Targeted Disruption of the GABAA Receptor delta  Subunit Gene Leads to an Up-regulation of gamma 2 Subunit-containing Receptors in Cerebellar Granule Cells*

Verena TretterDagger §, Birgit HauerDagger §, Zoltan Nusser||, Robert M. Mihalek**, Harald HögerDagger Dagger , Gregg E. Homanics**, Peter Somogyi, and Werner SieghartDagger §§

From the Dagger  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 Dagger Dagger  Research Institute for Laboratory Animal Breeding, Himberg A-2325, Austria

Received for publication, December 8, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 delta  subunit gene. In membranes and extracts of delta -/- 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 alpha 6 subunits but an increased expression of gamma 2 subunits in delta -/- cerebellum. Immunoaffinity chromatography of cerebellar extracts indicated there was an increased coassembly of alpha 6 and gamma 2 subunits and that 24% of all receptors in delta -/- cerebellum did not contain a gamma  subunit. Because 97% of delta  subunits are coassembled with alpha 6 subunits in the cerebellum of wild-type mice, these results indicated that, in delta -/- mice, alpha 6beta gamma 2 and alpha beta receptors replaced delta  subunit-containing receptors. The availability of the delta  subunit, thus, influences the level of expression or the extent of assembly of the gamma 2 subunit, although these two subunits do not occur in the same receptor.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

gamma -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).

GABAA receptors are composed of five subunits derived from different subunit classes. So far, six alpha , four beta , three gamma , one delta , one epsilon , one pi , one theta , and three rho  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).

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 (alpha 1, alpha 6, beta 2, beta 3, gamma 2, and delta ), forming alpha 1beta gamma 2, alpha 6beta gamma 2, alpha 1alpha 6beta gamma 2, alpha 6beta delta , and alpha 1alpha 6beta delta 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.

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 alpha 6 subunit gene, not only were the alpha 6 subunits completely eliminated from cerebellar granule cells but also the delta  subunits were dramatically reduced (16, 17). These results suggested that the alpha 6 subunit is necessary for the oligomerization and surface expression of the delta  subunit.

The recent generation of delta -/- mice (18) allowed to investigate whether the delta  subunit is also essential for the surface expression of the alpha 6 subunit. Therefore, in the present study, the abundance and subunit composition of GABAA receptors was compared in the cerebellum of delta +/+ and delta -/- mice. In contrast to alpha 6-/- mice (17), the total number of GABAA receptors was not reduced in the cerebellum of delta -/- mice, despite the complete loss of delta  subunits. Instead, an up-regulation of the gamma 2 subunit-containing receptors was observed. Furthermore, the composition of GABAA receptors in the cerebellum of delta -/- mice differed significantly from that of delta +/+ mice.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Generation of Mutant Mice-- Targeted disruption of the delta  subunit of the GABAA receptor in mouse embryonic stem cells and production of wild-type (delta +/+), and homozygous delta -/- 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 delta  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. delta +/+ mice were the F3 generation on a mixed C57BL/6J × strain 129Sv/SvJ genetic background.

Antibodies-- The generation of anti-peptide alpha 1-(1-9) (19), anti-peptide alpha 6-(317-371) (17), anti-peptide beta 1-(350-404) (13), anti-peptides beta 2-(351-405), beta 3-(345-408) and gamma 2-(319-366) (20), gamma 1-(324-366) or gamma 3-(322-372) antibodies (21), anti-peptide delta -(1-44) antibodies (16), or anti-peptide gamma 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).

Quantitative Immunoblot Analysis-- Membranes from a total of 12 delta +/+ and 12 delta -/- 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.

To test for equal protein loading, in some experiments a monoclonal anti-beta -actin antibody, was included in the antibody solution, and the amounts of endogenous beta -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 beta -actin neither changed the results nor reduced variability.

Preparation of Membrane Extracts, Affinity Chromatography, and Immunoprecipitation-- GABAA receptors were solubilized from cerebellar membranes of delta +/+ and delta -/- 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.

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 beta 1-(350-404), 18 µg of beta 2-(351-405), and 15 µg of beta 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 beta  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 beta 1-(350-404), beta 2-(351-405), and beta 3-(345-408) antibodies used were sufficient to maximally precipitate GABAA receptors containing the respective beta  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).

