QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

HOME  |   SEARCH  |   ARCHIVE  |   SUBSCRIBE  |   CONTACT  |   HELP

This Article Abstract Full Text (PDF) Submit an eLetter Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (107) Citing Articles via Google Scholar Google Scholar Articles by Jechlinger, M. Articles by Sieghart, W. Search for Related Content PubMed PubMed Citation Articles by Jechlinger, M. Articles by Sieghart, W.

 Previous Article  |  Next Article 

The Journal of Neuroscience, April 1, 1998, 18(7):2449-2457

Subunit Composition and Quantitative Importance of Hetero-oligomeric Receptors: GABA_A Receptors Containing ₆ Subunits

Martin Jechlinger, Robert Pelz, Verena Tretter, Thomas Klausberger, and Werner Sieghart

Section of Biochemical Psychiatry, University Clinic for Psychiatry, A-1090 Vienna, Austria

	ABSTRACT

Top Abstract Introduction Materials & Methods Results Discussion References

In cerebellum, GABA_A receptors containing ₆ subunits are expressed exclusively in granule cells. The number of ₆ receptor subtypes formed in these cells and their subunit composition presently are not known. Immunoaffinity chromatography on ₆ subunit-specific antibodies indicated that 45% of GABA_A receptors in cerebellar extracts contained ₆ subunits. Western blot analysis demonstrated that ₁, ₁, ₂, ₃, ₂, and  subunits co-purified with ₆ subunits, suggesting the existence of multiple ₆ receptor subtypes. These subtypes were identified using a new method based on the one-by-one immunochromatographic elimination of receptors containing the co-purifying subunits in parallel or subsequent experiments. By quantification and Western blot analysis of ₆ receptors remaining in the extract, the proportion of ₆ receptors containing the eliminated subunit could be calculated and the subunit composition of the remaining receptors could be determined. Results obtained indicated that ₆ receptors in cerebellum are composed predominantly of _6x2 (32%), _16x2 (37%), _6x (14%), or _16x (15%) subunits. Other experiments indicated that 10%, 51%, or 21% of ₆ receptors contained homogeneous ₁, ₂, or ₃ subunits, respectively, whereas two different  subunits were present in 18% of all ₆ receptors. The method presented can be used to resolve the total number, subunit composition, and abundancy of GABA_A receptor subtypes in the brain and can also be applied to the investigation of other hetero-oligomeric receptors.

Key words: GABA_A receptor; composition, ₆ subunit; granule cell; cerebellum; antibodies; immunoaffinity chromatography; immunoprecipitation; [³H]muscimol; [³H]Ro 15-4513; binding studies

	INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

GABA_A receptors are ligand-gated chloride ion channels and the site of action of various pharmacologically and clinically important drugs, such as benzodiazepines, barbiturates, steroids, anesthetics, and convulsants (Sieghart, 1995). So far six , four , three , one , one , and three  subunits have been cloned and sequenced from mammalian brain (Sieghart, 1995; Ogurusu and Shingai, 1996; Davies et al., 1997), and it is assumed that five subunits assemble to form functional GABA_A receptors (Nayeem et al., 1994; Tretter et al., 1997). Expression studies have indicated that , , and  subunits have to combine to form receptors closely resembling native receptors. Depending on the type of , , and  subunits used for transfection of cells, however, recombinant receptors with different pharmacological properties do arise (Sieghart, 1995). The distinct but overlapping regional and cellular expression of the individual subunits (Persohn et al., 1992; Wisden et al., 1992) raises the possibility of the existence of an extremely large variety of GABA_A receptor subtypes in the brain. So far the actual extent of GABA_A receptor heterogeneity is not known.

GABA_A receptors containing ₆ subunits are expressed in cerebellar granule cells and in the embryologically related granule cells of the cochlear nucleus only (Laurie et al., 1992; Persohn et al., 1992; Wisden et al., 1992; Varecka et al., 1994; Jones et al., 1997). Thus, all ₆ receptors from cerebellum are expressed in the same cell type. In addition, receptors consisting of _6x2 subunits have special properties because they exhibit a high affinity for the inverse benzodiazepine agonist Ro 15-4513 but no affinity for the benzodiazepine agonist diazepam (Sieghart, 1995).

Several studies have investigated the subunit composition of GABA_A receptors containing ₆ subunits. The results obtained, however, were partially conflicting. Whereas in one study (Quirk et al., 1994) ₆ subunits were not observed to occur in combination with other  subunits, other studies demonstrated a partial coexistence of ₆ and ₁ subunits in the same receptor (Pollard et al., 1993, 1995; Khan et al., 1994, 1996). Similarly, estimates of the abundancy of individual receptor subtypes differed between authors. Finally, because of the lack of suitable antibodies, not all ₆ subunit-containing receptors could be investigated.

