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The Journal of Neuroscience, December 1, 2001, 21(23):9124-9133
Alternate Use of Distinct Intersubunit Contacts Controls
GABAA Receptor Assembly and Stoichiometry
Thomas
Klausberger1, 3,
Isabella
Sarto1, 3,
Noosha
Ehya1, 3,
Karoline
Fuchs3,
Roman
Furtmüller3, 4,
Bernd
Mayer2,
Sigismund
Huck4, and
Werner
Sieghart1, 3
1 Section of Biochemical Psychiatry, University Clinic
for Psychiatry, 2 Institute for Theoretical Chemistry and
Molecular Structural Biology, and Divisions of
3 Biochemistry and Molecular Biology and
4 Cellular Physiology, Brain Research Institute, University
of Vienna, A-1090 Vienna, Austria
 |
ABSTRACT |
GABAA receptors are the major inhibitory transmitter
receptors in the CNS. Recombinant GABAA receptors
composed of 
1
3
2 subunits
have been demonstrated to assemble as pentamers consisting of two
1, two 3, and one
2 subunit. Using truncated and chimeric 1
subunits, we identified the 1(80-100) sequence as a
major binding site for 2 subunits. In addition, we
demonstrated its direct interaction with 2(91-104), a
sequence that previously has been identified to form the contact to
1 subunits. The observation that the amino acid residues
1P96 and 1H101, which can be photolabeled by [3H]flunitrazepam, are located within or
adjacent to the 1(80-100) sequence, indicates that the
benzodiazepine binding site of GABAA receptors is located
close to this intersubunit contact. The observation that
1(80-100) interacts with 2 but not with
3 subunits indicates the existence of an additional
3 binding site on 1 subunits. The
preferred alternate use of the 2 and 3
binding sites in two different 1 subunits of the same
receptor ensures the incorporation of only a single 2
subunit and thus, determines subunit stoichiometry of
132 receptors. Distinct
binding sites and their alternate use can therefore explain how
subunits of hetero-oligomeric transmembrane proteins assemble into a
defined protein complex.
Key words:
GABAA receptor; assembly; subunit interface; structure; subunit stoichiometry; benzodiazepine binding pocket
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INTRODUCTION |
Members of the ligand-gated ion
channel family, such as the nicotinic acetylcholine receptor (nAChR),
the GABAA receptor, the glycine receptor, or the
5-HT3 receptor, are heteromeric proteins composed
of five subunits (Bertrand and Changeux, 1995
). The subunits of these
proteins are cotranslationally inserted into the membrane, lumen, or
both, of the endoplasmic reticulum, after which the subunits fold and
oligomerize (Verrall and Hall, 1992; Green and Claudio, 1993; Connolly
et al., 1996a; Griffon et al., 1999). During these folding and
oligomerization events, each subunit must recognize its neighbors by
specific high-affinity interactions. To achieve the correct order of
subunits around the pore, in addition selective discriminations must be
made between different subunits. So far, little is known about the
molecular structures involved in these mechanisms.
GABAA receptors are the major inhibitory
neurotransmitter receptors in the CNS. These receptors are chloride ion
channels that can be opened by GABA (Macdonald and Olsen, 1994) and are the site of action of various pharmacologically and clinically important drugs, such as benzodiazepines, barbiturates, steroids, anesthetics, and convulsants. These drugs modulate GABA-induced chloride ion flux by interacting with separate and distinct allosteric binding sites (Sieghart, 1995).
GABAA receptors are hetero-oligomeric proteins
consisting of five subunits (Nayeem et al., 1994; Tretter et al.,
1997). 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 mammalian brain (Barnard et al., 1998; Sieghart et al.,
1999). In situ hybridization and immunocytochemical studies
indicate a distinct but overlapping temporal and regional expression of
these subunits. The finding that multiple receptor subunits are
expressed within single neurons (Fritschy et al., 1992; Pirker et al.,
2000) raises the possibility for the formation of an extremely large
variety of GABAA receptor subtypes. However, not
all receptors that can be formed theoretically are formed in the cells
(Sieghart et al., 1999). Thus, GABAA receptor heterogeneity is limited by the temporal and spatial pattern of subunit
expression and by the selective oligomerization mediated by receptor assembly.
Recently, the subunit stoichiometry and arrangement of
recombinant
132
GABAA receptors have been determined (Tretter et al., 1997). In this receptor only a single 2
subunit is present and is situated between an
1 and a 3 subunit. In
addition, the amino acid sequence 2(91-104)
was identified to form the binding site to 1
subunits (Klausberger et al., 2000).
In the present study truncated and chimeric
1 subunits were used to identify the
1 sequence mediating assembly with
2 subunits. It was demonstrated that the
sequence 1(80-100) directly interacts with
2(91-104) and forms part of the
1-2 interface. The
observation that 1(80-100) mediates assembly
with 2 but not with 3
subunits suggests the existence of an additional binding site for
3 subunits. The preferred alternate use of the
2 and 3 binding sites
in different 1 subunits of the same receptor
indicates that the 1-2 intersubunit
contact controls assembly and subunit stoichiometry of
GABAA receptors.
