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Volume 17, Number 17,
Issue of September 1, 1997
pp. 6587-6596
Copyright ©1997 Society for Neuroscience
Assembly of GABAA Receptors Composed of 
1 and 
2
Subunits in Both Cultured Neurons and Fibroblasts
George H. Gorrie1,
Yvonne Vallis1,
Anne Stephenson3,
Jonathan Whitfield2,
Brenda Browning1,
Trevor G. Smart3, and
Stephen J. Moss1
1 Medical Research Council Laboratory of Molecular Cell
Biology and Department of Pharmacology and 2 The Eisai
Research Labs, University College, London WC1E 6BT, United Kingdom, and
3 The School of Pharmacy, London WC1N 1AX, United Kingdom
ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
GABAA receptors are believed to be pentameric
hetero-oligomers, which can be constructed from six subunits (
, 
,
, 
, 
, and 
) with multiple members, generating a large
potential for receptor heterogeneity. The mechanisms used by neurons to
control the assembly of these receptors, however, remain unresolved.
Using Semliki Forest virus expression we have analyzed the assembly of
9E10 epitope-tagged receptors comprising 1 and 2 subunits in baby
hamster kidney cells and cultured superior cervical ganglia neurons.
Homomeric subunits were retained within the endoplasmic reticulum,
whereas heteromeric receptors were able to access the cell surface in
both cell types. Sucrose density gradient fractionation demonstrated
that the homomeric subunits were incapable of oligomerization, exhibiting 5 S sedimentation coefficients. Pulse-chase analysis revealed that homomers were degraded, with half-lives of ~2 hr for
both the 1(9E10) and
2(9E10) subunits. Oligomerization of the
1(9E10) and 2(9E10)
subunits was evident, as demonstrated by the formation of a stable 9 S
complex, but this process seemed inefficient. Interestingly the
appearance of cell surface receptors was slow, lagging up to 6 hr after
the formation of the 9 S receptor complex. Using metabolic labeling a
ratio of 1(9E10):2(9E10) of
1:1 was found in this 9 S fraction. Together the results suggest that
GABAA receptor assembly occurs by similar mechanisms in
both cell types, with retention in the endoplasmic reticulum featuring as a major control mechanism to prevent unassembled receptor subunits accessing the cell surface.
Key words:
Semliki Forest virus;
GABAA receptor;
density
gradients;
cultured neurons;
assembly;
endoplasmic reticulum
INTRODUCTION
GABAA receptors are the
major sites of fast inhibitory neurotransmission within the brain.
These receptors are believed to be hetero-oligomers constructed from
six classes of homologous subunits: (1-6), (1-4), (1-3),
, , and (1-3) (Macdonald and Olsen, 1994
; Davies et al.,
1997), generating a considerable potential for receptor heterogeneity.
Even within single neurons, there is likely to be heterogeneity of
structure, because many neurons often express multiple numbers of
receptor subunits (Wisden and Seeburg, 1992; Fritschy and Mohler,
1995). Therefore, to determine the true extent of GABAA
receptor heterogeneity, it is important to understand the mechanisms
that govern the assembly of these receptors. Selective oligomerization
and/or selective transport of defined subunit combinations to the cell
surface (Connolly et al., 1996a) may provide important mechanisms for
controlling receptor diversity.
To address these questions, the assembly of recombinant receptors has
been analyzed. These studies have focused on the minimal structural
requirement for the production of GABAA receptors
displaying the full physiological and pharmacological properties of
neuronal receptors. Parallel electrophysiological, biochemical, and
morphological studies have demonstrated that homomeric expression does
not result in surface expression for the 1, 2 or 2L subunits
in Xenopus oocytes, human embryonic kidney (A293) cells, or
Madin Darby canine kidney (MDCK) cells (Connolly et al., 1996a,b). In
common with the combinations 12L and 22L these homomeric
subunits are retained in the endoplasmic reticulum (ER) (Connolly et
al., 1996a,b). Expression of receptors composed of and subunits
produces GABA-gated chloride channels, but the co-expression of the
2 or 3 subunits is essential for modulation by benzodiazepines (Macdonald and Olsen 1994).
To explore receptor assembly further, we have developed Semliki
Forest viruses (Liljestrom and Garoff, 1991) that express receptor
subunits modified with the 9E10 epitope (Connolly et al., 1996a,b).
This expression system allows high levels of protein production in a
broad range of host cells, including neurons (Liljestrom and Garoff,
1991; deHoop et al., 1995). Here, we use this system to study the
assembly of GABAA receptors composed of
1(9E10) and 2(9E10)
subunits in both baby hamster kidney (BHK) cells and cultured superior
cervical ganglia (SCG) neurons.
MATERIALS AND METHODS
Construction and production of Semliki Forest viruses
expressing the 1(9E10) and
2(9E10) GABAA receptor subunits.
The murine 1(9E10) and
2(9E10) subunits modified with the 9E10 epitope
EDKLISEEDL, between amino acids 4 and 5 of the mature protein, have
been described previously (Connolly et al., 1996a,b). Addition of this
epitope has been demonstrated to be functionally silent with regard to
both receptor pharmacology and physiology (Connolly et al., 1996a,b).