The present study was performed to resolve these discrepancies. Using 13 highly specific antibodies directed against different GABA_A receptor subunits, we demonstrated that only ₁, ₁, ₂, ₃, ₂, and  subunits significantly co-purified with ₆ subunits. To determine the identity and quantitative importance of receptors formed from these subunits, a generally applicable method was developed that is based on a one-by-one elimination by immunoaffinity chromatography of receptors containing the co-purifying subunits. Quantification of the remaining ₆ receptors allowed us to estimate the proportion of ₆ receptors containing the eliminated subunit. Repeating this subtractive purification by eliminating another co-purifying subunit in a parallel or a subsequent experiment finally allowed us to identify the subunit composition of ₆ receptors and to determine their quantitative importance.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Generation and purification of antibodies. The antibodies anti-peptide ₁(1-9), anti-peptide ₂(416-424), and anti-peptide ₃(459-467) (Zezula et al., 1991), anti-peptide ₄(379-421) (Ebert et al., 1996), anti-peptide ₅(427-433) (Sieghart et al., 1993), anti-peptide ₃(345-408) (Slany et al., 1995), anti-peptide ₃(1-13), and anti-peptide ₂(319-366) (Tretter et al., 1997), anti-peptide ₁(324-366) (Mossier et al., 1994), anti-peptide ₃(322-372) (Tögel et al., 1994), and anti-peptide (1-44) (Jones et al., 1997) were generated and affinity-purified as described previously. Polyclonal anti-peptide ₁(350-404) and anti-peptide ₂(351-405) antibodies were generated in a way similar to that described (Mossier et al., 1994).

The N-terminal peptide ₆(1-15) (sequence QLEDEGNFYSENVSR-) or the C-terminal peptide ₆(429-434) (sequence -VSSTVE) were custom-synthesized with an additional C- or N-terminal cysteine, respectively (piChem, Graz, Austria) and were coupled to keyhole limpet hemocyanin. These adducts were then used for the immunization of rabbits. The antibodies were isolated from the sera of the immunized rabbits by affinity chromatography on thiopropyl-Sepharose 6B coupled to the cysteine residue of the respective peptide according to the recommendations of Pharmacia LKB Biotechnology.

Cloning of ₁, ₁, ₂, ₃, or ₂ subunits of GABA_A receptors. A rat brain cDNA library was constructed in ZAP (Stratagene, La Jolla, CA) from poly A⁺ mRNA isolated from the brains of 8- to 10-d-old rats as described in the protocol from Stratagene. ₁, ₁, ₂, ₃, and ₂ subunits of GABA_A receptors were cloned from this cDNA library (Fuchs et al., 1995; Slany et al., 1995), and their sequence proved to be identical to that of the respective sequence published previously.

Culture of human embryonic kidney (HEK) 293 cells and cDNA transfection. Transformed HEK 293 cells (CRL 1573; American Type Culture Collection, Rockville, MD) were grown in DMEM (Life Technologies, Grand Island, NY) supplemented with 10% fetal calf serum (JRH Biosciences, Lenexa, KS), 2 mM glutamine, 50 µM -mercaptoethanol, 100 U/ml penicillin G, and 100 µg/ml streptomycin in 75 cm² petri dishes using standard cell culture techniques.

HEK 293 cells were transfected with rat ₁, ₁, ₂, ₃, or ₂ subunit cDNAs subcloned individually into the expression vector pCDM8 (Invitrogen, San Diego, CA), using the calcium phosphate precipitation method (Chen and Okayama, 1988). The ratio for the , , and  subunits used for transfection of 3 × 10⁶ cells was 12:6:6 µg cDNA (Zezula et al., 1996). The cells were harvested 48 hr after transfection.

Extraction of GABA_A receptors. Membranes from the cerebellum of adult rats or membranes from HEK 293 cells transfected with various GABA_A receptor subunit cDNAs were extracted with a deoxycholate buffer containing 0.5% deoxycholate, 0.05% phosphatidylcholine, 10 mM Tris-chloride, pH 8.5, 150 mM NaCl, 500 µM benzamidine, 200 µg/ml bacitracin, and 300 µM phenylmethylsulfonylfluoride (PMSF).

Immunoaffinity chromatography of GABA_A receptors and Western blot analysis. The anti-peptide ₆(429-434) antibody was the first ₆ antibody available in our laboratory and therefore was used to prepare an immunoaffinity column. Immunoaffinity columns were prepared by coupling 3-5 mg of the purified antibodies to 1 ml of protein A-agarose using the ImmunoPure IgG Orientation Kit (Pierce Europe, Oud-Beijerland, The Netherlands) as described previously (Mossier et al., 1994).

The immunoaffinity columns were equilibrated in the deoxycholate extraction buffer. Deoxycholate extracts of cerebellar membranes were applied to the immunoaffinity column at a rate of 2 ml/h. To completely eliminate the respective subunit and its associated receptors from the extract, the extract was cycled three times through the respective immunoaffinity column. The column was washed twice with 4 ml of deoxycholate extraction buffer, twice with 4 ml of IP-high buffer (0.5% Triton X-100, 50 mM Tris-chloride, pH 8.3, 600 mM NaCl, 1 mM EDTA, 500 µM benzamidine, 200 µg/ml bacitracin, and 300 µM PMSF) and then twice with 4 ml of IP-low buffer (0.2% Triton X-100, 50 mM Tris-chloride, pH 8.3, 150 mM NaCl, 1 mM EDTA, 500 µM benzamidine, 200 µg/ml bacitracin, and 300 µM PMSF). Proteins bound to the column were eluted with a buffer containing 0.1 M glycine-HCl, pH 2.45, 150 mM NaCl, and 0.1% Triton X-100. The eluted proteins were precipitated with methanol/chloroform (Wessel and Flügge, 1984) and subjected to Western blot analysis (Fuchs and Sieghart, 1989). Proteins transferred to polyvinylidene difluoride (PVDF) membranes were detected with digoxigenated primary antibodies (Tögel et al., 1994), as indicated, and the anti-digoxigenin-alkaline phosphatase Fab fragments (Boehringer Mannheim, Mannheim, Germany) and the chemiluminescence substrate CSPD (Tropix, Bedford, MA) according to the instructions of the manufacturer.

