From the University Clinic for Psychiatry, Section of
Biochemical Psychiatry and Brain Research Institute of the University
of Vienna, Vienna A-1090, Austria, the ¶ Medical Research Council,
Anatomical Neuropharmacology Unit, Oxford OX1 3TH, United Kingdom, the
** Departments of Anesthesiology/Critical Care Medicine and
Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh,
Pennsylvania 15261, and the
Research
Institute for Laboratory Animal Breeding, Himberg A-2325,
Austria
Received for publication, December 8, 2000
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ABSTRACT |
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GABAA receptors are chloride
channels composed of five subunits. Cerebellar granule cells express
abundantly six subunits belonging to four subunit classes. These are
assembled into a number of distinct receptors, but the regulation of
their relative proportions is yet unknown. Here, we studied the
composition of cerebellar GABAA receptors after targeted
disruption of the GABAA receptors are composed of five subunits derived from
different subunit classes. So far, six The cerebellum is an excellent brain area for studying the composition
of GABAA receptors. The small number of cell types allows
an almost complete account of which cerebellar cell types express which
GABAA receptor subunit genes (8). For example, cerebellar
granule cells express six subunit genes abundantly ( Targeted disruption of genes by homologous recombination is widely used
to study the functional role of the respective gene product in the
organism. In receptors composed of multiple subunits, the deletion of
one subunit might also reduce the abundance of its subunit partners
present in the same receptors. Indeed, after the disruption of the
GABAA receptor The recent generation of Generation of Mutant Mice--
Targeted disruption of the Antibodies--
The generation of anti-peptide
Quantitative Immunoblot Analysis--
Membranes from a total of
12
To test for equal protein loading, in some experiments a monoclonal
anti- Preparation of Membrane Extracts, Affinity Chromatography, and
Immunoprecipitation--
GABAA receptors were solubilized
from cerebellar membranes of
For immunoprecipitation, 200 µl of the clear deoxycholate membrane
extract was mixed with 30 µl of antibody solution (20-45 µg of
antibody), and the mixture was incubated under gentle shaking at
4 °C overnight. Then 50 µl of pansorbin (Calbiochem, La Jolla, CA)
plus 100 µl of a low salt buffer for immunoprecipitation (50 mM Tris/HCl, 150 mM NaCl, 1 mM
EDTA, 0.2% Triton X-100, pH 8.0, 1 mM benzamidine, 200 µg/ml bacitracin, and 300 µM phenylmethylsulfonyl fluoride) containing 5% dry milk powder were added, and incubation was
continued for 2 h at 4 °C. The precipitate was centrifuged for
10 min at 10,000 × g, and the pellet was washed twice
with 500 µl of high salt buffer for immunoprecipitation (50 mM Tris/HCl, 600 mM NaCl, 1 mM
EDTA, 0.5% Triton X-100, pH 8.3, 1 mM benzamidine, 200 µg/ml bacitracin, and 300 µM phenylmethylsulfonyl
fluoride) and once with 500 µl of low salt buffer for immunoprecipitation.
For determination of the total amount of receptors present in the
extract, solubilized receptors were precipitated by a mixture containing 10 µg of Receptor Binding Studies--
Cerebellar membranes were
homogenized and washed three times with 50 mM Tris/citrate,
pH 7.1. Extracted receptors were immunoprecipitated, and the
precipitate was suspended in 1 ml of a solution containing 0.1% Triton
X-100, 50 mM Tris-citrate buffer, pH 7.1. A total of 1 ml
of a solution containing 100 µg of membrane protein or the
resuspended immunoprecipitate was incubated with 2-50 nM
of [3H]Ro15-4513 (21.7 Ci/mmol, PerkinElmer Life
Sciences) in the absence or presence of 100 µM diazepam
or 100 µM Ro15-1788, or with 2-50 nM
[3H]muscimol (20 Ci/mmol, PerkinElmer Life Sciences) in
the absence or presence of 1 mM GABA, for 90 min at
4 °C. Then the suspensions were filtered through Whatman GF/B
filters, and the filters were washed and subjected to liquid
scintillation counting.
Immunocytochemistry--
Two series of animals were processed.
In the first series, five adult Quantification of Immunoreactivity--
The animals were coded
and measurements were carried out according to a double-blind protocol.