These cDNAs were cloned as BamHI fragments into the vector
pSFV1 (Liljestrom and Garoff, 1991) using standard recombinant methods.
The respective plasmids were linearized with Nru1, and cRNA
was synthesized using SP6 polymerase. cRNA was synthesized from
SpeI linearized Helper I plasmid (Liljestrom and Garoff,
1991) also using SP6 polymerase. All manipulations were performed using
standard recombinant methods (Sambrook et al., 1989). To produce viral
stocks encoding the 1 and 2 subunits, cRNA transcribed from
receptor constructs and the helper plasmid were electroporated into BHK
cells at a 1:1 ratio. The supernatants from the cells were collected
after 2 d and frozen in 100 µl aliquots. Titers for
GABAA receptor subunit-expressing viruses were determined
by serial dilution on BHK cells. Expression was determined by
immunofluorescence as described below. The
1(9E10) and 2(9E10) viruses
had titers of 7 × 107-1 × 108 particles/ml.
Cell culture. BHK cells were maintained in BHK 21 medium
(Life Technologies) supplemented with 5% FCS, 20 mM HEPES,
pH 7.2, tryptose phosphate broth, penicillin, and streptomycin.
Cultured SCG neurons were prepared and maintained as described
previously (Krishek et al., 1994).
Infection of BHK cells and cultured SCG neurons. Infection
of BHK cells or neurons was conducted by diluting virus into binding medium (RPMI 1640 medium, pH 6.8, 20 mM HEPES, pH 6.8, and
20 mM BSA), and incubating the cells in this solution for 1 hr at 37°C. Normal culture medium was then reapplied after this
incubation. The 1(9E10) and
2(9E10) virus particles were used at
concentrations of ~10 infectious units per cell.
Immunocytochemistry. Immunofluorescence was performed as
described by Connolly et al., (1996a,b). BHK cells and neurons were fixed in 3% parafomaldehyde in PBS for 5 min and then quenched twice
with 50 mM glycine for 10 min. Cells were then blocked
using 1% BSA and 10% horse serum in PBS. The primary antibodies were applied for 1 hr at the following concentrations: BD17 (anti-2/3; Boehringer), 14 µg/ml; 9E10, 5 µg/ml; or a polyclonal antibody raised against a synthetic peptide corresponding to the C terminus of
the 1 subunit (amino acids 413-429) (Pollard et al., 1993) at 5 µg/ml. Secondary antibodies (Pierce, Rockford, IL), either fluorescein- or rhodamine-linked anti-mouse and anti-rabbit IgG, were
then applied as appropriate for 45 min in blocking solution. Permeabilization of samples was conducted by adding NP-40 to the blocking solution (0.05%). Fluorescence images were analyzed by confocal microscopy (Medical Research Council 1000). Images from SCG
neurons were generated by taking an optical Z section
through the cell at 1 µm intervals and then recombining the
images.
Quantification of fluorescence by confocal microscopy. The
level of surface membrane 9E10 staining on infected cells was
quantified using confocal microscopy as described by Bogler et al.
(1993) and Entwistle and Noble (1994). BHK cells were fixed at
differing time points after infection. Surface receptor populations
were then detected simultaneously via fluorescence using 9E10
antibodies without permeabilization. Individual cells at each time
point were selected at random, and the area command was used to collect brightness readings of the plasma membrane, using identical iris and
gain settings. The average fluorescence intensities were converted to
numerical readings of arbitrary values (pixels). This was performed on
at least five cells for each time point.
Sucrose density gradient fractionation. Receptor subunits
were subjected to sucrose density gradient fractionation using 5 and
20% linear sucrose density gradients in lysis buffer (Millar et al.,
1995). Before loading, solubilized cell extracts were clarified by
centrifugation (100,000 × g for 10 min). Gradients were calibrated via the inclusion of marker proteins (1 mg/ml) of known
sedimentation coefficients: BSA, 4.3 S; aldolase, 7.4 S; and catalase,
11.2 S. Gradients were run in a Beckman SW 55ti rotor at 40,000 rpm for
14.4 hr at 4°C. The gradients were then fractionated (14 350 µl
fractions), and receptor localization was analyzed by
immunoprecipitation or Western blotting.
Metabolic labeling and immunoprecipitation of GABAA
receptor subunits. For metabolic labeling studies infected BHK
cells were starved in methionine-free media for 30 min before labeling
with [35S]methionine (ICN/Flow) at 200 µCi/ml
for differing periods. Where appropriate, labeled cultures were chased
at 37°C with complete growth media. Total protein synthesis was
analyzed by SDS-PAGE, after cell lysis in SDS sample buffer. Receptor
subunits were isolated from cell extracts or sucrose density gradient
fractions via immunoprecipitation as described previously (Moss et al., 1995; Connolly et al., 1996b). Cells were lysed in a buffer containing 2% NP-40, 5 mM EDTA, 5 mM EGTA, 50 mM sodium fluoride, 50 mM sodium chloride, 1 mM sodium orthovanadate, 5 mM sodium
pyrophosphate, 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml antipain, 10 µg/ml pepstatin, and 0.1 mg/ml aprotinin. Receptor subunits were isolated with 9E10 antibody
coupled to protein G-Sepharose, followed by SDS-PAGE. In some
experiments 2%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)
was used in place of NP-40. For surface immunoprecipitations, expressing cells were chilled to 4°C and incubated in PBS containing 10 µg/ml BSA for 30 min. 9E10 antisera was then applied to the cells
(10 µg/ml) in the same buffer for 20 min, followed by 9E10 peptide at
1 µg/ml for 10 min. Surface receptor populations were then isolated
using protein G-Sepharose after cell lysis as described above, except
that all solutions contained 9E10 peptide at 1 µg/ml.