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MATERIALS AND METHODS |
Antibodies. The antibodies anti-peptide
1(1-9), anti-peptide
3(1-13), anti-peptide
2(319-366), and anti-peptide
2(1-33) were generated and affinity purified
as described previously (Tretter et al., 1997; Jechlinger et al., 1998;
Klausberger et al., 2000).
Generation of cDNA constructs. For the generation of
recombinant receptors, 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 (Tretter et al., 1997). Truncated subunits were
constructed by PCR amplification using the full-length subunit as a
template. The PCR 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. Chimeras were constructed using the
"gene splicing by overlap extension" technique
(Horton, 1993) and were cloned into pCDNAIAmp vectors using the
EcoRI and HindIII restriction sites of the primers.
Culture and transfection of human embryonic kidney
293 cells. Transformed human embryonic kidney (HEK 293)
cells (CRL 1573; American Type Culture Collection, Rockville, MD) were
cultured as described in Tretter et al. (1997). We transfected
3 × 106 cells with 20 µg of
subunit cDNA for single subunit transfection using the calcium
phosphate precipitation method (Chen and Okayama, 1988). After
cotransfection with two different subunits, for each subunit 10 µg of
cDNA was used. When cells were cotransfected with three different
subunits, 7 µg of cDNA was used per subunit. A total of ~20 µg of
cDNA per transfection and a cDNA ratio of 1:1:1 seemed to be optimal
for the expression of GABAA receptors under the
conditions used, as judged by receptor binding studies in cells
transfected with 1,
3, and 2 subunits.
Changing the subunit ratio by doubling the amount of a single subunit
at the cost of other subunits did not significantly change the number of [3H]Ro 15-1788 binding sites detected.
The cells were then harvested 36 hr after transfection. At this time
point the number of [3H]Ro 15-1788
binding sites formed per milligram of protein was at its maximum
for cells transfected with 1,
3, and 2 subunits. Results obtained, however, did not change when cells were harvested 34-48 hr after transfection. In addition, judged by Western blot analysis, expression levels of full-length, truncated, or chimeric subunits were comparable (see Figs. 1, 6) at all harvesting times.
Purification and immunoprecipitation of complete, truncated, and
chimeric subunits. The culture medium was removed from transfected HEK 293 cells, and cells from four culture dishes were extracted with
800 µl of a Lubrol extraction buffer (1% Lubrol PX, 0.18% phosphatidylcholine, 150 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl, pH 7.4, containing 0.3 mM PMSF, 1 mM benzamidine, and 100 µg/ml bacitracin) for 8 hr at 4°C. The extract was centrifuged for 40 min at 150,000 × g at 4°C, and the clear supernatant was incubated
overnight at 4°C under gentle shaking with 15 µg antibodies
directed against the full-length subunit. After addition of
Immunoprecipitin (Life Technologies, Gaithersburg, MD; for preparation,
see Tretter et al., 1997) and 0.5% nonfat dry milk powder and shaking
for additional 3 hr at 4°C, the precipitate was washed three times
with a low-salt buffer for immunoprecipitation (IP low buffer)
(50 mM Tris-HCl, 0.5% Triton X-100, 150 mM NaCl, and 1 mM EDTA, pH
8.0). The precipitated proteins were dissolved in sample buffer [108
mM Tris-sulfate, pH 8.2, 10 mM EDTA, 25% (w/v) glycerol, 2% SDS, and 3%
dithiothreitol]. SDS-PAGE and Western blot analysis with digoxygenized
antibodies was performed as described in Tretter et al. (1997).
All truncated or chimeric constructs used in this study could be
expressed to a comparable extent after single transfection into HEK
cells. After cotransfection of different constructs, however, the
stability of fragments that could not bind stably to each other was
reduced. This might have been caused by proteolytic degradation because
of an unstable or unproductive interaction of the fragments. In all
control experiments the extent of expression of fragments was therefore
determined in singly transfected HEK cells.
Immunoprecipitation of receptors expressed on the cell
surface. The culture medium was removed from HEK 293 cells
transfected with cDNA (21 µg per 3 × 106 cells) of GABAA
receptor subunits (cDNA ratio 1:1:1), and the cells were washed once
with PBS (in mM: 2.7 KCl, 1.5 KH2PO4, 140 NaCl, and 4.3 Na2HPO4, pH 7.3). Cells
were then detached from the culture dishes by incubating with 2.5 ml of
5 mM EDTA in PBS for 5 min at room temperature.