Neither the person taking the images nor the person making the
subsequent measurements knew the identity of the animals. The code was
opened only after completing the measurements. One digital image
(cooled charge-coupled device camera, Xilix Microimager, OpenLab
version 2.2.1, Improvision, Coventry, UK) was captured from each of
three sections per animal using a 20× objective, 100-watt mercury
lamp, and an L5 filter block (excitation filter, BP 480/40 nm; RKP 565 nm; suppression filter BP 610/75) of a Leitz DMRB fluorescence
microscope. The same exposure time was used for images from all animals
for a given antibody, and the images were treated in an identical way. The images were transferred to Adobe Photoshop (version 5.5) and the
mean gray scale pixel value (12-bit) was determined separately for the
molecular and granule cell layers for each section using the Histogram
command. Each animal was characterized by the mean average gray scale
pixel value obtained from the three sections. The values of control and
Immunoreactivity for the Immunoreactivity for
To quantify the change, light microscopic immunofluorescence labeling
for the
Membranes from the cerebellum of [3H]Ro15-4513 Binding in Cerebellum of Control and
These data were confirmed by binding studies using
[3H]flunitrazepam as a ligand. In contrast to
[3H]Ro15-4513, [3H]flunitrazepam
exclusively binds to DS sites (25). Scatchard analyses from two
separate experiments indicated that Bmax values observed in membranes from
In other experiments, GABAA receptors were extracted from
cerebellar membranes by a deoxycholate buffer and then
immunoprecipitated with a combination of antibodies directed against
the [3H]Muscimol Binding in the Cerebellum of Control and
A comparison of Bmax values in cerebellar
membranes and solubilized receptors indicated that ~52% and 61% of
GABAA receptors present in membranes of Subunit Composition of GABAA Receptors in the
Cerebellum of Control and
In other experiments, the extent of reduction of
The percentage of
In contrast, the percentage of
To further investigate the composition of
Quantification in the original membrane extract and column efflux of
receptor subtypes precipitated with subunit-specific antibodies
indicated that a comparable proportion of
Supporting previous conclusions (13, 28, 29), the percentage of
Summing of the proportions of No Reduction in the Number of GABAA Receptors in
Increase in GABAA Receptors Containing
This conclusion was supported by studies investigating subunit
partnerships in GABAA receptors: Immunoaffinity
chromatography using an anti- Increase in GABAA Receptors Composed of Changes in the Subunit Composition of GABAA Receptors
in
Although a significant increase in the expression of Molecular Mechanism Causing the Changes in GABAA
Receptor Subunit Composition in
Studies on GABAA receptor assembly in non-neuronal cells
have indicated that
The relatively stable Comparison with Previous Studies on Targeted Disruption of
GABAA Receptor Subunits--
Previous studies have
indicated that the targeted disruption of
Upon disruption of the
The likely presence of receptors composed of subunit gene. In membranes and extracts of
/
cerebellum, [3H]muscimol binding was not
significantly changed, whereas [3H]Ro15-4513 binding was
increased by 52% due to an increase in diazepam-insensitive binding.
Immunocytochemical and Western blot analysis revealed no change in
6 subunits but an increased expression of
2 subunits in
/
cerebellum. Immunoaffinity
chromatography of cerebellar extracts indicated there was an increased
coassembly of
6 and
2 subunits and that
24% of all receptors in
/
cerebellum did not contain a
subunit. Because 97% of
subunits are coassembled with
6 subunits in the cerebellum of wild-type mice, these
results indicated that, in
/
mice,
6
2 and
receptors replaced
subunit-containing receptors. The availability of the
subunit, thus, influences the level of expression or the extent of assembly of
the
2 subunit, although these two subunits do not occur
in the same receptor.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Aminobutyric acid A
(GABAA)1
receptors are ligand-gated anion channels that mediate the
majority of fast synaptic inhibition in the brain (1). A variety of
drugs, such as benzodiazepines, barbiturates, steroids, anesthetics,
and convulsants exert their pharmacologically and clinically important
actions by modulating the function of these receptors (2).
, four
, three
, one
, one
, one
, one
, and three
subunits have been cloned from mammalian brain (3, 4). From possible permutations of these
subunits, theoretically, an extremely large number of receptors with
distinct subunit composition can be formed. Due to restrictions in the
cellular expression of individual subunits and to mechanisms governing
the assembly of receptors, however, only a limited number of receptor
subtypes actually are present in the brain (5-7).
1,
6,
2,
3,
2,
and
), forming
1
2,
6
2,
1
6
2,
6
, and
1
6
GABAA receptor subtypes (9-13), and these receptors exhibit a distinct subcellular distribution (14, 15). The regulation of
the expression of the different receptors relative to each other is unknown.
6 subunit gene, not only were
the
6 subunits completely eliminated from cerebellar
granule cells but also the
subunits were dramatically reduced (16, 17). These results suggested that the
6 subunit is
necessary for the oligomerization and surface expression of the
subunit.
/
mice (18) allowed to investigate
whether the
subunit is also essential for the surface expression of
the
6 subunit. Therefore, in the present study, the
abundance and subunit composition of GABAA receptors was
compared in the cerebellum of
+/+ and
/
mice. In contrast to
6
/
mice (17), the total number of GABAA
receptors was not reduced in the cerebellum of
/
mice, despite
the complete loss of
subunits. Instead, an up-regulation of the
2 subunit-containing receptors was observed. Furthermore, the composition of GABAA receptors in the
cerebellum of
/
mice differed significantly from that of
+/+ mice.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit of the GABAA receptor in mouse embryonic stem cells
and production of wild-type (
+/+), and homozygous
/
mice is
described in detail in a previous study (18). The replacement-type DNA
targeting construct placed a selectable marker gene in exon 4, upstream
of the exons required for the putative transmembrane regions of this
receptor subunit. This targeting event prevented the production of
protein, i.e. produced a true null allele. The mice used for
the present studies were the F2-F5 generation on a mixed C57BL/6J × strain 129Sv/SvJ genetic background. These mice were normal in their
gross behavior.