Western blotting. Receptor subunits were detected in
gradient fractions using either 9E10 antibody or a rabbit polyclonal antibody against a peptide corresponding to amino acids of 326-343 of
murine 1 subunit (Macdonald and Olsen, 1994). Antibody production in
rabbits and Western blotting were performed as described by Krishek et
al. (1994) and Connolly et al. (1996a), respectively.
Electrophysiology. Whole-cell recordings from infected BHK
cells were conducted at 18 hr after infection as described previously (Krishek et al., 1994).
RESULTS
Production and characterization of Semliki Forest viruses
expressing GABAA receptor subunits
Co-electroporation of BHK cells with cRNA produced form either
plasmid pSFV11(9E10) or
PSFV12(9E10) with cRNA synthesized from the
Helper I plasmid produced high titers of infectious Semliki Forest
virus particles (Liljestrom and Garoff, 1991). Typical titers in excess
of 7 × 107/ml were obtained on electroporation
of 106 cells as determined by limiting dilution. To
characterize the virally expressed receptor subunits, infected cells
were pulse-labeled with [35S]methionine. Subunit
expression was then monitored with time by examining total protein
synthesis or via immunoprecipitation using 9E10 antibody. Infection of
BHK cells with 1(9E10) virus lead to the
time-dependent synthesis of a protein with an apparent molecular mass
of 52 kDa and a minor band at 50 kDa together with a significant
inhibition of host protein synthesis (Fig.
1A). In addition to
these, weaker bands of 65 and 40 kda were also evident. These bands
represent the replicase and capsid protein of the Semliki Forest virus
(Liljestrom and Garoff, 1991). Likewise, infection with the
2(9E10) virus resulted in the synthesis of a 56 kDa protein (Fig. 1B). Semliki Forest virus infection
of many cell types has previously been shown to inhibit protein
synthesis, via competition of the Semliki Forest virus-encoded RNAs
with host cell mRNAs for translation (Liljestrom and Garoff, 1991). To
confirm that these proteins represent receptor subunits,
immunoprecipitation with 9E10 antibody was performed on labeled cells
treated with or without tunicamycin to block N-glycosylation. From
cells infected with the 1(9E10) virus a protein
of 52 kDa was immunoprecipitated with a minor band of 50 kDa;
tunicamycin treatment resulted in the precipitation of a single band of
48 kDa (Fig. 1C). In contrast 9E10 antibody precipitated a
band of 56 kDa from cells infected with the
2(9E10) virus; treatment with tunicamycin reduced
the mass of this band to 50 kDa (Fig. 1D). The values
obtained for the molecular masses of the glycosylated and
unglycosylated forms of the 1(9E10) and
2(9E10) viruses produced in BHK cells are thus in
agreement with those observed on expression in A293 cells (Buller et
al., 1994; Connolly et al., 1996a).
Fig. 1.
Biochemical characterization of the
GABAA receptor 1(9E10) and
2(9E10) subunits produced on Semliki Forest virus
infection of BHK cells. BHK cells were infected with either the
1(9E10) (A) or
2(9E10) (B) subunit
virus. At defined periods (between 0.5 and 24 hr as indicated) after
infection the cells were labeled for 30 min with 500 µCi/ml
[35S]methionine. Infected cells were then lysed,
and labeled proteins were resolved by SDS-PAGE followed by
fluorography. The subunits were also immunoprecipitated from
infected cells labeled with [35S]methionine and
treated with (+) or without (
) tunicamycin (5 µg/ml), as shown
in C and D for the
1(9E10) and 2(9E10)
subunits, respectively. The migration of protein standards (Bio-Rad) is
indicated.
[View Larger Version of this Image (61K GIF file)]
Expression of homomeric and heteromeric GABAA receptors
in BHK cells using Semliki Forest virus
Expression of receptor subunits in BHK cells was analyzed by
immunofluorescence. Using subunit-specific antibodies, we failed to
detect expression of the 1, 6, 1-3, or 2 subunits in
uninfected BHK cells. Infection of cells with single
1(9E10) or 2(9E10) viruses
resulted in abundant intracellular staining using 9E10 antibody (Fig.
2A,B) on
permeabilization but no staining in nonpermeabilized cells (Fig.
2C). The staining pattern is consistent with ER retention of
these subunits on homomeric expression, as demonstrated previously in
A293 and MDCK cells (Connolly et al., 1996a,b). In contrast, co-infection with 1(9E10) and
2(9E10) viruses (1:1 ratio) resulted in high
levels of surface 9E10 staining (Fig. 2D) in ~80%
of all cells. The presence of both the 1(9E10)
and 2(9E10) subunits on the cell surface was also
demonstrated using subunit-specific antibodies (Fig.