Immunoprecipitation and receptor binding assay. For immunoprecipitation, 300 µl of the clear deoxycholate membrane extract were mixed with 30 µl of antibody solution (0-20 µg of antibody), and the mixture was incubated under gentle shaking at 4°C overnight. Then 50 µl of immunoprecipitin (Life Technologies, Gaithersburg, MD) plus 150 µl of an IP-low buffer containing 5% dry milk powder were added, and incubation was continued for 2 hr at 4°C. The precipitate was centrifuged for 10 min at 10,000 × g, and the pellet was washed twice with 500 µl IP-high and once with 500 µl IP-low buffer.

For [³H]Ro 15-4513 binding assays the precipitated receptors were suspended in 1 ml of a solution containing 0.1% Triton X-100, 50 mM Tris-citrate buffer, pH 7.1, 150 mM NaCl, and 10 or 20 nM [³H]Ro 15-4513 (20.9 Ci/mmol; DuPont NEN, Dreieich, Germany) in the absence or presence of 100 µM Ro 15-1788 or various concentrations of diazepam, and were incubated for 90 min at 4°C. For [³H]muscimol binding assays the precipitated receptors were suspended in 1 ml of a solution containing 0.1% Triton X-100, 50 mM Tris-citrate buffer, and 20 nM [³H]muscimol (17.1 Ci/mmol; DuPont NEN) in the absence or presence of 10 µM GABA, and were incubated for 60 min at 4°C (Zezula and Sieghart, 1991). The suspensions were then filtered through Whatman GF/B filters, and the filters were washed twice with 5 ml ([³H]Ro 15-4513 assay) or 3.5 ml ([³H]muscimol assay) of a 50 mM Tris-citrate buffer, pH 7.1. When the percentage of ₆ receptors retained by an immunoaffinity column had to be determined, immunoprecipitation with the ₆(1-15) antibody and the subsequent [³H]muscimol binding assays were performed in the same experiment with the original extract and the immunoaffinity column efflux.

Total [³H]Ro 15-4513 binding in the extract before or after immunoprecipitation of ₆ subunit-containing GABA_A receptors was measured using a polyethyleneglycol (PEG) precipitation assay as described (Zezula and Sieghart, 1991). For this, 100 µl of the deoxycholate extract (or of the supernatant from the immunoprecipitation with anti-₆ antibodies) was incubated for 90 min at 4°C in a total volume of 1 ml with a buffer containing 50 mM Tris-citrate, pH 7.1, 150 mM NaCl, 50 µg -globulin, 15% (wt/vol) PEG, and 10 or 20 nM [³H]Ro 15-4513 in the absence or presence of 100 µM Ro 15-1788. The suspension was then filtered through Whatman GF/B filters, and the filters were washed twice with 3.5 ml of an 8% PEG solution.

For the determination of total [³H]muscimol binding in the extract, the PEG precipitation assay could not be used. This was attributable to the relatively high viscosity of the PEG solutions, prolonging the time needed for filtration of the samples, and the rapid dissociation of [³H]muscimol from its binding site. Total [³H]muscimol binding therefore was determined after all GABA_A receptors present in the extract were precipitated with an antibody mixture containing 8 µg ₁(350-404), plus 8 µg ₂(351-407), plus 10 µg ₃(1-13) antibody, using the same assay as described above. The validity of this approach was demonstrated by the observation that [³H]Ro 15-4513 binding data were identical whether receptors were precipitated with PEG or with this antibody mixture.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Anti-₆ antibodies

The N- or C-terminal amino acid sequences ₆(1-15) or ₆(429-434) are unique for the ₆ subunit of GABA_A receptors (Lüddens et al., 1990). Antibodies generated against these sequences were able to immunoprecipitate native GABA_A receptors solubilized from rat cerebellar membranes in a dose-dependent manner (Fig. 1). Whereas anti-peptide ₆(1-15) antibodies precipitated up to 15 ± 4% (mean ± SD; n = 4) of all [³H]Ro 15-4513 binding sites present in the extract, anti-peptide ₆(429-434) antibodies precipitated only 5 ± 1% (mean ± SD; n = 4) of these sites. Of the [³H]Ro 15-4513 binding sites precipitated by these antibodies, 23 ± 2% were diazepam sensitive, whereas 77 ± 2% of these sites were diazepam insensitive.

View larger version (11K): [in this window] [in a new window]   Figure 1.   Immunoprecipitation of GABAA receptors solubilized from rat cerebellum. Solubilized receptors (470 fmol of [3H]Ro 15-4513 binding sites) were incubated with increasing amounts of 6(1-15) or 6(429-434) antibodies in a final volume of 350 µl. Receptors present in the pellets (solid symbols) or the supernatant (open symbols) were determined by specific [3H]Ro 15-4513 binding. Identical results were obtained when the supernatant from the 6(1-15) or 6(429-434) immunoprecipitation was investigated. The values are mean ± SD of four separate experiments performed in triplicates. SD bars that were smaller than the diameter of the symbols are not shown.