+/+ mice were the F3 generation on a mixed
C57BL/6J × strain 129Sv/SvJ genetic background.
1-(1-9) (19), anti-peptide
6-(317-371) (17), anti-peptide
1-(350-404) (13), anti-peptides
2-(351-405),
3-(345-408) and
2-(319-366) (20),
1-(324-366) or
3-(322-372) antibodies (21), anti-peptide
-(1-44)
antibodies (16), or anti-peptide
2-(1-29) antibodies
(22) has been described earlier. The antibodies were shown to
precipitate recombinant GABAA receptors containing the
respective subunit only and did not exhibit cross-reactivity with other
GABAA receptors (13, 17).
+/+ and 12
/
cerebella were isolated individually, and
equal amounts (15 µg) of protein per slot were subjected to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis in the same 10%
polyacrylamide gel (13). Proteins were blotted to polyvinylidene
difluoride membranes and detected by subunit specific antibodies.
Secondary antibodies (Fab'2 fragments of goat anti-rabbit
IgG coupled to alkaline phosphatase, Jackson ImmunoResearch Labs Inc.)
were visualized by the reaction of alkaline phosphatase with
CSPD (Tropix, Bedford, MA) and the chemiluminescence signal was
quantified by densitometry of Kodak X-Omat S films with the Docu Gel
2000i gel documentation system using RFLP 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 following 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.
-actin antibody, was included in the antibody solution, and the
amounts of endogenous
-actin were quantitatively determined in a way
analogous to GABAA receptor subunits. Protein loading was
comparable in different slots and referring the data to the amounts of
endogenous
-actin neither changed the results nor reduced variability.
+/+ and
/
mice using a
deoxycholate buffer (0.5% deoxycholate, 0.05% phosphatidylcholine, 10 mM Tris/HCl, pH 8.5, 150 mM NaCl, 1 mM benzamidine, 200 µg/ml bacitracin, and 300 µM phenylmethylsulfonyl fluoride), and immunoaffinity
chromatography was performed as described (13). Briefly, deoxycholate
extracts were cycled three times through the affinity column
(synthesized as described in Ref. 21) at a rate of 2 ml/h. To determine
the percentage of receptors retained by the column,
immunoprecipitations with subunit-specific antibodies and
[3H]muscimol binding assays were performed with the
original extract and the column efflux in parallel.
1-(350-404), 18 µg of
2-(351-405), and 15 µg of
3-(345-408)
antibody per 200 µl of extract. This antibody mixture was used,
because all functional GABAA receptors are supposed to
contain at least one of these three
subunits. Precipitation with
polyethylene glycol could not be used for the determination of total
[3H]muscimol binding due to the rapid dissociation of
[3H]muscimol from its binding site. The high viscosity of
the polyethylene glycol solution causes relatively long filtration and
washing times and, thus, significant losses of previously bound
[3H]muscimol. The amounts of
1-(350-404),
2-(351-405), and
3-(345-408) antibodies
used were sufficient to maximally precipitate GABAA receptors containing the respective
subunits from brain extracts. The total number of [3H]muscimol binding sites measured
in receptors precipitated by this antibody mixture was higher than that
precipitated by polyethylene glycol, whereas the total number of
[3H]Ro15-4513 binding sites measured was identical
whether receptors were precipitated by the antibody mixture or
polyethylene glycol (13, 17).
+/+ and five adult
/
mice
were anesthetized with Sagatal (pentobarbitone sodium, 220 mg/kg
intraperitoneal) and perfused through the heart first with 0.9%
saline, then with a fixative containing 4% paraformaldehyde, 0.05%
glutaraldehyde, and 0.2% picric acid in 0.1 M phosphate
buffer (pH 7.4; PB) for 7-15 min. After perfusion the brains were
removed, and blocks from the vermis of the cerebellum were cut out and
washed in PB before sectioning with a Vibratome. Normal goat
serum (NGS, 20%) was used in 50 mM Tris-HCl containing
0.9% NaCl (pH 7.4, TBS) as the blocking solution for 1 h, before
the sections were incubated in the primary antibody solution (in TBS
containing 1% NGS and 0.05% Triton X-100) overnight. Primary
antibodies were used at the following final protein concentrations (in
µg/ml):
1 = 0.9-1.3 (P16);
6 = 0.5-0.6 (P24);
2 = 0.9 (
2-(351-405)R23);
3 = 1.7-2.0 (
3-(345-408)R1);
2 = 1.0 (
2-(1-29), 22); and
= 1.1 (
-(1-44)R5). After washing, the sections were incubated in either
biotinylated goat anti-rabbit IgG or biotinylated goat anti-guinea pig
IgG (diluted 1:50 in TBS containing 1% NGS, Vector Laboratories,
Peterborough, UK) for 2 h. The sections were then incubated in
avidin biotinylated horseradish peroxidase complex (diluted 1:100 in
TBS) for 1.5 h before the peroxidase enzyme reaction was
visualized with 3,3'-diaminobenzidine tetrahydrochloride as chromogen
and H2O2 as oxidant. Sections were then
routinely processed for light microscopic examination. In the second
series, three adult
+/+ and three
/
mice were anesthetized
and perfused with the same fixative as above for 10 min. After
perfusion, 50-µm-thick sections were cut from the cerebellum on a
Vibratome. All solutions contained 0.1% Triton X-100. Normal goat
serum (NGS, 20%) in TBS was used as blocking solution, before the
sections were incubated in the primary antibody solution in TBS
containing 1% NGS overnight. Primary antibodies were used at the
following final protein concentrations:
1(P16), 0.6 µg/ml;
2-(319-366, T12/20/B9), 0.9 µg/ml. After
washing, the sections were incubated for 5 h in goat anti-rabbit
IgG coupled to Alexa488, diluted 1:1000 (Molecular Probes, Eugene, OR)
at room temperature. Sections were washed and mounted in Vectashield (Vector Laboratories).