2E,F). To attempt to quantify the level of
receptor production, whole-cell binding was performed using tritiated
muscimol. High-affinity binding was evident only in co-infected cells,
with a Bmax of 26 pmol/mg protein and
Kd values of 4 and 90 nM (results not shown). Similar Kd values for muscimol
binding have been reported for the expression of these subunits
previously (Pritchett et al., 1988; Moss et al., 1991; Hadingham et
al., 1992).
Fig. 2.
Surface expression of GABAA
receptor subunits in BHK cells. BHK cells were infected with either
single 1(9E10) or 2(9E10)
virus or co-infected with both, and expression was then determined by
immunofluorescence with (+) and without () permeabilization 20 hr
after infection. Images were then collected by confocal microscopy.
A, 1(9E10), 9E10 antibody (+);
B, 2(9E10), 9E10 antibody
(+); C, 1(9E10), 9E10
antibody (); D, 1(9E10)
and 2(9E10), 9E10 antibody (). Images were
collected from cells infected with both subunit viruses and co-stained
using a rabbit polyclonal antibody against the 1 subunit and a
monoclonal antibody (BD17) against the 2 subunit coupled to
fluorescein and rhodamine secondary antibodies, respectively. The
images collected for the two channels are shown in E and
F for the 1(9E10) and
2(9E10) subunits, respectively.
[View Larger Version of this Image (84K GIF file)]
Whole-cell recordings from BHK cells co-infected with
1(9E10) and 2(9E10) viruses
exhibited robust GABA-activated membrane currents (Fig. 3). The maximum current density evoked by
GABA (100 µM) was 3.5 ± 0.5 nA, significantly
higher than expected from conventional calcium phosphate or
electroporation studies using these subunits (Connolly et al., 1996a).
The membrane currents were sensitive to inhibition by 10 µM Zn+2 and were not enhanced by 1 µM flurazepam, and the GABA EC50 was 1.5 ± 0.12 µM (n = 8 cells), in agreement
with earlier studies (Macdonald and Olsen 1994). In contrast, infection
of BHK cells with either single 1(9E10) or
2(9E10) viruses resulted in complete
insensitivity to GABA (1 mM) and also pentobarbital (2 mM; n = 5). The latter was used because 1 and 3 homomeric ion channels are sensitive to pentobarbital in
preference to GABA (Sigel et al., 1989; Connolly et al., 1996b; Krishek
et al., 1996). These results suggests that only the
1(9E10)2(9E10) binary
combination forms functional ion channels as shown previously (Connolly et al., 1996a).
Fig. 3.
Electrophysiological properties of
GABAA receptors produced in BHK cells. A,
Whole-cell recordings form BHK cells infected with Semliki Forest
viruses expressing
1(9E10)2(9E10). Homomeric
1(9E10) and 2(9E10)
subunits were insensitive to GABA, and 2(9E10)
subunits were also insensitive to pentobarbital (PB).
The duration of the ligand application is indicated by solid
lines. Holding potential, 50mV. B, GABA (
,
, and 
) and pentobarbital (
) equilibrium concentration
responses for BHK cells expressing
1(9E10)2(9E10) or single
subunits. data were fitted by
I/Imax = [1/(1 + {[A]/EC50}n)], where
I and Imax represent
GABA-activated and maximally activated current, n is the
Hill coefficient, and A is the GABA concentration. EC50 = 1.47 ± 0.12 µM;
n = 1.0 ± 0.08. Data points indicate
mean ± SEM.
[View Larger Version of this Image (17K GIF file)]
Sucrose density fractionation of GABAA
receptor subunits
Receptor subunits expressed in BHK cells were fractionated on
sucrose density gradients. Receptors were solubilized in a range of
differing detergents. Up to 50% of the receptor subunits could be
solubilized in 2% NP-40, whereas <20% could be solubilized in 2%
CHAPS (data not shown). Therefore, the majority of experiments were
performed using solubilization in NP-40. Gradient fractions were then
subjected to SDS-PAGE followed by Western blotting with 9E10 antibody.
On homomeric expression a major band of 52 kDa corresponding to the
1(9E10) subunit could be detected, which
sedimented at 5 S (Fig.
4A). In some
experiments additional bands of 50 and 48 kDa could also be seen. These
bands presumably represent incompletely glycosylated forms of the 1
subunit (Fig. 1C). The sedimentation of the
1(9E10) subunit was also analyzed in cells
co-infected with the 2(9E10) virus using an
1-specific antibody that recognizes an epitope in the major
intracellular domain of this subunit. Co-expression with the
2(9E10) subunit resulted in a shift in the
sedimentation of the 1(9E10) subunit to 9 S (Fig.