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 delta +/+ and five adult delta -/- 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): alpha 1 = 0.9-1.3 (P16); alpha 6 = 0.5-0.6 (P24); beta 2 = 0.9 (beta 2-(351-405)R23); beta 3 = 1.7-2.0 (beta 3-(345-408)R1); gamma 2 = 1.0 (gamma 2-(1-29), 22); and delta  = 1.1 (delta -(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 delta +/+ and three delta -/- 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: alpha 1(P16), 0.6 µg/ml; gamma 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).

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 delta -/- animals were compared with the unpaired t test.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Immunoreactivity for the delta  Subunit in Control and delta -/- Mice-- In cerebellum, immunoreactivity for the delta  subunit is restricted to the granule cell layer (15-17, 23) (Fig. 1). After targeted disruption of the delta  subunit gene, staining of granule cells with the delta -(1-44) antibody (16, 17) completely disappeared (Fig. 1). In agreement with these immunocytochemical results, neither the complete delta  subunit (54-kDa protein band) nor a truncated delta  subunit protein were detectable in cerebellar membranes of delta -/- mice by Western blot analysis (18). Finally, in contrast to delta +/+ mice, where an immunoaffinity column containing the delta -(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 delta -/- mice, confirming the absence of delta  subunit-containing receptors in mice with a targeted disruption of the delta  subunit.


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Fig. 1.   Changes in the expression of the gamma 2 and delta  subunits of the GABAA receptor in the cerebellum of delta -/- mice. Pre-embedding immunoperoxidase reaction with antibodies gamma 2-(1-29) and delta -(1-44)R5. An increase in the immunoreactivity for the gamma 2 subunit was detected in the granule cell layer (gcl) of delta -/- mice, without any change in the molecular layer (ml). Immunoreactivity for the delta  subunit completely disappeared in delta -/- mice. +/+, control mice; Pcl, Purkinje cell layer; scale bar, 50 µm.

Immunoreactivity for alpha 1, alpha 6, beta 2, beta 3, and gamma 2 Subunits in Control and delta -/- Mice-- In addition to the delta  subunit, five other major subunits can be detected in cerebellar granule cells: alpha 1, alpha 6, beta 2, beta 3, and gamma 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 alpha 1, alpha 6, beta 2, and beta 3 subunits, but showed an up-regulation of immunoreactivity for the gamma 2 subunit in the cerebellar granule cell layer of delta -/- animals (Fig. 1).

To quantify the change, light microscopic immunofluorescence labeling for the gamma 2 and alpha 1 subunits was carried out and measured. For the gamma 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, delta -/-, p < 0.01, unpaired t test) in the granule cell layer of delta -/- mice. In addition, there was also a small (18%) but significant (p < 0.01, unpaired t test) increase in gamma 2 subunit labeling in the molecular layer of the delta -/- mice (49.1 ± 1.8, n = 3, control; 58.2 ± 2.4, n = 3, delta -/-). In agreement with the results of the peroxidase reactions, no significant change (p > 0.05, unpaired t test) was detected in immunoreactivity for the alpha 1 subunit in either the granule cell (81.0 ± 6.7, n = 3, control; 91.5 ± 4.7, n = 3, delta -/-) or the molecular layers (75.2 ± 6.0, n = 3, control; 84.3 ± 13.0, n = 3, delta -/-).

Membranes from the cerebellum of delta +/+ or delta -/- mice were then subjected to SDS-polyacrylamide gel electrophoresis and quantitative Western blot analysis, using subunit-specific antibodies directed against alpha 1, alpha 6, beta 2, beta 3, gamma 2, and delta  subunits (13, 17). As shown in Fig. 2, the expression of alpha 1 subunits was increased by 25% in the cerebellum of delta -/- mice, whereas the levels of alpha 6 or beta 2 subunits were not significantly different from those found in delta +/+ mice. The expression of beta 3 subunits was increased by 23% and that of gamma 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 delta +/+ and delta -/- membranes by Western blot analysis. A, representative blot comparing immunoreactivity for alpha 1, alpha 6, beta 2, beta 3, and gamma 2 subunits in three delta +/+ and three delta -/- cerebella. The chemiluminescence signals were quantified by densitometry. Results are expressed as percentage of the subunit level found in delta +/+ membranes ± S.E. B, results for alpha 1, alpha 6, beta 2, beta 3, and gamma 2 subunits were obtained from 12 delta +/+ and 12 delta -/- mice. Results for delta  subunits were from three delta +/+ and three delta -/- mice. No staining for the delta  subunit could be detected in cerebellar membranes of delta -/- mice. For statistical comparisons unpaired Student's t test was used. * p < 0.001; **p < 0.0001.