Interestingly, however, it was demonstrated that in the same experiment the percentage of total [³H]Ro 15-4513 binding sites eliminated from the supernatant was higher than that actually found in the precipitate. Thus, whether 15-20 µg of ₆(1-15) or ₆(429-434) antibodies was used for immunoprecipitation, the amount of [³H]Ro 15-4513 binding sites in the supernatant was reduced by 30 ± 3% (mean ± SD; n = 4) (Fig. 1). These results seem to indicate that each of these antibodies had a similar ability to bind to ₆ subunit-containing GABA_A receptors, but that part of the receptors presumably were lost during washing of the precipitate.

In other experiments the ability of ₆(1-15) antibodies to immunoprecipitate [³H]muscimol binding sites was investigated. As with [³H]Ro 15-4513 binding, the amount of [³H]muscimol binding sites retained in the pellet was smaller than that removed from the supernatant. Thus, ₆(1-15) antibodies precipitated 22 ± 3% (mean ± SD; n = 3) of all [³H]muscimol binding sites present in cerebellar extracts but eliminated 42 ± 3% of these binding sites from the supernatant. Overall, these data indicated that more [³H]muscimol than [³H]Ro 15-4513 binding sites were precipitated from cerebellar extracts (or removed from the supernatant) by ₆(1-15) antibodies.

Proteins precipitated from cerebellar extracts by ₆(1-15) antibodies were then subjected to SDS-PAGE and Western blot analysis. The ₆(429-434) antibody as well as two ₆(1-15) antibodies purified from the sera of different rabbits were able to identify a single protein band with an apparent molecular mass of 56-57 kDa (Fig. 2). The observation that three antibodies directed against two distinct epitopes of the ₆ subunit specifically identified the same protein supports the conclusion that the protein with apparent molecular mass of 56-57 kDa was the ₆ subunit of GABA_A receptors. This conclusion is in agreement with previous reports indicating that the ₆ subunit exhibits an apparent molecular mass of 56-57 kDa (Lüddens et al., 1990; Pollard et al., 1993; Quirk et al., 1994). The different signal intensity of the antibodies reacting with identical amounts of the immunoprecipitate indicates that these antibodies exhibited a differential affinity for ₆ subunits under Western blot conditions.

View larger version (45K): [in this window] [in a new window]   Figure 2.   Identification of 6 subunits in cerebellar membrane extracts. GABAA receptors were immunoprecipitated from cerebellar membrane extracts using 6(1-15) antibodies. Receptors were then subjected to SDS-PAGE and Western blot analysis, using digoxigenated antibodies. Lane 1, 6(429-434) antibodies; lane 2, 6(1-15) antibodies from rabbit 15; lane 3, 6(1-15) antibodies from rabbit 16. Antibodies bound to proteins were detected using anti-digoxigenin-alkaline phosphatase Fab fragments and a sensitive chemiluminescence detection system as described in Materials and Methods. The gel was calibrated with proteins of known molecular mass. The experiment was performed twice with similar results. The protein smear below the 56-57 kDa band was detected by all three antibodies and may have represented differentially glycosylated, partially degraded, or alternatively spliced 6 subunits.

Isolation, subunit composition, and quantitative importance of GABA_A receptors containing ₆ subunits

After solubilization of GABA_A receptors from cerebellar membranes, 67.8% of the [³H]Ro 15-4513 or [³H]muscimol binding sites present in the membranes could be recovered in the extract. This corresponded to 92.5% of the binding sites identified in the extract and in the 100,000 × g pellet after extraction. Because there was no significant difference in the efficiency of solubilization by detergent between [³H]muscimol binding sites or diazepam-sensitive or -insensitive [³H]Ro 15-4513 binding sites, it can be concluded that the extracted receptors were representative of the entire functional ₆ subunit-containing GABA_A receptor population.

To quantitatively isolate GABA_A receptors containing ₆ subunits, cerebellar extracts were cycled three times through an immunoaffinity column containing anti-peptide ₆(429-434) antibodies. In the final efflux of this column, anti-peptide ₆(1-15) antibodies no longer were able to precipitate GABA_A receptors, and ₆ subunits no longer could be demonstrated in Western blots, indicating that this procedure eliminated most if not all ₆ receptors from the extract. In the same efflux, [³H]Ro 15-4513 binding was reduced by 31 ± 1% (mean ± SD; n = 3), and [³H]muscimol binding was reduced by 45 ± 1% (mean ± SD; n = 3). These percentages correspond closely to the 30 ± 3% reduction of [³H]Ro 15-4513 and 42 ± 3% reduction of [³H]muscimol binding sites observed in cerebellar extracts after immunoprecipitation with ₆(1-15) antibodies (see above).

To identify GABA_A receptor subunits co-purifying with ₆ subunits, receptors bound to the ₆(429-434) immunoaffinity column were eluted by a change in the pH value of the buffer and were probed with 13 different antibodies, each of which specifically recognized a distinct GABA_A receptor subunit. As shown in Figure 3A (or Fig. 4A), in addition to the ₆ subunit, ₁, ₁, ₂, ₃, ₂, and  subunits were present in the ₆(429-434) column eluate. Thus, ₁(1-9), ₁(350-404), ₂(351-405), ₃(345-408), ₂(319-366), and (1-44) antibodies identified proteins with apparent molecular mass of 51 kDa, 51-54 kDa, 50-53 kDa, 51-56 kDa, 41-44 kDa, and 53 kDa, respectively. Proteins with identical apparent molecular mass could be identified by these antibodies in parallel control experiments investigating recombinant GABA_A receptors containing the respective subunits (experiments not shown). The ₃(345-408) antibody, in addition to the 51-56 kDa protein, identified a second protein with an apparent molecular mass of 42-47 kDa. The protein with lower molecular mass seemed to be a partially degraded ₃ subunit, because staining of this protein was variable in different experiments (compare Figs. 3 and 4), and increased with increasing time needed for the isolation of GABA_A receptors (compare Fig. 3A-C).