4B). This shift is consistent with oligomerization of
the 1(9E10) and 2(9E10)
subunits. An identical shift in sedimentation of the
1(9E10) subunit was seen using CHAPS in place of
NP-40. A parallel shift in sedimentation coefficient of the
1(9E10) subunit from 5 to 9 S was also seen on
co-expression with the 1(9E10) using Western
blotting with anti-2 subunit antibodies (data not shown). Similar
sedimentation coefficients have been previously demonstrated for
purified GABAA receptors and recombinant receptors composed
of both / and subunits (Mamalaki et al., 1987, 1989;
Hadingham et al., 1992) and for assembled pentameric muscle nicotinic
acetylcholine receptor (AChR) expressed in fibroblasts (Green and
Millar 1995). Our failure to detect homo-oligomerization of either the
1(9E10) or 2(9E10) subunit
may be attributable to the detergent used. Interestingly, substitution
of NP-40 for CHAPS did not result in the detection of
slower-sedimenting forms of either homomeric subunit. Furthermore, the
use of Lubrol for extraction does not lead to the detection of
homo-oligomers for the 1 subunit expressed in human embryonic kidney
293 (HEK) cells (Tretter et al., 1997).
Fig. 4.
Differential sedimentation of the
1(9E10) subunit dependent on coexpression with
the 2(9E10) subunit. Cells infected with the
1(9E10) (A) or co-infected
with both the 1(9E10) and 2(9E10)
(B) subunit viruses were lysed and subjected to
sucrose density gradient fractionation 16 hr after infection. Gradient
fractions were separated by SDS-PAGE, and the
1(9E10) subunit was detected via Western blotting
using 9E10 antibody (A) or a rabbit polyclonal
antibody against the 1 subunit (B). Sedimentation coefficients for the 1(9E10)
subunit were determined with reference to proteins with known sedimentation: BSA (4.3 S), aldolase (7.4 S), and catalase (11.2 S).
[View Larger Version of this Image (45K GIF file)]
To analyze the production and stability of this 9 S complex,
co-infected cells were labeled with
[35S]methionine for 1 hr and chased for defined
periods with excess cold methionine. Cell lysates were then prepared
and subjected to sucrose density gradient fractionation. Receptor
subunits were precipitated from gradient fractions using 9E10 antibody
(Fig. 5), and the levels of incorporated
methionine were quantified on a Bio-Rad phosphorimager (Fig.
6). Bands corresponding to 52 and 56 kDa
could be detected for the 1(9E10) and
2(9E10) subunits, respectively, in gradient
fractions (Fig. 5A-C). Immediately after a 1 hr labeling
period, the majority of synthesized subunits sedimented at 5 S. However a shoulder migrating at 9 S was also visible (Figs. 5A,
6A), which was not seen with either subunit expressed alone, using
identical conditions (data not shown). In contrast, at 6 hr the
majority of the labeled receptor subunits migrated at 9 S (Figs.
5B, 6B). There was also a drastic reduction in the total
number of receptor subunits at this 6 hr time point, with only ~36%
of the total counts present after labeling remaining (Fig.
6B). At 20 hr, most of the 9 S peak was still
evident, with ~31% of the starting counts remaining (Fig.
5C,F). The 9 S pool presumably forms quickly, because
it could be detected at zero time (after a 1 hr labeling period) but
was difficult to resolve from unassembled subunits at this time point
(Fig. 6A-C). Also, the 9 S fraction seemed
relatively stable, as demonstrated by only a small loss (~10%) of
this peak between 6 and 20 hr (Fig. 6B,C) suggesting
a half-life of ~40 hr for receptors composed of
1(9E10) and 2(9E10)
subunits. Collectively the results suggest that oligomerization of
receptor subunits occurs quickly but is relatively inefficient, and
that unassembled subunits are degraded. Pulse-chase and
immunoprecipitation were used to analyze the half-lives of the
1(9E10) and 2(9E10)
subunits on homomeric expression, as shown in Figure
7. At all time points a single band of 52 kDa was seen for the 1(9E10) subunit, whereas in
contrast two bands of 56 and 50 kDa were evident for the
2(9E10) subunit. The lower band for the
2(9E10) subunit presumably represents
unglycosylated material. These results were quantified using a
phosphorimager. In the case of the 2(9E10)
subunit the signals form both bands were pooled. Half-lives of ~2 hr
were determined for both the 1(9E10) and
2(9E10) subunits. Similar values were seen in
three separate experiments. These half-lives for homomeric receptor
subunits are consistent with the dramatic loss of signal seen during
the first 6 hr of the pulse-chase experiments shown in Figures 5 and
6.
Fig. 5.
Pulse-chase analysis of GABAA
receptor assembly. Cells co-infected with both subunit viruses were
labeled with 100 µCi/ml [35S]methionine 2 hr
after infection for 1 hr and chased for 0 (A), 6 (B), or 20 (C) hr excess
cold methionine. The cells were then lysed and subjected to sucrose
density gradient fractionation. Gradient fractions were then
immunoprecipitated with 9E10 antibody, and the
1(9E10) and 2(9E10)
subunits were resolved by SDS-PAGE. Sedimentation coefficients for the
1(9E10) and 2(9E10)
subunits were determined as described in Figure 4.
[View Larger Version of this Image (29K GIF file)]
Fig. 6.
Quantification of receptor assembly. The
levels of incorporated methionine in 1(9E10)
(
) and 2(9E10) () subunits were quantified
in gradient fractions for cells chased for 0 (A),
6 (B), and 20 (C) hr using
a Bio-Rad phosphorimager. Background was subtracted using the same
volume that was used to integrate the subunit signals.
