity-purified recombinant 132 receptors were subjected to
SDS-PAGE and Western blot analysis by using the same primary and
secondary antibodies and substrate. Chemiluminescence signals were
detected by exposure to x-ray film and were quantified with a gel
documentation system. The double band observed with the chimeric
protein might have resulted from differential glycosylation. Identical
antibody response factors were obtained when the signals from both
proteins or from the individual proteins were compared. Signals
obtained were subjected to linear regression analysis. Shown is a
typical experiment that was repeated a total of six times with
comparable results.
[View Larger Version of this Image (29K GIF file)]
Determination of subunit ratios using 1-2 chimeras
Finally, a chimeric protein was constructed consisting of the N
terminus and extracellular domain of the 1 and the four
transmembrane domains and the cytoplasmic loop of the 2 subunit. The
protein was expressed and purified; after SDS-PAGE and Western blot
analysis this protein was identified by the anti-1(1-9) as well as
by the anti-2(319-366) antibodies. As shown in Figure
4, the signal obtained from the anti-2(319-366)
antibodies was 6.16 times that obtained with the anti-1(1-9)
antibodies. When affinity-purified 132 receptor was
investigated in the same experiment, the signals obtained by the
reaction of the anti-2(319-366) antibodies with the 2 subunit
was 3.12 times larger than that obtained from the reaction of the
anti-1(1-9) antibodies with the 1 subunit. When these results
were corrected for the individual antibody response factor, an
1:2 subunit ratio in recombinant 132 receptors of 1:0.51
was obtained (Fig. 4). This experiment was performed independently a
total of six times, resulting in an average subunit ratio of
1:2 = 1.99 ± 0.51 (mean ± SD).
Fig. 4.
Determination of 1:2 subunit ratio using an
1-2 chimera. A chimeric protein consisting of the N terminus of
the 1 subunit and the four transmembrane domains plus the
cytoplasmic loop of the 2 subunit was expressed in HEK 293 cells.
The chimera was extracted and precipitated using anti-1(1-9)
antibodies; increasing amounts of this protein were subjected to
SDS-PAGE and Western blot analysis with digoxigenated anti-1(1-9)
or anti-2(319-366) antibodies, anti-digoxigenin F(ab)2
fragments coupled to alkaline phosphatase, and the chemiluminescence
substrate CDP-star. In the same experiment affinity-purified
recombinant 132 receptors were subjected to SDS-PAGE and
Western blot analysis by using the same primary and secondary
antibodies and substrate. Chemiluminescence signals were detected by
exposure to x-ray film and were quantified with a gel documentation
system. Signals obtained were subjected to linear regression analysis.
Shown is a typical experiment that was repeated a total of six times
with comparable results.
[View Larger Version of this Image (28K GIF file)]
Stoichiometry of recombinant 132 receptors
Combined results obtained from five to six separate experiments
for each chimera and subunit pair indicated a subunit stoichiometry for
1:3:2 of 2:2:1. The same subunit stoichiometry was obtained whether subunit cDNA ratios of 1:3:2 = 1:1:1 or 1:1:4
were used for transfection of HEK 293 cells, supporting previous
results (Chang et al., 1996) indicating that stoichiometry did not
depend on the relative availability of GABAA receptor
subunits.
A variety of control experiments was performed to account for factors
possibly influencing the results obtained. Thus, in the first
experiments of this study a less-sensitive immunological detection
system (chemiluminescence substrate CSPD instead of the ten times more
sensitive CDP-star) was used. Consequently, larger amounts of protein
had to be applied to the gels to detect the proteins. Nevertheless,
these experiments resulted in the same stoichiometry, indicating that
the results obtained were not influenced by the amount of receptor or
chimeric protein applied to the gel. In addition, results obtained were
independent from the receptor or chimera preparation used and were
identical whether chimeras were isolated by immunoprecipitation with
antibodies directed against the N terminus or against the cytoplasmic
loop. Because saturating concentrations of antibodies were used for the
detection of proteins in Western blots, results obtained were not
influenced by the antibody concentration. Finally, results obtained
were not influenced by the degree of digoxigenation of the antibodies.
Depending on the degree of digoxigenation, the reactivity of the
antibodies and, thus, the antibody response factors was different. In
the course of this study, antibody response factors of
anti-3:anti-1 antibodies between 1:2 and 1:5 were obtained.
Antibody response factors of anti-3:anti-2 antibodies were
between 1:3 and 1:8, and those for anti-1:anti-2 antibodies were
between 1:2 and 1:8. Nevertheless, the subunit ratios obtained were the
same as long as these ratios were determined with the same antibodies,
in the same experiment, and under the same conditions as the antibody
response factors.