View larger version (23K): [in this window] [in a new window]   Figure 3.   Subunit composition and quantification of 6 receptors before or after elimination of  or 1 subunit-containing receptors. Cerebellar extracts were chromatographed on an 6(429-434) immunoaffinity column either before (A) or after (B) chromatography on a (1-44) immunoaffinity column, or after (C) chromatography on both a (1-44) and an 1(1-9) immunoaffinity column. 6(429-434) column eluates were subjected to SDS-PAGE and Western blot analysis using the following digoxigenated antibodies: 1(1-9), 6(1-15), 1(350-404), 2(351-405), 3(345-408), 2(319-366), and (1-44). Western blots are from a typical experiment that was performed three times with similar results. In parallel experiments, the original extract as well as the efflux from the (1-44) or the 1(1-9) column were subjected to immunoprecipitation with 6(1-15) antibodies and subsequent [3H]muscimol binding assays. Data are presented as percentage of [3H]muscimol binding sites precipitated in the original extract and are means of a single experiment performed in triplicate. The experiment was repeated three times with similar results.

View larger version (25K): [in this window] [in a new window]   Figure 4.   Subunit composition and quantification of 6 receptors before or after elimination of 2 and 1 subunit-containing GABAA receptors. Cerebellar extracts were chromatographed on an 6(429-434) immunoaffinity column either before (A) or (B) after chromatography on a 2(319-366) immunoaffinity column, or after (C) chromatography on both a 2(319-366) and an 1(1-9) immunoaffinity column. 6(429-434) column eluates were subjected to SDS-PAGE and Western blot analysis using 1, 6, 1, 2, 3, 2, and  antibodies as described in Figure 3. Western blots are from a typical experiment that was performed three times with similar results. In parallel experiments, the original extract as well as the efflux from the 2(319-366) or the 1(1-9) column were subjected to immunoprecipitation with 6(1-15) antibodies and subsequent [3H]muscimol binding assays. Data are presented as percentage of [3H]muscimol binding sites precipitated in the original extract and are means of a single experiment performed in triplicate. The experiment was repeated three times with similar results.

The co-purification of ₁, ₁, ₂, ₃, ₂, and  subunits together with ₆ subunits was not caused by a cross-reactivity of ₆(429-434) antibodies with these subunits, because neither ₆(429-434) nor ₆(1-15) antibodies were able to precipitate [³H]muscimol binding sites or GABA_A receptor subunits from extracts of forebrain membranes, which do contain _1-5, _1-3, _1-3, and , but no ₆ subunits (Persohn et al., 1992; Wisden et al., 1992). These data therefore indicate that any one of the ₁, ₁, ₂, ₃, ₂, and  subunits can be colocalized with ₆ subunits in the same GABA_A receptor.

In contrast, the ₂, ₃, ₄, ₅, ₁, and ₃ subunits could not be detected in the eluate of the ₆(429-434) immunoaffinity column, although all of these subunits, except the ₃ and ₅ subunits, could be identified in cerebellar extracts by the respective antibodies (experiments not shown). This indicates that unspecific adsorption of receptors or an exchange of subunits during extraction was not a problem in this study.

Isolation, subunit composition, and quantitative importance of GABA_A receptors containing ₆ and ₂ subunits

Because GABA_A receptors are composed of five subunits, the co-purification of a total of seven different subunits by the ₆(429-434) immunoaffinity column indicated that a mixture of GABA_A receptor subtypes with different subunit composition was purified. To isolate GABA_A receptors containing ₆, _x, and ₂ subunits, GABA_A receptors containing any one of the other co-purifying subunits were quantitatively removed by immunoaffinity chromatography. In the first step, receptors containing  subunits were eliminated from cerebellar membrane extracts using a (1-44) column (Fig. 3). The (1-44) antibody specifically recognized the  but no other subunits of the GABA_A receptor (Jones et al., 1997). Interestingly, in the pH 2.45 eluate of the (1-44) column, ₁, ₆, ₁, ₂, ₃, , and other subunits, but no ₂ subunits, could be identified (R. Pelz, M. Jechlinger, and W. Sieghart, unpublished data).

To determine the composition of the remaining ₆ receptors, the efflux of the (1-44) column subsequently was chromatographed on the ₆(429-434) column. As shown in Figure 3B,  subunits could no longer be identified in the eluate of this column, indicating that these subunits had been completely eliminated by the (1-44) column. The presence of six different subunits (₁, ₆, ₁, ₂, ₃, ₂) in the eluate of the ₆(429-434) column indicates that GABA_A receptors retained by this column were still heterogeneous.

In the efflux of the (1-44) column, ₆(1-15) antibodies were able to precipitate 70% of the [³H]muscimol binding sites that could be precipitated by these antibodies in the original extract (Fig. 3B). This indicates that 30% of the ₆ subunit-containing GABA_A receptors were retained by the (1-44) column and contained the  subunit.