[View Larger Version of this Image (13K GIF file)]
Fig. 7.
Degradation of single GABAA receptor
subunits expressed in BHK cells. BHK cells were infected with Semliki
Forest viruses producing either the 1(9E10)
(A) or 2(9E10)
(B) subunit. Two hours after infection the cells
were labeled with [35S]methionine (400 µCi/ml)
for 20 min and then chased for differing periods. The cells were then
lysed, and receptor subunits were isolated by immunoprecipitation with
9E10 antibody. Precipitated material was then subjected to SDS-PAGE,
and the subunit levels were quantified using a Bio-Rad
phosphorimager.
[View Larger Version of this Image (62K GIF file)]
The time dependence of receptor surface expression
To examine the time dependence of surface expression, BHK cells
were co-infected with 1(9E10) and
2(9E10) viruses. Infected cells were then fixed
after differing periods, and the level of surface receptor expression
was determined by fluorescence. Surface receptor levels were then
quantified by confocal microscopy, as shown in Figure
8. Surface expression could be first
detected 8 hr after infection, which increased rapidly up to 10 hr and
leveled off after this period (Fig. 7). Interestingly these results
suggest a significant latency between subunit oligomerization, which
was evident within 1 hr (Fig. 5), and the appearance of cell surface
receptors, which could be detected 8 hr after infection, as determined
by fluorescence measurements.
Fig. 8.
Time dependence of surface expression of
receptors composed of 1(9E10) and
2(9E10) subunits. Co-infected cells were fixed at
differing periods after infection and processed simultaneously for
fluorescence with 9E10 antibody without permeabilization. Images were
collected from cells, and the average intensity of staining is shown.
Data were collected from at least five cells at each time point.
[View Larger Version of this Image (15K GIF file)]
Subunit ratio of GABAA receptors composed of
1(9E10) and 2(9E10)
subunits
The ratio of 1(9E10) and
2(9E10) subunits present in the 9 S receptor pool
was analyzed from expressing cells after labeling with
[35S]methionine for 1 hr followed by a 12 hr
chase. This will ensure degradation of all unassembled subunits
produced during the preceding 1 hr labeling period. Fractions
containing the 9 S receptor isolated by sucrose density gradient
fractionation were pooled and immunoprecipitated with 9E10 antibody
followed by SDS-PAGE. Incorporated counts were then quantified using a
Bio-Rad phosphorimager. Signals from the 1(9E10)
and 2(9E10) subunits were then normalized for
relative methionine content. A typical result is shown in Figure
9A, where an
1(9E10):2(9E10) subunit
ratio of 0.9 was determined. Ratios of 1.2, 1.1, 0.8, and 0.8 were
obtained in four separate experiments. To investigate this observation
further, the subunit ratio of cell surface receptors was analyzed.
Cells expressing both the 1(9E10) and
2(9E10) subunits were labeled with
[35S]methionine and exposed to 9E10 antibody at
4°C. The surface receptor population was then isolated using protein
G-Sepharose resolved by SDS-PAGE, and subunit levels were then
quantified. A ratio of 1.1 was found for the
1(9E10):2(9E10) subunits in the
experiment shown in Figure 9B. Similar ratios were found in
two other experiments. Together, these data are consistent with a 1:1
ratio of 1(9E10):2(9E10)
subunits, suggesting that receptors are composed of equal numbers of
subunits, e.g., a tetrameric or hexameric but, interestingly, not
pentameric tertiary structure.
Fig. 9.
Subunit ratio of GABAA receptors
composed of 1(9E10) and
2(9E10) subunits. A, BHK cells
expressing both receptor subunits were pulse-labeled with
[35S]methionine (200 µCi/ml) for 2 hr; 3 hr
after infection, the cells were then chased for 12 hr with excess cold
methionine. Cell lysates were then subjected to sucrose density
gradient fractionation, and the fractions corresponding to the 9 S peak
were pooled and immunoprecipitated with 9E10 antibody. Receptor
subunits were then resolved by SDS-PAGE and quantified using a Bio-Rad
phosphorimager. After correction for methionine content (1 = 9;
2 = 15) a ratio of 0.9 was found for the
1(9E10):2(9E10) subunits in
the experiment shown. B, Labeled cells were exposed to
9E10 antibody at 4°C for 30 min. The cell surface receptor population
was then isolated via immunoprecipitation with protein G in the
presence of excess 9E10 peptide and resolved by SDS-PAGE, and subunit
levels were quantified. A ratio of 1.1 was found for the
1(9E10):2(9E10) subunits in
the experiment shown.
[View Larger Version of this Image (28K GIF file)]
Surface expression of homomeric and heteromeric GABAA
receptors in cultured neurons using Semliki Forest virus
To determine whether GABAA receptor assembly in BHK
cells is a faithful indicator of neuronal events, cultured SCG neurons were infected with Semliki Forest viruses expressing 9E10-tagged receptor subunits. Infection of neurons with either single
1(9E10) or 2(9E10) virus
did not result in the detection of fluorescence in nonpermeabilized cells (Fig. 10A).