GABAA receptor assembly
132 coexpression
There are six possible subunit arrangements in pentameric
receptors consisting of two , two , and one subunit (Fig.
5). Arrangements 5A and 5B, as
well as 5C and 5D, are mirror images and are
characterized by subunits being in contact with as well as with
subunits. In arrangement 5E the subunit is in contact with two and in arrangement 5F with two subunits. To identify the subunit arrangement actually formed, we
investigated whether it was possible to isolate intermediates of the
assembly process of recombinant 132 receptors. For this, HEK
293 cells were transfected with 1, 3, and 2 subunits, and the
receptors that formed were extracted with an extraction buffer
containing Lubrol. Then receptors were subjected to density gradient
centrifugation on sucrose gradients. 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 the known s value added to each
gradient. As shown in Figure 6A, in
HEK cells transfected with 132 subunit cDNAs, the 1, 3,
as well as the 2 subunit protein sedimented at a single peak at 8.7 s. This sedimentation coefficient is identical with that of
GABAA receptors isolated from adult brain (experiments not
shown) and is comparable with that observed with the pentameric nicotinic ACh receptor (9 s; Green and Claudio, 1993). The
cosedimentation of 1, 3, and 2 subunits of GABAA
receptors in a single protein peak at 8.7 s thus indicates
the formation of pentameric GABAA receptors. The protein
shoulder above 8.7 s of the 1, 3, and 2 subunits
also has been observed with subunits of the nicotinic ACh receptors (Gu
et al., 1991; Green and Claudio, 1993) and presumably is caused by an
aggregation of pentameric receptors. The absence of 1, 3, and
2 protein peaks with lower s values (Fig.
6A) indicated that most of the GABAA
receptors formed in 132 transfected cells are pentamers
consisting of 132 subunits and that assembly intermediates
could not be identified under these conditions.
Fig. 5.
Possible subunit arrangements of receptors
composed of two subunits, two subunits, and one subunit.
[View Larger Version of this Image (20K GIF file)]
Fig. 6.
Sucrose density gradient centrifugation of
GABAA receptors extracted from HEK cells transfected with
various combinations of 1, 3, and 2 subunits. HEK 293 cells
were transfected with (A) 132,
(B) 13, (C) 12, or
(D) 32 subunits. Receptors were extracted and
size-fractionated on 5-20% linear sucrose density gradients.
Gradients were fractionated, and protein in individual fractions was
precipitated and subjected to SDS-PAGE and Western blot analysis with
anti-1(1-9), anti-3(1-13), or anti-2(319-366) antibodies.
s values were measured by including digoxigenated standard proteins with known s values in each gradient.
OD, Optical density (arbitrary units). Symbols used:
open squares, 1; filled circles, 3;
filled triangles, 2. The experiments were performed three to four times with comparable results.
[View Larger Version of this Image (16K GIF file)]
13 coexpression
In a further attempt to identify possible assembly intermediates,
we transfected HEK 293 cells with 1 and 3 subunits of GABAA receptors only. Density gradient centrifugation
indicated that under these conditions 1 as well as 3 subunit
proteins again sedimented at a peak of 8.7 s, indicating the
formation of pentameric 13 receptors (Fig.
6B).
In addition to the 13 pentamers, however, especially 3
subunits were able to form subunit complexes with lower sedimentation coefficients. This is indicated by the presence of additional protein
peaks at 7.4, 6.7, 5.5, and 3.3 s in the gradient shown in
Figure 6B. In studies investigating the nicotinic ACh
receptor, it has been demonstrated that monomeric subunits of this
receptor show sedimentation between 3 and 4 s (Green and
Millar, 1995). Sedimentation of subunit dimers was observed at 6 s, trimers sedimented at 7 s, and tetramers at 8 s (Green and Claudio, 1993). Given the similarity of the
sedimentation coefficients observed in experiments investigating the
structurally similar GABAA receptors and nicotinic receptors, it is reasonable to assume that the peaks with 3.3, 5.5, 6.7, and 7.4 s represented mono-, di-, tri-, and tetramers of GABAA receptor 3 subunits, respectively. The
difference in the sedimentation coefficients between subunit oligomers
of nACh receptors and of GABAA receptors might have been
attributable to differences in the apparent molecular mass of nACh
receptor and GABAA receptor subunits. The comparatively
broad peak of the 1 subunit sedimentation and its overlap with the
7.4 s peak of the 3 subunit might indicate that, in
addition to 3 homotetramers, 13 tetramers had been formed to a
significant extent under these conditions (Fig. 6B).