[3H]Ro15-4513 Binding in Cerebellum of Control and delta -/- Mice-- To investigate whether the additional gamma 2 subunits expressed in delta -/- cerebellum were part of functional receptors, [3H]Ro15-4513 binding studies were performed in membranes from delta +/+ and delta -/- cerebellum. [3H]Ro15-4513 is a ligand for the benzodiazepine binding site of GABAA receptors, which are only formed by receptors containing a gamma  subunit (2). Scatchard analysis of specific [3H]Ro15-4513 binding indicated that the total number (Bmax) of sites was increased by 52% in delta -/- 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 delta +/+ 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 alpha 6 subunits (27) was increased by 180% in delta -/- mice. The diazepam-sensitive (DS) [3H]Ro15-4513 binding, however, was comparable in delta -/- and delta +/+ mice (Table I).

                              
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Table I
Comparison of benzodiazepine binding sites by Scatchard analysis of [3H]Ro15-4513 binding
Cerebellar membranes from delta +/+ and delta -/- 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.

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 delta +/+ cerebella (2072 and 1640 fmol/mg of protein) were comparable to those observed in delta -/- 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).

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 beta 1, beta 2, and beta 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 delta +/+ and 57%, 53%, and 60% in delta -/- 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 delta -/- mice were increased by 55% as observed in the corresponding membranes. Whereas DS binding was comparable in the extracts of delta +/+ and delta -/- cerebella, DIS binding in delta -/- extracts was increased by 216% relative to that in delta +/+ extracts (Table I).

[3H]Muscimol Binding in the Cerebellum of Control and delta -/- Mice-- To investigate the abundance of GABAA receptors, Scatchard analysis of [3H]muscimol binding to cerebellar membranes of delta +/+ and delta -/- mice was carried out. Comparable Kd values and a slight increase in the Bmax value in delta -/- 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 beta 1, beta 2, and beta 3 subunits, which should precipitate the vast majority of GABAA receptors (13, 17): Bmax values measured in delta -/- mice were higher than those found in delta +/+ mice, but the difference again did not reach statistical significance.

                              
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Table II
Comparison of total GABAA receptors by Scatchard analysis of [3H]muscimol binding
Cerebellar membranes from delta +/+ and delta -/- 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.

A comparison of Bmax values in cerebellar membranes and solubilized receptors indicated that ~52% and 61% of GABAA receptors present in membranes of delta +/+ and delta -/- 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 delta -/- mice, indicate that the receptors recovered in the extract were representative of the entire functional GABAA receptor population.

Subunit Composition of GABAA Receptors in the Cerebellum of Control and delta -/- Mice-- Cerebellar membrane extracts from delta +/+ and delta -/- mice were cycled on an alpha 6-(317-371) immunoaffinity column until alpha 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 beta 1, beta 2, and beta 3 subunit-specific antibodies. The total amount of GABAA receptors in the column efflux was reduced by 55.7% in delta +/+ and by 57.5% in delta -/- mice, indicating that the proportion of alpha 6 subunit-containing receptors present in the extracts from these tissues was similar (Table III).

                              
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Table III
Extent of copurification of GABAA receptor subunits with alpha 6 subunits from delta +/+ and delta -/- mice
GABAA receptors were extracted from the cerebellum of delta +/+ and delta -/- mice, and alpha 6 subunit-containing receptors were completely eliminated from the extract by chromatography on an immunoaffinity column containing the antibody alpha 6-(317-371). GABAA receptors containing alpha 1, beta 2, beta 3, gamma 2, or delta  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 alpha 6 subunits in the same receptors. Data are mean values ± S.E. with the numbers of experiments given in parentheses. Results from delta -/- mice were statistically compared with results from delta +/+ mice using Student's unpaired t test.