The resulting cell suspension was diluted in 6.5 ml of cold DMEM and
centrifuged for 5 min at 1000 × g. The pellet from two
dishes was incubated with 30 µg of 1(1-9) antibodies in 3 ml of the same medium for 30 min at 37°C. Cells were
again pelleted, and free antibodies were removed by washing twice with
10 ml of PBS buffer. Then receptors were extracted with IP low buffer
containing 1% Triton X-100 for 1 hr under gentle shaking. Cell debris
was removed by centrifugation (30 min; 150,000 × g;
4°C). After addition of Immunoprecipitin and 0.5% nonfat dry milk
powder and shaking for 3 hr at 4°C, the precipitate was centrifuged
for 10 min at 10,000 × g and washed three times with IP low buffer. The precipitated proteins were dissolved in sample buffer and subjected to SDS-PAGE and Western blot analysis using digoxygenized antibodies. Secondary antibodies (anti-digoxygenin-AP, Fab fragments; Roche Diagnostics GmbH, Mannheim, Germany) were visualized by the reaction of alkaline phosphatase with CSPD
(Tropix, Bedford, MA). Protein bands were quantified by densitometry of Kodak X-Omat S films with the Docu Gel 2000i gel documentation system
using restriction fragment length polymorphism scan software (MWG Biotech, Ebersberg, Germany). The linear range of the detection system was established by determining the antibody response to a range
of antigen concentrations after immunoblotting. The experimental conditions were designed such that immunoreactivities obtained in the
assay were within this linear range, thus permitting a direct
comparison of the amount of antigen applied per gel lane between
samples. Different exposures of the same membrane were used to ensure
that the measured signal was in the linear range of the x-ray film.
To verify that only receptors on the cell surface were labeled by the
antibodies, parallel samples were incubated with antibodies directed
against the intracellular loop of GABAA receptor
subunits (data not shown). These antibodies could not precipitate any
GABAA receptor subunits under the conditions
used. A possible redistribution of the antibodies during the extraction
procedure could be excluded by an experiment performed analogous to
that described in Klausberger et al. (2000).
Immunofluorescence. HEK cells were fixed with 2%
paraformaldehyde in PBS 30-35 hr after transfection, followed by a 10 min wash in 50 mM NH4Cl in
PBS. Washes between incubation steps were performed in PBS. For
detection of intracellular receptors, cells were permeabilized with
0.1% Triton X-100 for 5 min. Blocking was performed in 5% bovine
serum albumin (BSA) in PBS for 10 min, followed by an incubation with
primary antibody in 1% BSA in PBS. Primary antibodies were detected
with goat anti-rabbit IgG(H+L) bodipy FL
(Molecular Probes, Eugene, OR) in 1% BSA in PBS. Labeling was
visualized using a Zeiss Axiovert 135 M microscope attached to a
confocal laser system (Carl Zeiss LSM 410, BRD), equipped with an argon
laser and a helium-neon laser and suitable filter sets. To verify that
labeling of cells without permeabilization was restricted to the cell
surface, parallel samples were stained with antibodies directed against
the intracellular loop of GABAA receptor subunits
(data not shown). These antibodies detected GABAA
receptor subunits only after permeabilization of transfected cells.
Electrophysiological investigations. HEK cells were
cotransfected with GABAA receptor subunits
together with pEGFP-N1 (Clontech, Palo Alto, CA) as a transfection
marker. Electrophysiologic recordings were performed at room
temperature 1-2 d after transfection using the perforated patch
technique (Rae et al., 1991). GABA and ZnCl2 were
applied using a DAD-12 superfusion system (Adams and List Associates
Ltd., Westbury, NY). Extracellular solution contained (in
mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 5 glucose,
and 10 HEPES, pH 7.4. The pipette solution contained (in
mM): 140 KCl, 11 EGTA, 1 CaCl2, 1 MgCl2, 10 HEPES,
and 0.2 amphotericin B, pH 7.2. The cells were clamped at 
60 mV, and
currents were filtered at 1 kHz, recorded with an Axopatch 200A
amplifier (Axon Instruments, Foster City, CA) and analyzed with
Clampfit software (Axon Instruments).
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RESULTS |
Truncated 1 constructs are able to assemble with
full-length 2 subunits
In the present study C-terminally truncated
1 subunits (Fig.
1A) were cloned, and it
was investigated which of these fragments could assemble with
full-length 2 subunits. For this, HEK cells were cotransfected with 2 subunits and either
full-length or truncated 1 subunits. Expressed
subunits were extracted from these cells and were immunoprecipitated
with 2(319-366) antibodies. The precipitate
was subjected to SDS-PAGE and Western blot analysis using digoxygenized
1(1-9) antibodies. As shown in Figure
1B, full-length 1 subunits
(protein band of 51 kDa), as well as the fragments
1(1-249) (two bands of 39 and 41 kDa),
1(1-221) (three bands of 26, 28, 30 kDa),
1(1-134) (three bands of 17, 19, 21 kDa), and
1(1-117) [three bands of 14, 16 (very weak),
and 18 kDa] could be coimmunoprecipitated with full-length
2 subunits from appropriately transfected HEK
cells. Because all 1 fragments investigated
contained two glycosylation sites, the three bands presumably
represented unglycosylated, partially, and fully glycosylated fragments. The observation that only one or two protein bands could be
observed for the full-length 1 subunit or the
1(1-249) construct might indicate that these
subunits predominantly occur in the double-glycosylated or double- and
mono-glycosylated state, respectively. This conclusion is supported by
the apparent molecular mass of these proteins that amounted to 51 kDa
or 41 kDa for the full-length 1 subunit or the
1(1-249) construct, respectively, although
the ungycosylated mass of these proteins can be calculated to be 47 or
37 kDa. Alternatively, the differentially glycosylated protein bands
with higher molecular mass might not have been resolved by the 15%
polyacrylamide gel used in this investigation.