In the next step, the efflux of the (1-44) column was chromatographed on an ₁(1-9) immunoaffinity column. The ₁(1-9) antibody has been demonstrated to selectively identify only ₁ but no other GABA_A receptor subunits (Nusser et al., 1996; Zezula et al., 1991). The ₆ subunit-containing receptors remaining in the efflux of the ₁(1-9) column were then collected by the ₆(429-434) column. In the pH 2.45 eluate of this column, only ₆, ₁, ₂, ₃, and ₂ subunits, but no ₁ subunits, could be detected (Fig. 3C). The five subunits present in this eluate still could have been combined in a variety of different ways, resulting in a multiplicity of pentameric or receptors with different subunit composition and stoichiometry. At this point, therefore, no conclusion on the identity and composition of the receptors isolated by this procedure could be made.

As expected, the intensity of the individual signals for ₆, ₁, ₂, ₃, and ₂ subunits was lower in Figure 3C than in 3A or B. In the efflux of the ₁(1-9) column, 32 ± 3% (mean ± SD; n = 3) of the ₆ subunit-containing receptors present in the original extract could be precipitated by ₆(1-15) antibodies (Fig. 3C). Thus, 32% of ₆ receptors were composed of ₆, ₁, ₂, ₃, and ₂ subunits. The observation that 70% of the ₆ receptors could be precipitated before and only 32% after the ₁(1-9) column additionally indicates that 38% of ₆ receptors were removed by the ₁(1-9) column and thus contained ₁ as well as ₆ subunits.

All of these percentages were obtained by investigating binding of [³H]muscimol to the precipitated receptors. Because [³H]muscimol binding sites can be demonstrated only on receptors containing  and , or , , and  subunits (Zezula et al., 1996), these experiments indicate that the 32% of ₆ and 38% of ₁₆ receptors so far discussed must also have contained  subunits. Whether all or only some of these receptors additionally contained ₂ subunits cannot be answered at this time.

Isolation, subunit composition, and quantitative importance of GABA_A receptors containing ₆ and  subunits

In another experiment (Fig. 4), GABA_A receptors containing ₂ subunits were eliminated from cerebellar membrane extracts using a ₂(319-366) column. The high specificity of this immunoaffinity column has been demonstrated previously (Mossier et al., 1994). In the pH 2.45 eluate of the ₂(319-366) column, ₁, ₆, ₁, ₂, ₃, ₂, and other subunits, but no  subunits, could be identified (experiments not shown). This again supports the conclusion that ₂ and  subunits, at least in the cerebellum, seem not to be present in the same GABA_A receptors.

Receptors remaining in the efflux of the ₂(319-366) column were then chromatographed on the ₆(429-434) column. In the eluate of this column, ₁, ₆, ₁, ₂, ₃, and  subunits, but no ₂ subunits, could be detected (Fig. 4B). Immunoprecipitation with ₆(1-15) antibodies in the efflux of the ₂(319-366) column indicated that receptors composed of these subunits represented 30% of the ₆ receptors present in the original extract (Fig. 4B). All of these receptors contained the  subunit, because 30% of all ₆-containing GABA_A receptors could also be bound to the (1-44) immunoaffinity column, as discussed above (Fig. 3B).

The identification of only 30% of the ₆ receptors in the efflux of the ₂(319-366) column indicates that 70% of these receptors were retained by this column and thus contained ₂ subunits. Combined with the above observation (Fig. 3B) that 70% of all ₆ receptors could be precipitated in the efflux of the (1-44) column and were composed of ₁, ₆, ₁, ₂, _3, and ₂ subunits, these data suggest that ₆ receptors contain either ₂ or  subunits.

In the next step, the efflux of the ₂(319-366) column was chromatographed on the ₁(1-9) column, and ₆ receptors remaining in the efflux of this column were then either collected by a subsequent ₆(429-434) immunoaffinity chromatography or precipitated by ₆(1-15) antibodies (Fig. 4C). In the eluate of the ₆(429-434) column, ₆, ₁, ₂, ₃, and  subunits, but no ₁ subunits, could be identified. Immunoprecipitation experiments indicated that 15% of all ₆ subunit-containing GABA_A receptors could still be precipitated in the efflux of the ₁(1-9) immunoaffinity column (Fig. 4C) and thus were composed of _6x subunits.

Because 30% of all ₆ (and ) subunit-containing receptors could be precipitated before and only about 15% after chromatography on the ₁(1-9) column, these results additionally indicate that 15% of all ₆ subunit-containing receptors are composed of _16x subunits. Thus, the ₆ and  subunit-containing receptors _16x and _6x obviously are present in cerebellum at a 1:1 ratio. As expected, the signal strength of the individual protein bands was reduced according to the receptors removed by the various immunoaffinity columns (compare Fig. 4A-C). In this experiment the staining of the ₃ subunit was quite prominent. Because staining intensity depends on the individual properties of the digoxigenated antibody batch used, different staining intensities obtained with different antibodies do not necessarily reflect differences in the amount of protein present in the extract.

Results so far presented indicate the existence of at least four ₆ subunit-containing GABA_A receptor subtypes in cerebellum that are composed of _6x2, _16x2, _6x, and _16x subunits. The same four ₆ receptor subtypes were also identified when the sequence of columns was changed, and an ₁(1-9) column was used before the ₂(319-366) column to eliminate receptors containing the respective subunits from cerebellar extracts. In addition, the quantitative data obtained were consistent with each other and not dependent on the sequence of columns used (experiments not shown). These results strongly suggest that none of the antibodies used for immunochromatography exhibited a significant cross-reactivity and that the ₆(1-15) or ₆(429-434) antibodies were able to recognize or precipitate these four 6 subunit-containing GABA_A receptor subtypes with comparable efficiency. The experiments described were repeated several times, and the average proportion of the four GABA_A receptor subtypes calculated from the individual experiments is given in Table 1. In addition, taking into account that only 45 ± 1% of all GABA_A receptors in the cerebellum contained the ₆ subunit, the absolute contribution of the various ₆ receptors to total GABA_A receptors present in cerebellum was calculated (Table 1).