/
animals were compared with the unpaired t test.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Subunit in Control and
/
Mice--
In cerebellum, immunoreactivity for the
subunit is
restricted to the granule cell layer (15-17, 23) (Fig.
1). After targeted disruption of the
subunit gene, staining of granule cells with the
-(1-44) antibody
(16, 17) completely disappeared (Fig. 1). In agreement with these
immunocytochemical results, neither the complete
subunit (54-kDa
protein band) nor a truncated
subunit protein were detectable in
cerebellar membranes of
/
mice by Western blot analysis (18).
Finally, in contrast to
+/+ mice, where an immunoaffinity column
containing the
-(1-44) antibody was able to remove 28.8 ± 4.2% (n = 3, mean ± S.E.) of all
[3H]muscimol binding sites from cerebellar extracts, no
[3H]muscimol binding sites could be removed by this
column from cerebellar extracts of
/
mice, confirming the
absence of
subunit-containing receptors in mice with a targeted
disruption of the
subunit.
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Fig. 1.
Changes in the expression of the
2 and
subunits of the GABAA receptor in the cerebellum
of
/
mice. Pre-embedding
immunoperoxidase reaction with antibodies
2-(1-29) and
-(1-44)R5. An increase in the immunoreactivity for the
2 subunit was detected in the granule cell layer
(gcl) of
/
mice, without any change in the molecular
layer (ml). Immunoreactivity for the
subunit completely
disappeared in
/
mice. +/+, control mice; Pcl,
Purkinje cell layer; scale bar, 50 µm.
1,
6,
2,
3, and
2 Subunits in
Control and
/
Mice--
In addition to the
subunit, five
other major subunits can be detected in cerebellar granule cells:
1,
6,
2,
3,
and
2 (15, 17). We applied light microscopic
immunolabeling to assess possible alterations in the expression of
these subunits and to determine the cellular distribution of any
change. Immunoperoxidase reactions indicated no change in
immunoreactivity for the
1,
6,
2, and
3 subunits, but showed an
up-regulation of immunoreactivity for the
2 subunit in
the cerebellar granule cell layer of
/
animals (Fig. 1).
2 and
1 subunits was carried out
and measured. For the
2 subunit, the mean gray scale
pixel value increased by 82% (53.5 ± 7.9, mean ± S.D.,
n = 3, control; 97.2 ± 5.9, n = 3,
/
, p < 0.01, unpaired t test) in
the granule cell layer of
/
mice. In addition, there was also a
small (18%) but significant (p < 0.01, unpaired
t test) increase in
2 subunit labeling in the
molecular layer of the
/
mice (49.1 ± 1.8, n = 3, control; 58.2 ± 2.4, n = 3,
/
). In agreement with the results of the peroxidase
reactions, no significant change (p > 0.05, unpaired t test) was detected in immunoreactivity for the
1 subunit in either the granule cell (81.0 ± 6.7, n = 3, control; 91.5 ± 4.7, n = 3,
/
) or the molecular layers (75.2 ± 6.0, n = 3, control; 84.3 ± 13.0, n = 3,
/
).
+/+ or
/
mice were then
subjected to SDS-polyacrylamide gel electrophoresis and quantitative Western blot analysis, using subunit-specific antibodies directed against
1,
6,
2,
3,
2, and
subunits (13, 17). As shown in Fig. 2, the expression of
1 subunits was increased by 25% in the cerebellum of
/
mice, whereas the levels of
6 or
2 subunits were not significantly different from those
found in
+/+ mice. The expression of
3 subunits was
increased by 23% and that of
2 subunits was increased
by 45% (Fig. 2).
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Fig. 2.
Quantification of GABAA receptor
subunit proteins in cerebellar membranes by Western blot analysis.
Equal amounts of cerebellar membrane proteins were separated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis, and subunit levels
were compared in +/+ and
/
membranes by Western blot
analysis. A, representative blot comparing immunoreactivity
for
1,
6,
2,
3, and
2 subunits in three
+/+ and
three
/
cerebella. The chemiluminescence signals were quantified
by densitometry. Results are expressed as percentage of the subunit
level found in
+/+ membranes ± S.E. B, results for
1,
6,
2,
3,
and
2 subunits were obtained from 12
+/+ and 12
/
mice. Results for
subunits were from three
+/+ and
three
/
mice. No staining for the
subunit could be detected
in cerebellar membranes of
/
mice. For statistical comparisons
unpaired Student's t test was used. * p < 0.001; **p < 0.0001.