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Figure 1.
Coimmunoprecipitation of truncated
1 with full-length 2 subunits.
A, Schematic drawing of the 1 subunit and
of C-terminally truncated 1 constructs. The
1 subunit consists of the N-terminal extracellular
domain with the typical cysteine loop, of four transmembrane domains
(TM1-4), and the large cytoplasmic loop between TM3 and TM4. The
sequences of the C-terminally truncated 1 constructs are
indicated by the amino acid numbers given in
parentheses. A 1 represents the first
amino acid of the mature subunit. B, HEK cells were
transfected with truncated 1 constructs together with
full-length 2 subunits, as indicated. Cell extracts were
immunoprecipitated with 2(319-366) antibodies.
1 fragments coprecipitated were identified by SDS-PAGE
and Western blot analysis using digoxygenized 1(1-9)
antibodies (lanes 1-6). In control experiments
(lanes 7, 8), truncated 1(1-117) and
1(1-100) constructs were transfected separately into
HEK cells, and the fragments formed were precipitated with
1(1-9) antibodies and subjected to SDS-PAGE and Western
blot analysis using digoxygenized 1(1-9) antibodies.
The protein fragments formed from these constructs [apparent molecular
mass: 14, 16, and 18 kDa for 1(1-117) and apparent
molecular mass: 12 and 14 kDa for 1(1-100)] were
expressed to a similar extent. Interestingly, the relative abundance of
the unglycosylated, monoglycosylated, and diglycosylated
1(1-117) fragments differed when these fragments were
expressed in the absence or presence of 2 subunits,
possibly suggesting that 2 subunits preferentially
assemble with fully glycosylated 1(1-117) fragments.
All experiments were performed three times with comparable
results.
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Binding between 2 subunits and these fragments
seemed to be the result of a specific assembly process because after
cotransfection of HEK cells with full-length 2
subunits and the fragment 1(1-221), high-affinity binding sites for the benzodiazepine
[3H]Ro15-1788 were formed. These sites
are assumed to be located at the interface of
1 and 2 subunits in
GABAA receptors (Sigel and Buhr, 1997). The
number of [3H]Ro15-1788 binding sites
formed (16.8 ± 2.3 fmol/mg protein) was comparable with that
observed after transfection of HEK cells with full-length
1 and 2 subunits
(17.6 ± 1.2 fmol/mg protein) but was significantly smaller
(p < 0.001; unpaired Student's t test) than that of HEK cells transfected with
1, 3, and
2 subunits in parallel experiments (874 ± 19 fmol/mg of protein). Data given are mean values ± SEM from
three different experiments performed in triplicate.
In contrast to the protein fragments formed from
1(1-117), the fragments formed from
1(1-100) could not be coprecipitated with
2 subunits from appropriately cotransfected
HEK cells (Fig. 1B), although the extent of
expression of these fragments (12 and 14 kDa) was similar to that of
the fragments derived from the 1(1-117)
construct (Fig. 1B, lanes 7, 8). The inability of 2(319-366)
antibodies to coprecipitate the fragment
1(1-100) confirmed previous conclusions
(Jechlinger et al., 1998) that these antibodies did not cross-react
with 1 subunits. These results indicate that
the amino acid sequence of the 1 subunit that
is responsible for binding to 2 subunits is
located in the N-terminal 117 amino acids of the
1 subunit.
Amino acid sequence 1(80-100) mediates binding to
2 subunits
To identify this contact site, it was investigated which
1 amino acid sequence could induce binding to
2 subunits after incorporation into a fragment
that originally could not bind to these subunits. The fragment
3(1-115) seemed to be suitable for this
purpose because it is homologous to 1(1-117)
but could not be coprecipitated with 2
subunits (or 3 subunits) after coexpression in HEK cells (Fig.
2). To incorporate binding sites of the
1 subunit, several chimeras were constructed
by replacing the C-terminal part of the
3(1-115) fragment with the corresponding
1 sequences (Fig. 2). These chimeras were
transfected into HEK cells together with full-length
2 subunits. Expressed subunits were
precipitated from cell extracts with
2(319-366) antibodies. The precipitate was
subjected to SDS-PAGE, and the proteins were detected with digoxygenized 3(1-13) antibodies in Western
blots. The actual expression of the chimeras was confirmed by
precipitation and detection with 3(1-13)
antibodies (data not shown).
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Figure 2.