View this table: [in this window] [in a new window]   Table 1.   Relative and absolute abundancy of 6 receptor subtypes in rat cerebellum

Isolation, subunit composition, and quantitative importance of GABA_A receptors containing ₆ and distinct  subunits

The low number of ₆ receptors remaining in the extract after complete removal of ₂ and ₁ (_6x, 15% of all ₆ receptors) or of  and ₁ subunits (_6x2, 32% of all ₆ receptors) prevented a direct investigation of the  subunit composition of these receptors, even more so because each immunoaffinity chromatography step is time consuming and enhances degradation and inactivation of receptors. Therefore, the  subunit-composition of ₆ receptors was investigated in the original extract from cerebellum only.

For this, cerebellum extracts were first chromatographed on a ₁(350-404) immunoaffinity column (Fig. 5A). In the efflux of this column, ₁ subunits no longer could be demonstrated (experiments not shown), indicating that receptors containing this subunit had been removed completely. Precipitation with ₆(1-15) antibodies indicated that 85 ± 1% (mean ± SD; n = 4) of the original ₆ receptors were still present after removal of the ₁ subunit-containing receptors and suggested that 15% of all ₆ receptors contained ₁ subunits (Fig. 5A).

View larger version (27K): [in this window] [in a new window]   Figure 5.   Quantification of 6 receptors containing different  subunits. [3H]muscimol binding to GABAA receptors immunoprecipitated with 6(1-15) antibodies was determined in cerebellar membrane extracts before or after chromatography on a 1(350-404), 2(351-405), or 3(345-408) immunoaffinity column as indicated. Data are presented as percentage of [3H]muscimol binding sites precipitated by 6(1-15) antibodies in the original extract and are mean values ± SD of three to four experiments performed in triplicate. The proportion of [3H]muscimol binding sites retained on the initial anti- immunoaffinity columns (A, C, E) was significantly different (Student's t test) from that remaining in the extract after the other two  subunits had been removed (efflux, B, D, F): A, efflux D (p = 0.007); C, efflux F (p = 0.002); E, efflux B (p = 0.001). [3H]muscimol binding sites present in the original extract were significantly different (p = 0.0001) from the sum of the [3H]muscimol binding sites retained on the initial anti- immunoaffinity columns (A + C + E) and were also significantly different (p = 0.007) from the sum of the [3H]muscimol binding sites found in the efflux of B, D, and F.

The efflux of the ₁(350-404) column was then chromatographed on a ₂(351-405) immunoaffinity column (Fig. 5B). On this second column all receptors containing ₂ subunits were adsorbed, as indicated by the absence of ₂ subunits in the column efflux (experiments not shown). In the same efflux, however, 21 ± 7% (mean ± SD; n = 3) of the original ₆ receptors could be precipitated using ₆(1-15) antibodies. Because GABA_A receptors containing ₁ as well as those containing ₂ subunits now had been completely removed from the extract, the remaining 21% of the ₆ receptors thus contained only ₃ subunits.

In other experiments, all receptors containing ₂ subunits were first removed from the cerebellum extract using a ₂(351-405) immunoaffinity column (Fig. 5C). In the efflux of this column, only 34 ± 2% (mean ± SD; n = 4) of the original ₆ receptors were present. From this it can be concluded that 66% of all ₆ receptors contained a ₂ subunit. A subsequent chromatography on a ₃(345-408) column (Fig. 5D) eliminated an additional 24% of the ₆ receptors. The remaining 10 ± 1% (mean ± SD; n = 3) of receptors thus contained only ₁ subunits.

Finally, the cerebellum extract was chromatographed first on a ₃(345-408) column. In the efflux of this column, 63 ± 2% (mean ± SD; n = 4) of the ₆ receptors were still present (Fig. 5E), indicating that ~37% of all ₆ receptors contained a ₃ subunit. A subsequent chromatography on a ₁(350-404) column removed an additional 12% of ₆ receptors. The remaining 51 ± 8% (mean ± SD; n = 3) of ₆ receptors thus contained only ₂ subunits.

Interestingly, a comparison of the proportion of ₆ receptors retained by the  subunit-specific columns from the original extract with that remaining in the extract after removal of the other two  subunits revealed striking and statistically significant differences (see legend to Fig. 5 ). Although 15% of all ₆ receptors were removed by the ₁ column from the original extract (Fig. 5A), only 10% of ₆ receptors were left after elimination of all ₂ and ₃ subunits (Fig. 5D). Although 66% of all ₆ receptors were removed by the ₂ column from the original extract (Fig. 5C), only 51% of these receptors were left after removal of ₁ and ₃ receptors (Fig. 5F). Finally, although 37% of all ₆ receptors were removed by the ₃ column from the original extract (Fig. 5E), only 21% of these receptors were left after removal of ₁ and ₂ subunits (Fig. 5B).