Abundant intracellular staining could be seen for both subunits (Fig.
10C). This perinuclear and tubular staining (Fig.
9A,B) is consistent with localization of these subunits to
the ER in neurons (Krijnse-Locker et al., 1995). In contrast, co-infection of SCG neurons with both viruses led to the detection of
robust staining in the absence of detergent (Fig.
10D). Staining of 9E10 was evident in neuronal cell
bodies, as well as in the neuronal processes. Interestingly "hot
spots" (indicated in Fig. 10D by the
arrow) of fluorescence were often detected in the neuronal processes. This presumably represent clustering of the virally encoded
receptors.
Fig. 10.
Infection of cultured SCG neurons with
Semliki Forest viruses expressing GABAA receptor subunits.
Cultured SCG neurons were infected after 3 d in culture, and
receptor expression was determined by immunofluorescence using 9E10
antibody 12 hr after infection with (+) or without ()
permeabilization. Images were then collected using confocal microscopy
from cells infected with A,
1(9E10) (+); B,
2(9E10) (+); C,
1(9E10) (); and D,
1(9E10) and 2(9E10) ().
[View Larger Version of this Image (98K GIF file)]
DISCUSSION
Molecular cloning has revealed a large multiplicity of
GABAA receptor subunits, with some neurons, such in the
hippocampal dentate gyrus expressing up to 12 differing subunits.
Clearly to establish the extent of GABAA receptor
heterogeneity it is important to understand the mechanisms that control
receptor assembly.
We have engineered Semliki Forest viruses (Liljestrom and Garoff, 1991)
to express epitope-tagged GABAA receptor subunits (Connolly
et al., 1996a,b). Using this system, we have compared the assembly of
GABAA receptors composed of 1(9E10)
and 2(9E10) subunits in BHK cells and cultured
SCG neurons. In BHK cells, homomeric expression resulted in the
synthesis of large amounts of protein for each of these respective
subunits. However, the subunits were unable to access the cell surface.
In common with studies in A293 and MDCK cells these subunits were
ER-retained in BHK cells (Connolly et al., 1996b). Given the high
levels of protein produced on viral expression, our results highlight
the efficiency and capacity of the ER retention system in controlling GABAA receptor expression. Homomeric expression of receptor
subunits has proven controversial. In the case of the 1 and 3
subunits functional Cl
channels have been reported
on expression in a range of heterologous systems (Sigel et al., 1989;
Connolly et al., 1996b; Krishek et al., 1996). In contrast, homomeric
expression of the 1 or 2 subunits produces functional expression
in some cases (Blair et al., 1988) but not others (Sigel et al., 1990;
Connolly et al., 1996b). The reasons for these discrepancies remain
unclear; differences in the species of cDNAs used may be of
significance. Another explanation is that some expression systems may
produce trace levels of GABAA receptor subunits, which may
complicate the interpretation of homomeric expression (Ueno et al.,
1996).
In contrast, co-infection of BHK cells with both
1(9E10) and 2(9E10) viruses
leads to robust surface expression. Sucrose density gradient fractionation was used to analyze the ability and efficiency of receptor subunits to oligomerization. 1(9E10) or
2(9E10) subunits expressed as homomers exhibited
sedimentation coefficients of 5 S. In contrast, the sedimentation
coefficients of both the 1(9E10) and
2(9E10) subunits increased to 9 S on
co-expression. This shift in sedimentation is indicative of subunit
oligomerization. The inability of 1(9E10) and
2(9E10) to homo-oligomerize presumably results in
ER retention and subsequent degradation, with similar half-lives of 120 min. ER retention of these subunits is presumably mediated via
interaction with the chaperone proteins heavy-chain binding protein and
calnexin, respectively (Ou et al., 1993; Hammond et al., 1994; Pelham,
1995). Both of these proteins have been previously shown to interact with the 1(9E10) and
2(9E10) subunits, respectively (Connolly et al.,
1996a). To study the efficiency of receptor oligomerization the
appearance of the 9 S complex containing both subunits was analyzed.
Thirty-six percent of available 1(9E10) and
2(9E10) subunits attained this 9 S sedimentation
coefficient; the remainder of the subunits were degraded. The majority
of the unassembled subunits were degraded within 6 hr, consistent with
the short half-lives of either the 1(9E10) or
2(9E10) subunit found on homomeric expression. In
contrast, assembled receptors seemed relatively stable, with only
~10% of this population being degraded over a 14 hr period. Semliki
Forest virus infection results in suppression of host synthesis, and
therefore our result may overestimate subunit half-lives. However,
receptors composed of 1 and 2 have half-lives of in excess of 45 hr in transfected HEK A293 cells (S. J. Moss and T. G. Smart,
unpublished observations), consistent with our findings using Semliki
Forest virus expression. The appearance of
1(9E10) and 2(9E10)
receptors at the cell surface was slow, lagging considerably after the
appearance of 9 S receptor complexes. The slow transport of these
receptors to the cell surface may be attributable to the absence of
2 subunits, or alternatively this may reflect a difference in
cellular environment.