1(80-100) forms the contact site
to 2 subunits. C-terminal sequences of the fragments
1(1-117), 3(1-115), and of different
chimeras are shown. Amino acid sequences of the 1
subunit are boxed. HEK cells were cotransfected with
these constructs together with 2 subunits, and a
possible coimmunoprecipitation was investigated, as described in
Results. + indicates binding, and indicates absence of
binding between these constructs and full-length 2
subunits. The experiments were performed three times with similar
results.
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In
3(1-115)chim1(101-117)
the 17 C-terminal amino acids of the 3(1-115)
fragment were replaced by amino acids 101-117 of the
1 subunit. As indicated in Figure 2, this
chimera could not be coprecipitated with full-length
2 subunits from appropriately cotransfected
HEK cells, demonstrating the specificity of the 2(319-366) antibodies used and indicating
that amino acids 1(101-117) are not able to
induce binding to 2 subunits. In
3(1-115)chim1(80-117), the amino acid sequence 3(78-115) was
replaced by 1(80-117). This construct was
able to bind to full-length 2 subunits (Fig. 2), but not to full-length 3 subunits (data
not shown). Because amino acids 1(101-117)
were not sufficient to induce binding to 2
subunits as discussed above, this indicated that amino acids 80-100 of
the 1 subunit are important for binding to
2 subunits. To directly confirm this
conclusion, the construct
3(1-115)chim1(80-100) was generated (Fig. 2), in which amino acids
1(80-100) were incorporated into
3(1-115), replacing amino acids
3(78-98). As expected, this chimera was able
to bind to 2 subunits.
To investigate which part of the 1(80-100)
sequence is responsible for binding to 2
subunits, four additional chimeras were constructed. In
3(1-115)chim1(80-86),
amino acids 3(78-84) were replaced by the
amino acids 1(80-86), in
3(1-115)chim1(87-93) the sequence 3(85-91) was replaced by
1(87-93), in
3(1-115)chim1(94-100) the sequence 3(92-98) was replaced by amino
acids 1(94-100), and in
3(1-115)chim1(80-93)
the sequence 3(78-91) was replaced by
1(80-93) in the
3(1-115) fragment. None of these chimeras was
able to bind to 2 subunits. These results
indicate that the whole 1(80-100) sequence is
necessary for binding to 2 subunits.
The sequence 1(80-100) is important for the
assembly of GABAA receptors composed of
132 subunits
To investigate the importance of the
1(80-100) sequence not only for the assembly
of truncated subunits and dimers, but also for assembly of full-length
subunits and pentameric receptors, a full-length
1 chimera (1*) was
constructed in which the sequence 1(79-100)
was replaced by the sequence 3(77-98). The
additional exchange of the amino acid 79 of the
1 subunit in 1* was
necessary to avoid the generation of two adjacent prolines that could
have destroyed the conformation of the resulting chimera (Fig. 2). In
control experiments, it was demonstrated that the extent of expression
of the 1* chimera was similar to that of the
1 subunit in HEK cells (data not shown).
HEK cells were then cotransfected with 1*,
3, and 2 subunits and
subunits expressed were investigated by immunofluorescence and confocal
laser microscopy. As shown in Figure 3,
1* (Fig. 3A) and
3 subunits (Fig. 3B) could be
easily detected on the surface of intact cells, but for the
2 subunit only a weak labeling was observed
(Fig. 3C), although the labeling of the
2 subunit in permeabilized cells (Fig.
3F) was comparable with that of
1 (Fig. 3D) and
3 (Fig. 3E) subunits. In HEK cells
cotransfected with 1,
3, and 2 subunits,
all three subunits could be detected on the cell surface (Fig.
3G-I). Because previous results have indicated that
1 subunits alone in contrast to
13 subunit
combinations do not form receptors that are incorporated into the
plasma membrane to a significant extent, these results suggested that
1* predominantly formed receptors with
3 subunits that are expressed on the cell surface, but the ability to form receptors containing
2 subunits was significantly reduced.
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Figure 3.
Immunofluorescence of HEK cells cotransfected with
GABAA receptor subunits. HEK cells were cotransfected with
1*, 3, and 2
subunits (A-F) or with 1,
3, and 2 subunits
(G-I). Immunofluorescence was performed using
1(1-9) antibodies (A, D, G),
3(1-13) antibodies (B, E, H), or
2(1-33) antibodies (C, F, I) on
the cell surface (A-C, G-I) or in permeabilized cells
(D-F) by confocal laser microscopy (single sections).
Scale bar, 10 µm. The experiment was performed four times with
similar results.
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To quantify this phenomenon, HEK cells were cotransfected with
1, 3, and
2 subunits or with
1*, 3, and
2 subunits. GABAA receptors expressed on the surface of the cells were labeled by an
incubation of intact cells with 1(1-9)
antibodies. Antibody labeled receptors were extracted and precipitated
by addition of Immunoprecipitin. The precipitate was subjected to
SDS-PAGE and Western blot analysis using digoxygenized
1(1-9) antibodies (Fig.