In other experiments, the extent of reduction of alpha 1, beta 2, beta 3, gamma 2, or delta  subunit-containing receptors after chromatography on the anti-alpha 6 column was determined. For this, receptors containing these subunits were immunoprecipitated in the original extract and the anti-alpha 6 column efflux with alpha 1, beta 2, beta 3, gamma 2, or delta  subunit-specific antibodies and were quantitatively determined by [3H]muscimol binding. In the absence of any cross-reactivity of the anti-alpha 6 antibodies with other GABAA receptor subunits (13, 17), the percentage of reduction indicates the percentage of these receptors containing alpha 6 subunits. As shown in Table III, the percentage of alpha 1, beta 2, beta 3, gamma 2, or delta  subunit-containing receptors containing alpha 6 subunits in the cerebellum of delta +/+ mice was similar to that reported previously (17) for the cerebellum of wild-type mice.

The percentage of alpha 1, beta 2, and beta 3 subunit-containing receptors removed by the anti-alpha 6 column from extracts of delta -/- cerebella was comparable, indicating that a similar proportion of these receptors contained alpha 6 subunits in control and delta -/- cerebella (Table III).

In contrast, the percentage of gamma 2 receptors eliminated by the anti-alpha 6 column was significantly larger in the cerebellum of delta -/- mice, suggesting an increased formation of GABAA receptors containing both alpha 6 and gamma 2 subunits. The actual increase in the number of receptors containing these subunits, however, was even larger, because of the increased expression of gamma 2 subunits (Fig. 2) and the increased number of gamma 2 subunit-containing receptors in delta -/- cerebellum (Table I).

To further investigate the composition of gamma 2-containing receptors, these receptors were completely eliminated from cerebellar extracts of delta +/+ and delta -/- mice using an immunoaffinity column containing the gamma 2-(319-366) antibody. This column eliminated 68.3% of all receptors in delta +/+ and 76.5% of all receptors in delta -/- mice. These proportions were not significantly different (Table IV). It has to be kept in mind, however, that when comparing the number of gamma 2-containing receptors present in delta +/+ and delta -/- mice, the total number of GABAA receptors in these tissues has to be considered (Table II).

                              
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Table IV
Extent of copurification of GABAA receptor subunits with gamma 2 subunits from delta +/+ and delta -/- mice
GABAA receptors were extracted from the cerebellum of delta +/+ and delta -/- mice, and gamma 2 subunit-containing receptors were completely eliminated from the extract by chromatography on an immunoaffinity column containing the antibody gamma 2-(319-366). GABAA receptors containing alpha 1, alpha 6, beta 2, beta 3, or delta  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 gamma 2 subunits in the same receptor. Data are mean values ± S.E. with the numbers of experiments given in parentheses. Results from delta -/- mice were statistically compared with results from delta +/+ mice using Student's unpaired t test.

Quantification in the original membrane extract and column efflux of receptor subtypes precipitated with subunit-specific antibodies indicated that a comparable proportion of alpha 1 subunit-containing receptors was removed by the anti-gamma 2 affinity column in delta +/+ and delta -/- extracts. In agreement with the results from the anti-alpha 6 column (Table III) a significantly higher percentage of alpha 6 receptors was associated with gamma 2 subunits in extracts from delta -/- cerebella (Table IV). Although only 52.4% of alpha 6 receptors were eliminated by the anti-gamma 2 subunit column from membrane extracts of delta +/+ mice, 70.2% of alpha 6 receptors were eliminated from those of delta -/- mice. The extent of association with gamma 2 subunits was also slightly elevated for beta 2 subunits: 67% of beta 2 subunit-containing receptors contained gamma 2 subunits in delta +/+ and 75.2% in delta -/- cerebella. The degree of association of beta 3 and gamma 2 subunits, however, was comparable in the cerebellum of delta +/+ and delta -/- mice (Table IV).

Supporting previous conclusions (13, 28, 29), the percentage of delta  subunit-containing receptors present in cerebellar extracts from delta +/+ mice (28.8 ± 4.2% of all receptors) was not reduced in the efflux of the anti-gamma 2 subunit column (Table IV). This demonstrates that GABAA receptors containing both gamma 2 and delta  subunits are not present in the cerebellum.