/
Mice--
To investigate whether the additional
2 subunits expressed in
/
cerebellum were part of
functional receptors, [3H]Ro15-4513 binding studies were
performed in membranes from
+/+ and
/
cerebellum.
[3H]Ro15-4513 is a ligand for the benzodiazepine binding
site of GABAA receptors, which are only formed by receptors
containing a
subunit (2). Scatchard analysis of specific
[3H]Ro15-4513 binding indicated that the total number
(Bmax) of sites was increased by 52% in
/
mice, whereas the binding affinity was not significantly
different (Table I). In agreement with previous reports (24-26), 31% of total binding in the cerebellum of
+/+ mice was not displaced by 100 µM diazepam, but
could be displaced by 100 µM Ro15-1788, a benzodiazepine
site antagonist. This diazepam-insensitive (DIS) binding, which is
attributed to receptors containing
6 subunits (27) was
increased by 180% in
/
mice. The diazepam-sensitive (DS)
[3H]Ro15-4513 binding, however, was comparable in
/
and
+/+ mice (Table I).
Comparison of benzodiazepine binding sites by Scatchard analysis of
[3H]Ro15-4513 binding
+/+ and
/
mice (A) or GABAA
receptors extracted from these membranes and precipitated as described
under "Experimental Procedures" (B) were incubated with various
concentrations of [3H]Ro15-4513 in the absence or presence
of 100 µM diazepam or Ro15-1788. Membranes or
precipitated receptors were then filtered through Whatman GF/B filters
and washed as described. Scatchard analysis was performed from
[3H]Ro15-4513 binding data that could be displaced by 100 µM Ro15-1788 (total specific binding) or by 100 µM diazepam (diazepam sensitive = DS binding). Total
specific binding minus DS binding resulted in diazepam insensitive
(DIS) binding. Values are means ± S.E. from three separate
experiments (A). For statistical comparisons unpaired Student's
t test was used. In B only two experiments were performed
due to the limited amounts of antibody available.
+/+ cerebella (2072 and 1640 fmol/mg of
protein) were comparable to those observed in
/
cerebella (2103 and 2126 fmol/mg of protein) and were also comparable to the
Bmax values of the DS
[3H]Ro15-4513 binding sites in these tissues (Table
I).
1,
2, and
3 subunits
of GABAA receptors (13, 17). Scatchard analysis of
[3H]Ro15-4513 binding to the precipitated receptors
indicated that the recovery of total, DS and DIS binding was 56%,
57%, and 53% in
+/+ and 57%, 53%, and 60% in
/
cerebella, respectively. Receptors recovered in the extract, however,
represented 93% of all binding sites detectable in cerebellar membrane
homogenates treated with detergent, because part of the receptors
present in the membranes became inactivated during solubilization.
Total Bmax values measured in cerebellar
extracts from
/
mice were increased by 55% as observed in the
corresponding membranes. Whereas DS binding was comparable in the
extracts of
+/+ and
/
cerebella, DIS binding in
/
extracts was increased by 216% relative to that in
+/+ extracts
(Table I).
/
Mice--
To investigate the abundance of GABAA
receptors, Scatchard analysis of [3H]muscimol binding to
cerebellar membranes of
+/+ and
/
mice was carried out.
Comparable Kd values and a slight increase in
the Bmax value in
/
mice were found, but
this increase in the Bmax value did not reach
statistical significance (Table II). Similar results were obtained from Scatchard analysis of
[3H]muscimol binding to receptors extracted and
immunoprecipitated with a mixture of antibodies directed against the
1,
2, and
3 subunits,
which should precipitate the vast majority of GABAA receptors (13, 17): Bmax values measured in
/
mice were higher than those found in
+/+ mice, but the
difference again did not reach statistical significance.
Comparison of total GABAA receptors by Scatchard analysis of
[3H]muscimol binding
+/+ and
/
mice (A) or GABAA
receptors extracted from these membranes and precipitated as described
under "Experimental Procedures" (B) were incubated with various
concentrations of [3H]muscimol in the absence or presence of
1 mM GABA. Membranes or precipitated receptors were then
filtered through Whatman GF/B filters and washed as described under
"Experimental Procedures." Scatchard analysis was performed from
[3H]muscimol binding data that could be displaced by 1 mM GABA (total specific binding). Data are mean values ± S.E. from three experiments performed in triplicate. For statistical
comparisons unpaired Student's t tests were used.
+/+ and
/
cerebella, respectively, could be recovered as binding sites in the
membrane extract (Table II). The recovery of [3H]muscimol
binding sites was therefore comparable to that of total, diazepam-sensitive, or diazepam-insensitive [3H]Ro15-4513
binding sites. The similar proportions of different binding sites
recovered, as well as the parallel changes in membrane-bound and
-extracted binding sites in
/
mice, indicate that the receptors recovered in the extract were representative of the entire functional GABAA receptor population.