4). In contrast to
1 subunits (51 kDa, the weak 46 kDa band
presumably represents a degradation product), the protein band of
1* exhibited an apparent molecular mass of 53 kDa because of an additional glycosylated asparagine at position 80 of
the newly introduced 3 subunit insert. The
protein bands were quantified, and results obtained indicated that
1* and 1 subunits
were expressed to a similar extent on the surface of transfected cells.
Then, the Western blot was stripped and analyzed using digoxygenized 3(1-13) antibodies (Fig. 4). Finally, blots
were again stripped and were probed with
2(1-33) antibodies. Whereas similar amounts of 3 subunits (54 kDa) were coprecipitated
with 1 subunits from 132
or
1*32
transfected cells, the amount of 2 subunits (49 kDa) coprecipitated with 1* subunits was
only 32 ± 3% (mean ± SEM, n = 3; from
three different transfections) of that coprecipitated with
1 subunits. Similar results were obtained when
the order of detection of subunits was changed and Western blots were
first probed with 2(1-33) antibodies and
after stripping were re-analyzed with 1(1-9)
or 3(1-13) antibodies. These results indicate
that 1* was able to form receptors with
3 subunits, but that the ability to form
receptors containing 2 subunits was reduced by 68%.
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Figure 4.
Western blot analysis of GABAA
receptors labeled on the surface of HEK cells. HEK cells were
cotransfected with 1, 3,
and 2 subunits or with 1*,
3, and 2 subunits.
GABAA receptors expressed on the cell surface were
immunolabeled by adding 1(1-9) antibodies to intact
cells, and were then extracted, immunoprecipitated, and analyzed by
SDS-PAGE and Western blots using digoxygenized 1(1-9),
3(1-13), or 2(1-33) antibodies.
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Properties of GABAA receptors composed of
1*32 or
1*3 subunits
To investigate the properties of the receptors formed, HEK cells
cotransfected with 1*,
3, and 2 subunits
were subjected to patch-clamp analysis, and whole-cell recordings were
compared with those from cells transfected with
1, 3, and
2 subunits. GABA exhibited an apparent
EC50 of 68 ± 10 µM (mean ± SEM; n = 11 cells from different plates; total of
four transfections) (Fig. 5E)
in HEK cells transfected with 1*,
3, and 2 subunits and elicited a maximal current of 713 ± 170 pA at a GABA
concentration of 1000 µM (Fig. 5A).
In contrast, GABA exhibited an EC50 of 7.7 ± 2.3 µM (mean ± SEM; n = 8 cells from different plates; total of four transfections) (Fig.
5E) in HEK cells transfected with 1, 3, and
2 subunits and elicited a maximal current of
2988 ± 469 pA at a concentration of 300 µM (Fig. 5B). These data not only
indicated that GABA exhibited a 10-fold reduced potency for activating
1*32
receptors, but also that the maximal current of cells transfected with
1*32
subunits was only ~24% of that transfected with
132
subunits.
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Figure 5.
Functional properties of GABAA
receptors containing the 1* subunit.
A D, Whole-cell recordings from HEK cells cotransfected
with 1*, 3, and
2, or 1,
3, and 2, 1*
and 3, or 1 and 3
subunits after application of GABA concentrations producing maximal
currents. Shown are single experiments that were reproduced with
similar results 8-11 times in different cells. E,
Relative currents induced by various GABA concentrations in cells
transfected with subunit combinations as indicated. Data shown are mean
values ± SEM of relative currents from 8-11 individual
dose-response curves obtained from different cells derived from a
total of four transfections. F, Bar graph showing the
effect of 100 µM Zn2+ on currents
activated by 100 µM GABA. HEK cells were transfected, as
indicated. Height of bars indicates fraction of control current
remaining in the presence of Zn2+, measured 1.5 sec
after application was started; error bars indicate ± SEM
(n = 6-8).
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Because surface expression studies indicated a significant formation of
1*3 receptors in
1*32
transfected cells, the properties of receptors in
1*3 transfected cells
were also investigated. Although homo-oligomeric receptors composed of
3 subunits could also have been formed under
the conditions used, they would not have contributed to the GABA evoked
current because these receptors apparently are not gated by GABA
(Connolly et al., 1996b). Using various GABA concentrations, it was
demonstrated that GABA exhibited an EC50 of
10.5 ± 2.1 µM (mean ± SEM; n = 8 cells from different plates; total of four transfections) (Fig.
5E) in HEK cells transfected with
1* and 3 subunits and
elicited a maximal current of 270 ± 63 pA at a concentration of
300 µM (Fig. 5C). In contrast, GABA exhibited an EC50 of 3.0 ± 1.2 µM (mean ± SEM; n = 9 cells from different plates; total of three transfections) (Fig.