Summing of the proportions of delta  (28.8%)- and gamma 2 (68.3%)-containing receptors in delta +/+ mice indicates that 97.1% of all receptors in the cerebellum are assemblies of alpha beta gamma 2 or alpha beta delta combinations. Interestingly, despite the absence of delta  subunits, only 76.5% of all receptors in delta -/- mice contained gamma 2 subunits. This raised the possibility that the remaining GABAA receptors contained up-regulated gamma 1 or gamma 3 subunits. However, no significant amounts of gamma 1 or gamma 3 subunit-containing GABAA receptors could be immunoprecipitated in cerebellar extracts from delta +/+ and delta -/- mice using gamma 1-(324-366) or gamma 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 delta -/- mice did not contain gamma 1, gamma 2, gamma 3, or delta  subunits. Because significant amounts of alpha 1, alpha 6, beta 2, or beta 3 subunit-containing receptors remained in the extract after complete elimination of gamma 2 subunit-containing receptors (Table IV), it can be concluded that the remaining 23.5% of GABAA receptors were composed of combinations of alpha 1, alpha 6, beta 2, and beta 3 subunits, or additionally contained as yet unidentified subunits.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

No Reduction in the Number of GABAA Receptors in delta -/- 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 delta  subunit expression. Because the delta  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 delta  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 delta -/- 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 delta -/- 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.

Increase in GABAA Receptors Containing gamma 2 Subunits in delta -/- Cerebellum-- Western blot analysis demonstrated a 45% increase in the expression of gamma 2 subunits in the cerebellum of delta -/- 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 delta -/- 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 delta -/- cerebellum. Because DIS binding sites are formed by receptors containing alpha 6beta gamma 2 or alpha 1alpha 6beta gamma 2 subunits (12, 25, 27), these data suggested a significant increase in the formation of receptors containing alpha 6 as well as gamma 2 subunits in the cerebellum of delta -/- mice.

This conclusion was supported by studies investigating subunit partnerships in GABAA receptors: Immunoaffinity chromatography using an anti-alpha 6 subunit column showed that the proportion of gamma 2 receptors containing alpha 6 subunits was significantly larger in membrane extracts from delta -/- than in those from delta +/+ mice. Similarly, the use of an anti-gamma 2 subunit column showed the increased coassembly of the gamma 2 and alpha 6 subunits but not the gamma 2 and alpha 1 subunits in extracts from delta -/- cerebella. These data indicated an increased formation of alpha 6beta gamma 2 but not of alpha 1alpha 6beta gamma 2 receptors in delta -/- cerebellum.

Increase in GABAA Receptors Composed of alpha beta Subunits in delta -/- Cerebellum-- In wild-type cerebellum, 68.3% and 28.8% of GABAA receptors contained gamma 2 and delta  subunits, respectively. Because gamma 2 and delta  subunits are not colocalized in the same GABAA receptors in this tissue (13, 28) 97% of all cerebellar GABAA receptors contained gamma 2 or delta  subunits. This is consistent with the finding that receptors containing gamma 1 or gamma 3 subunits are not very abundant in the cerebellum (30, 31). After the complete loss of delta  receptors, most of the remaining receptors might be expected to contain gamma 2 subunits. However, only 76.5% of all receptors in the extract of delta -/- cerebella contained gamma 2 subunits. Because gamma 1 or gamma 3 receptors were not up-regulated in the cerebellum of delta -/- mice, the remaining receptors must have been composed of either alpha  and beta  subunits only, or these subunits may have been associated with as yet unidentified subunits. Because comparable proportions of alpha 1, alpha 6, beta 2, or beta 3 subunit-containing receptors remained in the extracts of delta -/- cerebellum after the removal of gamma 2 subunit-containing receptors, the remaining receptors were probably composed of various combinations of alpha 1, alpha 6, beta 2, and beta 3 subunits. The exact composition of receptors formed, however, cannot be deduced from the data available.

Changes in the Subunit Composition of GABAA Receptors in delta -/- Cerebella Mostly Occur in Granule Cells-- Immunohistochemical observations indicated a large increase in labeling for the gamma 2 subunit in the cerebellar granule cell layer and a small increase in the molecular layer of delta -/- mice. Combined with the fact that in cerebellum alpha 6 subunits are exclusively expressed in granule cells (where they are colocalized with delta  subunits in wild-type mice) and with the observation suggesting an increased formation of alpha 6beta gamma 2 receptors in delta -/- mice, this indicates that most of the changes in receptor composition observed in mice with a targeted disruption of the delta  subunit were induced in granule cells.