There seem to be parallels between the assembly of GABAA
receptors and the muscle AChR subunits stably expressed in fibroblasts. These studies have demonstrated that single AChR , , , or subunits are incapable of oligomerization and are unable to access the
cell surface (Claudio et al., 1989; Paulson et al., 1991). Stable cell
surface receptors are only produced on co-expression of all four
subunits (Claudio et al., 1989; Paulson et al., 1991; Green and Millar,
1995). Again, in common with GABAA receptor assembly there
is a significant lag time (up to 4 hr) between the appearance of AChR
receptors at the cell surface and subunit oligomerization. Likewise,
the assembly of the AChR seems to be an inefficient process, with only
20-30% of translated subunits being assembled into surface receptors
(Ross et al., 1991; Green and Millar 1995).
GABAA receptors composed of subunits differ from
receptors composed of and 2 subunits with regard to
Zn2+ and benzodiazepine sensitivity, in addition to
single-channel conductance (Draguhn et al., 1990; Verdoorn et al.,
1990; Moss et al., 1991; Smart et al., 1991; Angelotti et al., 1993).
The existence of receptors composed of subunits in neurons has not been established. However, the high Zn2+
sensitivity (Draguhn et al., 1990; Smart et al., 1991) of these receptors coupled with low single-channel conductance (Moss et al.,
1991; Angelotti et al., 1993) is reminiscent of some populations of
neuronal GABAA receptors (Smart, 1992; Kaneda et al.,
1995). The tertiary structure of these differing receptor isoforms has been examined. Mutagenic and biochemical approaches have suggested that
receptors composed of 22 or 32 subunits are
pentameric (Backus et al., 1993; Chang et al., 1996; Tretter et al.,
1997). The structure of / receptors is more controversial.
Receptors composed of 13 receptors expressed in
Xenopus oocytes have been reported to be tetrameric
(Kellenberger et al., 1996) whereas the same receptor population
expressed in A293 cells has been reported to be pentameric (Tretter et
al., 1997). These studies may be complicated by the presence of
receptors composed of homomeric 3 subunits (Connolly et al., 1996b)
in addition to receptors containing both the 1 and 3 subunits.
Studying receptors composed of 12 subunits may give more
consistent results, because neither of these subunits can access the
cell surface on homomeric expression because of ER retention (Connolly
et al., 1996a,b). An alternate explanation for the observations on
/ receptors is that two populations of receptors exist, which are
pentameric but differ in their subunit ratios, e.g., 23 and
32. Given the unitary dose-response curves for /
receptors, the single IC50 value for
Zn2+ inhibition, and unitary single-channel
properties, this seems unlikely (Macdonald and Olsen, 1994).
A tetrameric rather than pentameric structure of receptors composed of
subunits compared with may possibly explain the lower
single-channel conductance of 15-16 and 28-32 pS, respectively. (Moss
et al., 1991; Angelotti and Macdonald, 1993). Analysis of the channel
permeability of these two receptor types may help resolve this
intriguing possibility. It will be of interest to study the assembly of
GABAA receptors composed of and subunits using
Semliki Forest virus. Routine triple infection of cells with viruses
encoding 1, 2, and 2 subunits has proven difficult.
We exploited the broad host range of the Semliki Forest virus
(Liljestrom and Garoff, 1991) to examine whether the assembly of
GABAA receptors seen in heterologous systems is a faithful model of neuronal events. The use of receptor cDNAs modified with reporter epitopes (Connolly et al., 1996b), allowed the discrimination between recombinant and neuronal receptor pools to be made. Infection of SCG neurons with either single 1(9E10) or
2(9E10) subunit viruses did not result in surface
expression, because these subunits were ER-retained. Co-infection
resulted in robust surface expression. SCG neurons express
benzodiazepine-sensitive GABAA receptors, suggesting the
presence of , , and receptor subunits of unknown identity.
Presumably, the failure of single subunits to reach the cell surface on
viral infection is attributable to low levels of endogenous subunits
available for oligomerization. Suppression of host protein synthesis on
Semliki infection will exaggerate this problem (Liljestrom and Garrof,
1991). An alternative explanation is that the virally expressed
subunits are unable to assemble with the endogenous receptor subunits.
Taken together the ER retention of single subunits and robust surface
expression seen of the
1(9E10)2(9E10) combination
in both cell types suggest that receptor assembly occurs via similar
pathways in BHK cells and SCG neurons. Whether the efficiency of
assembly is similar in BHK cells and SCG neurons remains to be
established. Neuronal-specific chaperone proteins that enhance
expression of red and green opsins have been reported (Ferreria et al.,
1996). Whether a similar mechanism exists to enhance the assembly of
GABAA receptors is unknown. Finally, the ability to infect
neurons with multiple Semliki Forest viruses expressing defined
epitope-tagged receptor subunits should allow the assembly and
trafficking of GABAA receptors to be studied in their
native environments.
FOOTNOTES
Received April 11, 1997; revised June 12, 1997; accepted June 17, 1997.
This work was supported by the Medical Research Council (UK) and the
Wellcome trust.
Correspondence should be addressed to Dr Stephen J. Moss, Medical
Research Council Laboratory of Molecular Cell Biology, University College London, Gordon Street, London WC1E 6BT, UK.
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