5E) in cells transfected with 1 and
3 subunits and elicited a maximal current of
426 ± 146 pA at a concentration of 100 µM
(Fig. 5D). These data supported the conclusion that the
1* construct was able to form functional
receptors with 3 subunits. The potency of GABA
for activating 1*3
receptors, however, was significantly (p < 0.05; Student's t test) reduced compared with receptors
composed of 13
subunits. Similarly, the maximal currents elicited by GABA in
1*3 transfected cells
were significantly smaller than those in
13 transfected cells
(p < 0.05).
Although 1*3
receptors significantly contribute to receptors formed in
1*32
transfected HEK cells, as indicated by surface expression studies,
because of the low maximum currents observed in
1*3 receptors (Fig.
5C), these receptors overall have a comparatively small
contribution to currents elicited in
1*32
transfected cells (Fig. 5A) that is apparent only as a
slightly increased range of GABA concentrations that are able to elicit
currents in these cells (Fig. 5E). Thus, most of the current
elicited in the cells investigated was produced by
1*32
receptors. The low apparent potency of GABA to activate currents in
these cells as well as the increased dose range of GABA for stimulation
of currents clearly indicated the formation of
1*32
receptors in addition to
1*3 receptors.
This conclusion was supported by investigating the effects of 100 µM Zn2+ on whole-cell
currents stimulated by 100 µM GABA. In agreement with
previous results (Draguhn et al., 1990; Gingrich and Burkat, 1998),
currents mediated by the wild-type
132
receptors were only weakly reduced (86 ± 4% of control;
n = 6; total of four transfections), whereas currents
mediated by 13
receptor were reduced to 7 ± 3% (n = 6; total of
three transfections) (Fig. 5F) in the presence of
Zn2+. For HEK cells transfected with
1*, 3, and
2 subunits, currents mediated by 100 µM GABA were reduced to 50 ± 4%
(n = 7; total of four transfections), and for cells
transfected with 1* and 3 subunits, GABA-mediated currents were
reduced to 8 ± 2% (n = 8; total of four
transfections) in the presence of 100 µM
Zn2+ (Fig. 5F). Because
13 and
1*3 receptors exhibit
a comparable Zn2+ sensitivity, it is
reasonable to assume that the Zn2+
sensitivity of
1*32
and
132
receptors was also comparable. The 36% increase in
Zn2+ sensitivity of
1*32-transfected
cells therefore indicated that ~40% of the
1*32
current was mediated by the additionally formed
1*3 receptors.
Combined with the observation that the main conductance level of
receptors (15-18 pS) is only half of that of receptors
(~30 pS; Hevers and Lüddens, 1998), and assuming that the same
holds true for 1*3
and
1*32
receptors, a ratio of
1*3:1*32
receptors of 80:60 can be calculated, indicating that
1*32
receptors represented 43% of receptors formed in these cells. Given
the many assumptions that had to be made in the course of this
calculation, this percentage is in good agreement with the data from
the immunoprecipitation experiments shown in Figure 4.
Amino acid sequence 1(80-100) binds to the
2(91-104) sequence
Recently it has been demonstrated that incorporation of the amino
acid sequence 2(91-104) into the fragment
1(1-100), that per se could not bind to
1 subunits, resulted in the chimera 1(1-100)chim2(91-104)
that was able to bind to 1 subunits. From this
it was concluded that the amino acid sequence
2(91-104) forms the contact site to
1 subunits (Klausberger et al., 2000). It
therefore seemed interesting to investigate whether the
2(91-104) sequence directly interacts with
the 1(80-100) sequence.
To clarify this question, it first was investigated whether the
1(1-100) fragment and the fragment
3(1-115), which was used to identify the
1(80-100) contact site (Fig. 2), could bind
to each other. For this, fragments 3(1-115)
and 1(1-100) were cotransfected into HEK
cells. The extract of HEK cells expressing
3(1-115) and
1(1-100) fragments was then
immunoprecipitated with 3(1-13) antibodies,
and the precipitate was subjected to SDS-PAGE and Western blot analysis
using digoxygenized 1(1-9) antibodies. As
shown in Figure 6, A and
B, 1(1-100) fragments were not
coprecipitated by 3(1-13) antibodies,
confirming the absence of cross-reactivity of these antibodies with the
1(1-100) fragments and indicating that
3(1-115) could not bind to
1(1-100) fragments. Similarly, the construct
3(1-115)chim1(80-100),
which contains the putative binding site for 2
subunits, was unable to bind to 1(1-100) fragments after cotransfection into HEK cells (Fig.
6A,B). In the reverse experiment, the construct
1(1-100)chim2(91-104), containing the binding site for 1 subunits,
could also not bind to the 3(1-115) fragment.
Only when the 1(80-100) sequence was incorporated into the 3(1-115) fragment and
the 2(91-104) sequence was incorporated into
the 1(1-100) fragment, the resulting chimeras could bind to each other (Fig. 6A,B). These results
indicate that the 1(80-100) and the
2(91-104) sequences can directly bind to each
other.
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|
Figure 6.