Although a significant increase in the expression of alpha 1 and beta 3 subunits was observed in Western blot studies, no apparent change in the expression of these subunits was observed in immunocytochemical studies in delta -/- cerebella. Because changes in alpha 1 and beta 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 delta -/- mice, and because the extent of coassembly of alpha 1 and beta 3 subunits with gamma 2 receptors was comparable in extracts from delta +/+ and delta -/- cerebella, the increased abundance of alpha 1 and beta 3 subunits did not result in an increased formation of alpha 1beta 3gamma 2 receptors. These subunits, therefore, either formed alpha 1beta 3 receptors that contributed to the number of [3H]muscimol but not of [3H]Ro15-4513 binding sites (32) or represented unassembled alpha 1 and beta 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 delta -/- mice.

Molecular Mechanism Causing the Changes in GABAA Receptor Subunit Composition in delta -/- Cerebellum-- Immunoaffinity purification on an anti-alpha 6 subunit column showed that delta  subunits almost exclusively are coassembled with alpha 6 subunits. The corresponding GABAA receptors are composed of alpha 6beta delta and alpha 1alpha 6beta delta subunits in the rat (13). Because there was no change in the expression of alpha 6 subunits in the cerebellum of delta -/- mice, alpha 6beta delta and alpha 1alpha 6beta delta receptors presumably were converted into alpha 6beta , alpha 1alpha 6beta , and alpha 6beta gamma 2 receptors.

Studies on GABAA receptor assembly in non-neuronal cells have indicated that alpha  and beta  subunits form subunit tetramers and pentamers, whereas alpha , beta , and gamma  subunits exclusively form pentamers (20). The rate of formation of alpha beta pentamers, therefore, presumably is significantly slower than that of alpha beta gamma (and possibly also of alpha beta delta ) pentamers (33, 34). Once formed, however, alpha beta pentamers are transported to the cell surface (34) and form functional receptors (35). It is, therefore, possible that most of the alpha beta subunit combinations that do not assemble with gamma 2 subunits in the cerebellum of delta -/- mice represented completely assembled and functional receptors. However, the presence of functional alpha beta receptor on the surface of these cells needs to be directly demonstrated.

The relatively stable alpha beta tetramers, however, presumably also act as a trap for gamma  subunits. Any gamma  subunit newly synthesized at sites where preformed alpha beta tetramers are located may preferentially combine with the tetramers to complete the assembly of receptors composed of alpha beta gamma subunits (33). Such a mechanism may explain the presence of alpha 6beta gamma 2, alpha 1alpha 6beta gamma 2, alpha 6beta , and alpha 1alpha 6beta receptors in the cerebellum of delta -/- mice.

Comparison with Previous Studies on Targeted Disruption of GABAA Receptor Subunits-- Previous studies have indicated that the targeted disruption of gamma 2, beta 3, or alpha 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 alpha  and beta  subunits was essential for receptor formation. Thus, upon disruption of the alpha 6 or beta 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).

Upon disruption of the gamma 2 subunit expression, however, neither the number of GABAA receptors nor the protein level of the subunits alpha 1, alpha 2, alpha 3, beta 2/3, were reduced, indicating the predominant formation of receptors composed of alpha beta subunits (36). In addition, the protein level of gamma 1 and gamma 3 subunits remained unaltered in gamma 2-/- mice as shown by Western blotting. This may indicate that the supply of gamma 1 and gamma 3 subunits is not linked to the availability of alpha beta subunits, because receptors containing these subunits either are not synthesized in the same cells or the same subcellular compartments as those containing gamma 2 subunits. The extent of expression of delta  subunits was not investigated in this study.

The likely presence of receptors composed of alpha beta subunits without additional subunit classes in the cerebellum of delta -/- mice is consistent with the expression of these receptors in gamma 2-/- mice (36). The additional up-regulation of alpha 6beta gamma 2 receptors in delta -/- mice can be explained if the level of gamma 2 subunit expression is linked to the function of the granule cells. The latter mechanism might be unique for gamma 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 gamma 2 and delta  subunits compete for alpha 6 and beta  subunits during assembly of receptors and that subunits unable to find assembly partners are degraded (34). A lack of delta  subunits in delta -/- mice might then reduce degradation of the surplus gamma 2 subunits by increasing their chances to find assembly partners, thus leading to an increased formation of alpha 6beta gamma 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 gamma 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, gamma -aminobutyric acid; NGS, normal goat serum; PB, phosphate buffer; TBS, Tris-buffered saline; DS, diazepam-sensitive; DIS, diazepam-insensitive.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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