The peaks at 6.7 and 5.5 s, however, seem to have consisted
predominantly of 3 homo-oligomers.
Stoichiometry of recombinant 13 receptors expressed at the
cell surface
The actual assembly of pentameric 13 receptors, their
incorporation into the cell membrane, and their stoichiometry were investigated by incubating intact HEK cells transfected with 1 and
3 subunits with anti-1(1-9) antibodies. Under these conditions these N-terminal antibodies interacted only with receptors expressed on
the cell surface. Then cells were washed to remove excess of antibodies, and the receptors were extracted with a buffer containing 1% Triton X-100. Under these conditions the interaction between antibodies and receptors was reasonably stable. Receptors previously labeled by the antibody on the cell surface thus could be precipitated by the addition of Immunoprecipitin. Then the pellet was subjected to
SDS-PAGE and Western blot analysis, and the ratio of the 1 and 3
subunits in these receptors was investigated by using the 3-1
chimera as described above (experiments not shown). Results from two
separate experiments indicated that the ratio of 1:3 subunits was
2:2.8 and 2:3.4, indicating that these receptors consisted of three
3 and two 1 subunits.
12 or 32 coexpression
When HEK cells were transfected with 1 and 2 subunits,
protein complexes containing 1 subunits sedimented at 6.7 and 5.5 s, whereas those containing 2 subunits sedimented as a
single protein peak at 5.5 s (Fig. 6C). This
seems to indicate that 1 and 2 subunits readily can form dimers,
whereas predominantly the 1 subunits seem to be able to form
trimers. Similarly, after transfection of HEK cells with 3 and 2
subunits, the largest part of the 3 and 2 subunits sedimented at
5.5 s, whereas a small proportion of predominantly the 3
subunit sedimented with a higher sedimentation coefficient (Fig.
6D). This again seems to indicate that 3 and 2
subunits readily form dimers but rather inefficiently form higher
molecular mass complexes. Other experiments demonstrated that, in
contrast to 132 and 13 receptors, 12 or 32
subunit complexes could not be identified on the surface of HEK 293 cells (experiments not shown).
Subunit arrangement of recombinant 132 receptors
The present results are in agreement with experiments from the
nicotinic ACh receptor, indicating that the assembly of multimeric receptors is an ordered and step-wise process in which each newly added
subunit causes a conformational change in the assembly complex that is
necessary for the addition of further subunits. Failure to associate
with the right subunit then might prevent proper folding and thereby
lead to degradation (Green and Millar, 1995).
The formation of pentamers in cells cotransfected with 1 and 3
subunits is consistent with all subunit arrangements shown in Figure 5.
Each of these arrangements allows for an uninterrupted assembly of two
and two subunits. The addition of a further subunit
finalizes the pentameric receptor that then is transported to the cell
surface.
The observation that 1 and 2 or 3 and 2 subunits
predominantly form dimers, however, is consistent only with the subunit arrangements shown in Figure 5, A or B. Only
these subunit arrangements predict an interruption of the assembly
process at the level of an 12 or 32 dimer, because at this
stage the presence of a 3 or 1 subunit, respectively, is
necessary to induce the proper conformational change for an efficient
further assembly. Subunit arrangements 5C or 5D,
in the absence of a 3 or 1 subunit, predict an interruption of
the assembly process at the level of trimers consisting of one 2 and
two 1 or of one 2 and two 3 subunits, respectively. Similarly,
subunit arrangement 5E in the absence of a 3 subunit
predicts an interruption of the assembly process at the level of
trimers composed of one 2 and two 1 subunits but, in addition,
suggests that 2 and 3 subunits do not combine with each other.
Thus, in the absence of 1 subunits, arrangement 5E
predicts the formation of 3 homodimers and of monomeric 2 subunits. Finally, arrangement 5F in the absence of an 1
subunit predicts the formation of trimers composed of one 2 and two
3 subunits and, in addition, suggests that 2 and 1 subunits do not combine with each other. In the absence of a 3 subunit,
arrangement 5F predicts the formation 1 homodimers and of
2 subunit monomers. Because previous studies have demonstrated the
formation of receptors consisting of 12 subunits (Sigel et al.,
1990; Knoflach et al., 1992) and of 22 subunits (Draguhn et al.,
1990; Sigel et al., 1990; Verdoorn et al., 1990), in agreement with the
present conclusion the actual formation of arrangement 5E
and 5F seems not to be very probable.