Amino acid sequence 1(80-100)
directly binds to 2(91-104). A, B, HEK
cells were cotransfected with the constructs as indicated. Cell
extracts were immunoprecipitated with 3(1-13)
antibodies, and the precipitate was subjected to SDS-PAGE and Western
Blot analysis using digoxygenized 1(1-9) antibodies.
A, Schematic representation of the experiment. + indicates binding, and indicates absence of binding between the
cotransfected constructs. B, Western blots demonstrating
binding between constructs under condition
"4". C, Western blots
demonstrating the extent of expression of the indicated constructs on
single transfection into HEK cells. All experiments were performed
three times with similar results.
|
|
In control experiments (Fig. 6C) it was demonstrated that
each of the constructs used in this experiment was expressed to a
similar extent after single transfection into HEK cells. Constructs 1(1-100) and
1(1-100)chim2(91-104)
each contained a single glycosylation site and thus, gave rise to two
fragments: a weakly labeled of 12 kDa and a strongly labeled of 14 kDa.
Construct 3(1-115) contained two
glycosylation sites and thus, formed three fragments, two strongly
labeled of 16 and 18 kDa and a weakly labeled fragment of 14 kDa.
Construct
3(1-115)chim1(80-100) contained only one glycosylation site and formed a strongly labeled protein band of 14 and weakly labeled band of 16 kDa.
Interestingly, predominantly the unglycosylated
1(1-100)chim2(91-104)
fragment of 12 kDa seemed to assemble with
3(1-115)chim1(80-100) on cotransfection of these fragments into HEK cells, although the
glycosylated fragment of 14 kDa was the predominant one expressed after
single transfection of HEK cells (Figs. 1B,
6C). This suggests that assembly of subunit fragments
already starts when subunits are not fully glycosylated. This
conclusion is supported by previous observations (Klausberger et al.,
2000, 2001) as well as by observations with other constructs (Fig.
1B).
| |
DISCUSSION |
Amino acid sequence 1(80-100) forms the binding
site to 2 but not to 3 subunits
The present study demonstrated that the N-terminal extracellular
domain of the 1 subunit
[1(1-221)] could bind to full-length 2 subunits after coexpression in HEK cells, as
indicated by coimmunoprecipitation with subunit-specific antibodies.
Binding between 1(1-221) and 2 subunits represented a specific assembly
process, as indicated by the formation of specific
[3H]Ro15-1788 binding sites that are
assumed to be formed on the interface of 1 and
2 subunits of GABAA
receptors. These results are consistent with previous studies
indicating that N-terminal sequences of GABAA
receptor (Hackam et al., 1997; Klausberger et al., 2000) or
K+ channel (Shen et al., 1993) subunits
can assemble with full-length subunits.
A subsequent reduction in the size of the truncated subunit indicated
that the 1(1-117), but not the
1(1-100) construct was still able to bind to
2 subunits. The respective binding site was
then identified by incorporating various 1
sequences into the 3(1-115) fragment. This
fragment is homologous to 1(1-117) but in
contrast to the latter construct could not bind to
2 subunits after coexpression in HEK cells.
The incorporation of the sequence 1(80-100)
into the 3(1-115) fragment was sufficient to
induce binding to 2 but not to
3 subunits, suggesting that the
1 binding sites for 2
and 3 subunits are different.
The observation that the 1(1-100) fragment
was unable to bind to 2 subunits although it
contained the 1(80-100) sequence is
consistent with previous results indicating that
2(1-113) was the smallest fragment that could
bind to 1 subunits, although the respective
binding site was identified to be formed by the 2(91-104) sequence (Klausberger et al.,
2000). The additional length of the fragments presumably is required
for stabilizing the conformation of the actual binding sites located in
a more N-terminal position.
In other experiments a chimeric 1 subunit
(1*) was constructed in which the
1(79-100) sequence was replaced by the
homologous 3(77-98) sequence. Chimera
1* was then coexpressed with
3 and 2 subunits in
HEK cells. Confocal immunofluorescence microscopy, whole-cell
patch-clamp experiments, as well as immunolabeling and quantification
of receptors on the cell surface indicated a 60-70% reduction in
receptors containing 1*,
3, and 2 subunits, although the level of expression of 1*
subunits and its extent of assembly with 3
subunits was unimpaired. These results confirmed the importance of the
1(80-100) sequence for assembly with
2 but not with 3
subunits. The remaining formation of
1*32
receptors can be explained by the existence of additional binding sites between 1 and 2
subunits that partially can compensate for the absence of the
1(80-100) sequence in
1* subunits.
Amino acid sequences 1(80-100) and
2(91-104) form part of the - interface and are
located close to the benzodiazepine binding site of GABAA
receptors
Recently it was demonstrated that the sequence
2(91-104) forms the contact site to
1 subunits (Klausberger et al., 2000). To
investigate whether the sequences 1(80-100)
and 2(91-104) directly interact with each
other, these sequences were incorporated into
GABAA receptor fragments
3(1-115) and
1(1-100), respectively, which could not bind
to each other. The observation that 1(80-100) had to be incorporated into
TOP
ABSTRACT
INTRODUCTION