/
Mice--
Cerebellar membrane
extracts from
+/+ and
/
mice were cycled on an
6-(317-371) immunoaffinity column until
6 subunits were no longer detectable in the efflux of
the column using immunoprecipitation experiments or Western blot
analysis. Total GABAA receptors present in the original
membrane extract and in the column efflux were then determined by
[3H]muscimol binding studies after precipitation with a
mixture of
1,
2, and
3
subunit-specific antibodies. The total amount of GABAA
receptors in the column efflux was reduced by 55.7% in
+/+ and by
57.5% in
/
mice, indicating that the proportion of
6 subunit-containing receptors present in the extracts
from these tissues was similar (Table
III).
Extent of copurification of GABAA receptor subunits with
6 subunits from
+/+ and
/
mice
+/+
and
/
mice, and
6 subunit-containing receptors were
completely eliminated from the extract by chromatography on an
immunoaffinity column containing the antibody
6-(317-371).
GABAA receptors containing
1,
2,
3,
2, or
subunits were precipitated in the
original extract and in the column efflux using the respective
subunit-specific antibodies. Precipitated receptor subtypes were
quantified using [3H]muscimol binding. The percentage of
reduction in the column efflux of receptors containing the respective
subunit indicated their extent of colocalization with
6
subunits in the same receptors. Data are mean values ± S.E. with
the numbers of experiments given in parentheses. Results from
/
mice were statistically compared with results from
+/+ mice using
Student's unpaired t test.
1,
2,
3,
2, or
subunit-containing receptors after chromatography on the
anti-
6 column was determined. For this, receptors
containing these subunits were immunoprecipitated in the original
extract and the anti-
6 column efflux with
1,
2,
3,
2,
or
subunit-specific antibodies and were quantitatively determined
by [3H]muscimol binding. In the absence of any
cross-reactivity of the anti-
6 antibodies with other
GABAA receptor subunits (13, 17), the percentage of
reduction indicates the percentage of these receptors containing
6 subunits. As shown in Table III, the percentage of
1,
2,
3,
2,
or
subunit-containing receptors containing
6
subunits in the cerebellum of
+/+ mice was similar to that reported
previously (17) for the cerebellum of wild-type mice.
1,
2, and
3 subunit-containing receptors removed by the
anti-
6 column from extracts of
/
cerebella was
comparable, indicating that a similar proportion of these receptors
contained
6 subunits in control and
/
cerebella (Table III).
2 receptors eliminated by
the anti-
6 column was significantly larger in the
cerebellum of
/
mice, suggesting an increased formation of
GABAA receptors containing both
6 and
2 subunits. The actual increase in the number of
receptors containing these subunits, however, was even larger, because
of the increased expression of
2 subunits (Fig. 2) and
the increased number of
2 subunit-containing receptors in
/
cerebellum (Table I).
2-containing
receptors, these receptors were completely eliminated from cerebellar extracts of
+/+ and
/
mice using an immunoaffinity column containing the
2-(319-366) antibody. This column
eliminated 68.3% of all receptors in
+/+ and 76.5% of all
receptors in
/
mice. These proportions were not significantly
different (Table IV). It has to be kept
in mind, however, that when comparing the number of
2-containing receptors present in
+/+ and
/
mice, the total number of GABAA receptors in these tissues
has to be considered (Table II).
Extent of copurification of GABAA receptor subunits with
2 subunits from
+/+ and
/
mice
+/+
and
/
mice, and
2 subunit-containing receptors were
completely eliminated from the extract by chromatography on an
immunoaffinity column containing the antibody
2-(319-366).
GABAA receptors containing
1,
6,
2,
3, or
subunits were precipitated in the
original extract and in the column efflux using the respective
subunit-specific antibodies. Precipitated receptor subtypes were
quantified using [3H]muscimol binding. The percentage of
reduction in the column efflux of receptors containing the respective
subunit indicated their extent of colocalization with
2
subunits in the same receptor. Data are mean values ± S.E. with
the numbers of experiments given in parentheses. Results from
/
mice were statistically compared with results from
+/+ mice using
Student's unpaired t test.
1
subunit-containing receptors was removed by the anti-
2
affinity column in
+/+ and
/
extracts. In agreement with the
results from the anti-
6 column (Table III) a
significantly higher percentage of
6 receptors was
associated with
2 subunits in extracts from
/
cerebella (Table IV). Although only 52.4% of
6
receptors were eliminated by the anti-
2 subunit column
from membrane extracts of
+/+ mice, 70.2% of
6
receptors were eliminated from those of
/
mice. The extent of
association with
2 subunits was also slightly elevated for
2 subunits: 67% of
2
subunit-containing receptors contained
2 subunits in
+/+ and 75.2% in
/
cerebella. The degree of association of
3 and
2 subunits, however, was comparable
in the cerebellum of
+/+ and
/
mice (Table IV).
subunit-containing receptors present in cerebellar extracts from
+/+
mice (28.8 ± 4.2% of all receptors) was not reduced in the
efflux of the anti-
2 subunit column (Table IV). This
demonstrates that GABAA receptors containing both
2 and
subunits are not present in the cerebellum.