DISCUSSION
Stoichiometry of recombinant 132 receptors
In the present study chimeric proteins composed of the complete
N-terminal domain of one GABAA receptor subunit and of the four transmembrane domains plus the cytoplasmic loop of another subunit
were used to compare the reactivity of subunit-specific N-terminal or
cytoplasmic loop antibodies in Western blots. Using this information,
we could determine the ratio of the respective subunits in purified
GABAA receptors from the relative signal intensities of the
antibodies in Western blots. Results obtained indicated that
recombinant 132 GABAA receptors are composed of
two , two , and one subunit.
Our conclusions are tied strongly to the assumption that the antibodies
exhibited the same reactivity toward the chimeric proteins and the
respective subunits. Theoretical considerations do support this
assumption. Thus, the epitopes identified by the antibodies used are
located at the very N-terminal end or at the cytoplasmic loop of the
subunits and are surrounded by amino acid domains that are identical in
the original as well as in the chimeric subunits. An identical
reactivity of the antibodies with original and chimeric subunits,
therefore, is highly likely.
This conclusion is strengthened by the observation that, overall,
results on 132 receptors obtained from the investigation of
three different chimeras and subunit ratios with four different antibodies were highly consistent. Thus, subunit ratios 1:3 = 1:1 and 3:2 = 2:1 are consistent with the ratio
1:2 = 2:1 and support the observation that native
GABAA receptors do form subunit pentamers (Nayeem et al.,
1994). A differential reactivity of the antibodies for receptor
subunits and chimeras would have resulted in discrepant subunit
ratios.
Finally, assembly studies indicating that 1 and 3 subunits can
form pentamers (and possibly significant amounts of tetrameric intermediate products) whereas 1 and 2 or 3 and 2 subunits can form dimers only with each other (Fig. 6) support a subunit stoichiometry of 1:3:2 = 2:2:1. Any other subunit
stoichiometry would have resulted in 13 complexes smaller than
tetramers and in 12 or 32 complexes larger than dimers.
Comparison with other studies on recombinant
GABAA receptors
The present results on the subunit stoichiometry of recombinant
GABAA receptors are supported partially by an
electrophysiological study (Backus et al., 1993) investigating the
degree of outward rectification of the GABA-evoked current in
recombinant 322 receptors induced by charged substitutions in
homologous positions of the putative pore-forming domain of the
individual subunits. Although this study favored a subunit
stoichiometry of two , one , and two subunits, a subunit
composition of two , two , and one subunit was only slightly
less probable. Given the possibility that the degree of the effect on
outward rectification of changing the charge might not be identical in
different subunits, as is assumed in this study (Backus et al., 1993),
the agreement of this study with the present investigation is
reasonable.
Two other electrophysiological studies, however, fully support the
present conclusions on the subunit stoichiometry of 132 receptors. Thus, Im et al. (1995), investigating ion channels formed
from GABAA receptor subunits and tandem constructs of
6-2 subunits, and Chang et al. (1996), using site-directed
mutagenesis to increase the GABA sensitivity of recombinant receptors
in proportion to the number of incorporated mutant subunits, concluded
that recombinant 622 and 122 GABAA
receptors, respectively, are pentamers composed of two subunits,
two subunits, and one subunit. The observation that several
different groups investigating distinct recombinant GABAA
receptor subtypes with different methods end up with the same subunit
stoichiometry strongly supports the conclusion that GABAA
receptors in most cases are composed of two subunits, two subunits, and one subunit.
Comparison with native GABAA receptors
This conclusion is in agreement with results obtained with native
GABAA receptors. Several investigations have indicated that two different subunits can be present in native GABAA
receptors (Duggan et al., 1991; Lüddens et al., 1991; Zezula and
Sieghart, 1991; Mertens et al., 1993; Pollard et al., 1995), whereas
only a single type of subunit was detected in these receptors
isolated by highly specific anti- antibodies, although the other subunits could be identified easily in the original extracts (Mossier
et al., 1994).
A stoichiometry of two subunits, two subunits, and one subunit, however, is in apparent contrast to experiments from Benke et
al. (1994), suggesting that native GABAA receptors may contain only a single type of subunit. This conclusion was reached from the observation that native GABAA receptors
immunoprecipitated by anti-1, anti-2, or anti-3 antibodies sum
up to ~100% of all receptors present in the brain extract. This
interpretation, however, did not take into account a possible
incomplete immunoprecipitation of receptor subtypes by the antibodies
used or a possible predominant presence of two subunits of the same
isoform in GABAA receptors.