(28.8%)- and
2
(68.3%)-containing receptors in
+/+ mice indicates that 97.1% of
all receptors in the cerebellum are assemblies of
2 or
combinations. Interestingly,
despite the absence of
subunits, only 76.5% of all receptors in
/
mice contained
2 subunits. This raised the
possibility that the remaining GABAA receptors contained
up-regulated
1 or
3 subunits. However, no
significant amounts of
1 or
3 subunit-containing GABAA receptors could be
immunoprecipitated in cerebellar extracts from
+/+ and
/
mice
using
1-(324-366) or
3-(322-372)
antibodies under conditions where these antibodies precipitated
significant amounts of receptors in forebrain extracts (experiments not
shown, Ref. 21). This indicated that 23.5% of all GABAA
receptors in cerebellum extracts from
/
mice did not contain
1,
2,
3, or
subunits.
Because significant amounts of
1,
6,
2, or
3 subunit-containing receptors
remained in the extract after complete elimination of
2
subunit-containing receptors (Table IV), it can be concluded that
the remaining 23.5% of GABAA receptors were composed of
combinations of
1,
6,
2, and
3 subunits, or additionally contained as yet
unidentified subunits.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
Cerebellum--
In the present study possible changes in the
abundance and subunit composition of GABAA receptors were
investigated in the brain of mice with a targeted disruption of the
subunit expression. Because the
subunit is much more abundant in
the cerebellum than in the forebrain (30, 31), we focused our
investigation on this brain region. Despite the loss of
subunit-containing receptors, which comprise nearly 30% of all
GABAA receptors in the cerebellum, the number of
GABAA receptors was not reduced in membranes or membrane
extracts of
/
cerebellum, as demonstrated by Scatchard analysis
of [3H]muscimol binding. These data are in contrast to
those of a previous study (18) in which a significant reduction in
[3H]muscimol binding was observed in whole brain
homogenates of
/
mice. The different tissues used, as well as
differences in the tissue preparation and [3H]muscimol
binding assay conditions, might have contributed to the different results.
2
Subunits in
/
Cerebellum--
Western blot analysis
demonstrated a 45% increase in the expression of
2
subunits in the cerebellum of
/
mice, which corresponded to a
52% increase in GABAA receptors containing
[3H]Ro15-4513 binding sites. This finding is consistent
with the elevated [3H]Ro15-4513 binding in cerebellum and
several other brain tissues of
/
mice observed in
autoradiographic studies (18). The increase in
[3H]Ro15-4513 binding sites was due to a dramatic
increase in DIS binding sites in membranes and extracts from
/
cerebellum. Because DIS binding sites are formed by receptors
containing
6
2 or
1
6
2 subunits (12, 25,
27), these data suggested a significant increase in the formation of
receptors containing
6 as well as
2
subunits in the cerebellum of
/
mice.
6 subunit column showed
that the proportion of
2 receptors containing
6 subunits was significantly larger in membrane extracts
from
/
than in those from
+/+ mice. Similarly, the use of an
anti-
2 subunit column showed the increased coassembly of
the
2 and
6 subunits but not the
2 and
1 subunits in extracts from
/
cerebella. These data indicated an increased formation of
6
2 but not of
1
6
2 receptors in
/
cerebellum.
Subunits
in
/
Cerebellum--
In wild-type cerebellum, 68.3% and 28.8%
of GABAA receptors contained
2 and
subunits, respectively. Because
2 and
subunits are
not colocalized in the same GABAA receptors in this tissue (13, 28) 97% of all cerebellar GABAA receptors contained
2 or
subunits. This is consistent with the finding
that receptors containing
1 or
3 subunits
are not very abundant in the cerebellum (30, 31). After the complete
loss of
receptors, most of the remaining receptors might be
expected to contain
2 subunits. However, only 76.5% of
all receptors in the extract of
/
cerebella contained
2 subunits. Because
1 or
3
receptors were not up-regulated in the cerebellum of
/
mice, the
remaining receptors must have been composed of either
and
subunits only, or these subunits may have been associated with as yet
unidentified subunits. Because comparable proportions of
1,
6,
2, or
3 subunit-containing receptors remained in the extracts
of
/
cerebellum after the removal of
2
subunit-containing receptors, the remaining receptors were probably
composed of various combinations of
1,
6,
2, and
3 subunits. The exact composition
of receptors formed, however, cannot be deduced from the data available.
/
Cerebella Mostly Occur in Granule
Cells--
Immunohistochemical observations indicated a large increase
in labeling for the
2 subunit in the cerebellar granule
cell layer and a small increase in the molecular layer of
/
mice. Combined with the fact that in cerebellum
6
subunits are exclusively expressed in granule cells (where they are
colocalized with
subunits in wild-type mice) and with the
observation suggesting an increased formation of
6
2 receptors in
/
mice, this
indicates that most of the changes in receptor composition observed in
mice with a targeted disruption of the
subunit were induced in
granule cells.
1
and
3 subunits was observed in Western blot studies, no
apparent change in the expression of these subunits was observed in
immunocytochemical studies in
/
cerebella. Because changes in
1 and
3 subunit expression were small and
varied in different animals, they might have escaped detection in
immunocytochemical studies. Because there was no change in the number
of DS [3H]Ro15-4513 binding sites in the cerebellum of
/
mice, and because the extent of coassembly of
1
and
3 subunits with
2 receptors was
comparable in extracts from
+/+ and
/
cerebella, the
increased abundance of
1 and
3 subunits
did not result in an increased formation of
1
3
2 receptors. These
subunits, therefore, either formed
1
3
receptors that contributed to the number of [3H]muscimol
but not of [3H]Ro15-4513 binding sites (32) or
represented unassembled
1 and
3 subunits.
The first possibility seems to be more likely, because specific
[3H]muscimol binding in cerebellar membranes or membrane
extracts was slightly, although not significantly, increased in
/
mice.
/
Cerebellum--
Immunoaffinity purification on an
anti-
6 subunit column showed that
subunits almost
exclusively are coassembled with
6 subunits. The
corresponding GABAA receptors are composed of
6
and
1
6
subunits in the rat (13). Because there was no change in the expression
of
6 subunits in the cerebellum of
/
mice,
6
and
1
6
receptors presumably were converted into
6
,
1
6
, and
6
2 receptors.
and
subunits form subunit tetramers and
pentamers, whereas
,
, and
subunits exclusively form
pentamers (20). The rate of formation of
pentamers, therefore,
presumably is significantly slower than that of
(and possibly
also of
) pentamers (33, 34). Once formed, however,
pentamers are transported to the cell surface (34) and form functional
receptors (35). It is, therefore, possible that most of the
subunit combinations that do not assemble with
2
subunits in the cerebellum of
/
mice represented completely
assembled and functional receptors. However, the presence of functional
receptor on the surface of these cells needs to be directly demonstrated.
tetramers, however, presumably also act as
a trap for
subunits. Any
subunit newly synthesized at sites
where preformed
tetramers are located may preferentially combine
with the tetramers to complete the assembly of receptors composed of
subunits (33). Such a mechanism may explain the presence of
6
2,
1
6
2,
6
, and
1
6
receptors
in the cerebellum of
/
mice.
2,
3, or
6 subunits did not induce a
compensatory up-regulation of the transcription of other
GABAA-receptor subunit genes in mutant mouse brain (16,
36-38). But these studies also indicated that the availability of
and
subunits was essential for receptor formation. Thus, upon
disruption of the
6 or
3 subunit
expression, the extent of reduction in the number of the remaining
receptors indicated a complete loss of receptors containing the
respective subunit and a corresponding reduction in the number of the
remaining GABAA receptors (39).
2 subunit expression, however,
neither the number of GABAA receptors nor the protein level
of the subunits
1,
2,
3,
2/3, were reduced, indicating the predominant formation
of receptors composed of
subunits (36). In addition, the protein
level of
1 and
3 subunits remained
unaltered in
2
/
mice as shown by Western blotting.
This may indicate that the supply of
1 and
3 subunits is not linked to the availability of
subunits, because receptors containing these subunits either are not
synthesized in the same cells or the same subcellular compartments as
those containing
2 subunits. The extent of expression of
subunits was not investigated in this study.
subunits without
additional subunit classes in the cerebellum of
/
mice is
consistent with the expression of these receptors in
2
/
mice (36). The additional up-regulation of
6
2 receptors in
/
mice can be
explained if the level of
2 subunit expression is linked
to the function of the granule cells. The latter mechanism might be
unique for
2 subunits, which are the most abundant
GABAA receptor subunits in the brain and seem to be
responsible for receptor anchoring (40, 41). Alternatively, it is
possible that in wild-type mice
2 and
subunits
compete for
6 and
subunits during assembly of
receptors and that subunits unable to find assembly partners are
degraded (34). A lack of
subunits in
/
mice might then
reduce degradation of the surplus
2 subunits by
increasing their chances to find assembly partners, thus leading to an
increased formation of
6
2 receptors.
Future studies will have to decide which of these possibilities most
likely occur.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to J. M. Fritschy, Dept.
Pharmacology, University of Zurich, Switzerland, for generously
providing the antibody 2-(1-29).
![]() |
FOOTNOTES |
---|
* This study was supported by a European Commission Shared Cost RTD Programme Grant (ERBBIO4CT960585) and by National Institutes of Health Grants AA10422 and GM52035.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
Present address: Institute of Experimental Medicine, Hungarian
Academy of Sciences, Budapest H-1450, Hungary.
§§ To whom correspondence should be addressed: Brain Research Institute of the University of Vienna, Spitalgasse 4, Vienna A-1090, Austria. Tel.: 43-1-4277-62950; Fax: 43-1-4277-62959, E-mail: Werner.Sieghart@univie.ac.at.
Published, JBC Papers in Press, January 1, 2001, DOI 10.1074/jbc.M011054200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
GABA, -aminobutyric acid;
NGS, normal goat serum;
PB, phosphate buffer;
TBS, Tris-buffered saline;
DS, diazepam-sensitive;
DIS, diazepam-insensitive.
![]() |
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