Quirk et al. (1994) or Khan et al. (1994) observed that the sum of the
percentages of native GABAA receptors immunoprecipitated by
anti-2 and anti-3 or by anti-2S and anti-2L antibodies, respectively, was larger than that obtained by a combination of the
antibodies. In both cases the data were used to conclude that a minor
fraction of the GABAA receptors can contain more than one
type of subunit. In addition, Quirk et al. (1994), using Western
blot analysis, were able to demonstrate the presence of 2 subunits
in GABAA receptors precipitated by anti-3
antibodies, again suggesting that 2 and 3 subunits can coexist in
the same GABAA receptor. The discrepancy of these studies
with that mentioned above (Mossier et al., 1994) is not clear at
present. It should be kept in mind, however, that well characterized
and initially very specific antibodies might become unspecific in the
course of immunization and that the addition of too much antibody (for instance when receptors are precipitated simultaneously with several antibodies directed against different subunits) often decreases the
overall efficiency of immunoprecipitation. In addition, it is possible
that the stoichiometry of GABAA receptors depends on the
particular , , and subunit isoform.
Stoichiometry of recombinant 13 receptors
Via an investigation of 13 receptors incorporated into the
cell membranes, it was demonstrated that these receptors are composed
of two and three subunits. Interestingly, from the observation
that the 6-2 tandem constructs are able to form GABA-activated
channels with 6 or 2, but not with 2 subunits, Im et al.
(1995) concluded that 62 receptors are composed of three 6 and
two 2 subunits. The discrepancy between this conclusion and the
present results on the composition of 13 receptors again could
indicate that the stoichiometry of receptors depends on the
specific subunits present in these receptors. Alternatively, it might
have been attributable to changes in the structure of subunits induced
by the 10 glutamine residues linking the C-terminal of the 6 to the
N-terminal of the 2 subunit in the 6-2 tandem construct (Im
et al., 1995).
GABAA receptor assembly
The present results support previous conclusions (Connolly et al.,
1996a) that only 132 or 13 subunits, but not 12 or
32 subunits, give rise to completely assembled pentameric GABAA receptors that then are transported to the cell
surface. Whereas in cells transfected with 132 subunits no
significant accumulation of assembly intermediates was observed, in
cells transfected with 13 subunits lower oligomers of 3
subunits and possibly 13 tetramers do accumulate within the cell.
In cells transfected with 12 or 32 subunit combinations,
however, predominantly heterodimers consisting of 12 or 32
subunits are formed.
The present data are consistent with the hypothesis that the assembly
of GABAA receptors starts with the formation of 13, 12, and/or 32 dimers. In the presence of the respective
third subunit, 132 pentamers are formed efficiently. Whereas
further assembly of 12 or 32 dimers is reduced dramatically
in the absence of 3 or 1 subunits, respectively, the assembly of
13 subunits presumably can continue with reduced efficiency in
the absence of the 2 subunit, resulting in 13 tetramers and
pentamers. Because of the reduced assembly efficiency of 13
subunits, homo-oligomers of 3 subunits, which also can be formed
with low efficiency (Slany et al., 1995; Connolly et al., 1996b), might
become relatively enriched in cells transfected with 13 subunits.
Further experiments will have to confirm this hypothesis.
Arrangement of the subunits
The formation of tetramers and pentamers from 13 subunit
combinations and of dimers from 12 or 32 combinations
suggests that GABAA receptors are pentamers composed of a
total of four alternating and subunits connected by a subunit (Fig. 5A or 5B). Results obtained,
however, cannot distinguish between these two mirror image
arrangements. Cross-linking or mutagenesis experiments will have to be
performed to confirm this subunit arrangement and to decide whether it
corresponds with that shown in Figure 5A or 5B.
In any case, knowledge on the subunit stoichiometry and arrangement is
essential for further studies on the structure and function of
GABAA receptors.
FOOTNOTES
Received Dec. 10, 1996; revised Feb. 4, 1997; accepted Feb. 6, 1997.
This work was supported by Grant P9828-Med from the Austrian Science
Foundation.
Correspondence should be addressed to Dr. Werner Sieghart, Section of
Biochemical Psychiatry, University Clinic for Psychiatry, Währinger Gürtel 18-20, A-1090 Vienna, Austria.
REFERENCES
-
Anand R,
Conroy W,
Schoepfer R,
Whiting P,
Lindstrom J
(1991)
Neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes have a pentameric quaternary structure.
J Biol Chem
266:11192-11198 .
[Abstract/Free Full Text]
-
Backus KH,
Arigoni M,
Dresch
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