In addition, the sum of ₆ receptors retained by the ₁, ₂, and ₃ columns from the original extract was 118% (Fig. 5A,C,E), whereas the sum of the receptors remaining in the extract after two of the three  subunits had been removed was 82% (Fig. 5B,D,F). These differences could not be explained by a cross-reactivity of the antibodies, because ₁, ₂, or ₃ antibodies were unable to precipitate recombinant _1x2 receptors containing the wrong  subunit (experiments not shown). These data therefore suggest that 18% of the ₆ receptors in cerebellum contain more than one type of  subunit. Because of the variability of binding data, however, a further calculation of the proportion of receptors containing ₁₂, ₁₃, or ₂₃ subunit combinations does not provide reliable results.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

Composition and quantitative importance of GABA_A receptors containing ₆ subunits

In the present investigation, 13 antibodies, each one highly specific for a different GABA_A receptor subunit, were used to investigate the subunit composition and quantitative importance of GABA_A receptors containing ₆ subunits. Chromatography on an ₆(429-434) immunoaffinity column quantitatively removed ₆ subunits and 45 ± 1% of all GABA_A receptors from cerebellar extracts, supporting previous conclusions (Khan et al., 1996; Jones et al., 1997) that 45% of all GABA_A receptors in the cerebellum contain the ₆ subunit. In the eluate of this column, in addition to the ₆ subunit, only ₁, ₁, ₂, ₃, ₂, and  subunits of GABA_A receptors could be demonstrated, suggesting that any one of these subunits can be colocalized with ₆ subunits in native GABA_A receptors.

In contrast, ₂, ₃, ₄, _{5, 1}, or ₃ subunits did not co-purify with ₆ subunits. This is to be expected for ₂, ₃, ₅, or ₁ subunits, which are not expressed in the granule cells of cerebellum (Persohn et al., 1992; Wisden et al., 1992). The existence of minor amounts of receptors containing ₃ and ₆ subunits has been demonstrated previously after purification of GABA_A receptors by a ₃ subunit-specific immunoaffinity column (Tögel et al., 1994). The observation that ₄ subunits did not co-purify with ₆ subunits, although these subunits are expressed in cerebellar granule cells and could be identified in cerebellar extracts (E. Bencsits, V. Ebert, and W. Sieghart, unpublished data), indicates that receptors containing ₄ as well as ₆ subunits, if they exist at all, are quantitatively not important. Thus, the great majority of ₆ subunit-containing GABA_A receptors is composed of ₆ and ₁, ₁, ₂, ₃, ₂, or  subunits.

A new strategy for the determination of the subunit composition and quantitative importance of hetero-oligomeric receptors

A random assembly of ₆ subunits with six other subunits into pentameric receptors (Nayeem et al., 1994; Tretter et al., 1997) would result in a total of 210 GABA_A receptor subtypes with distinct subunit composition. It is impossible to isolate a single receptor subtype from an even much less heterogeneous mixture by immunoenrichment. In the present study, therefore, immunodepletion was used to purify and characterize GABA_A receptors. Receptors containing one of the co-purifying subunits were eliminated from extracts by chromatography on subunit-specific antibodies. Quantification and Western blot analysis of ₆ receptors remaining in the extract then allowed us to estimate the proportion of ₆ receptors containing the eliminated subunit and to determine the composition of the remaining receptors. Repeating this procedure by eliminating all co-purifying subunits in parallel or subsequent experiments finally allowed us to identify the subunit composition of ₆ receptor subtypes and to determine their quantitative importance.

₁, ₂, or  subunit-containing ₆ receptors

In agreement with previous studies (Khan et al., 1994, 1996; Pollard et al., 1995), 52% of the [³H]muscimol binding sites precipitated by ₆(1-15) antibodies could be eliminated from cerebellar extracts by an ₁ subunit-specific column, indicating that ₁₆ receptors are as abundant as receptors containing homogeneous ₆ subunits (Table 1). Other experiments indicated that 70% of ₆ receptors could be eliminated from cerebellar membrane extracts by a ₂ subunit-specific (Fig. 4) and 30% by a  subunit-specific column (Fig. 3). In addition, it was demonstrated that ₂ and  subunits did not co-purify with each other, supporting the conclusion that these subunits do not co-exist in the same GABA_A receptor (Quirk et al., 1995).

Furthermore, the number of [³H]Ro 15-4513 binding sites removed from cerebellar extracts by ₆(429-434) or ₆(1-15) antibodies was 69% or 71% of the [³H]muscimol binding sites eliminated by these antibodies, respectively. Because [³H]Ro 15-4513 binding sites are present on GABA_A receptors containing or subunits and [³H]muscimol binding sites are present on receptors composed of , , and subunits (Quirk et al., 1995; Sieghart, 1995; Zezula et al., 1996), these data agree with the conclusion that 70% of the ₆ receptors contained a ₂ subunit. The observation that the [³H]muscimol binding sites of ₂ or  subunit-containing ₆ receptors add up to 100% additionally indicates that all ₆ receptors contain either a ₂ or a  subunit. From this it can be concluded that receptors composed of _6x subunits, and consequently also those composed of ₆₂ subunits, which would contribute to [³H]Ro 15-4513 but not to [³H]muscimol binding sites, are not significantly expressed in cerebellum.

Further fractionation of the 70% ₆ receptors containing ₂ subunits using an ₁ subunit-specific column indicated that 37 ± 3% of ₆ receptors are composed of _16x2 and 32 ± 3% of _6x2 subunits. _16x2 receptors have been identified previously (Khan et al., 1994, 1996; Pollard et al., 1995), and quantification of these receptors led to comparable results (Khan et al., 1994).

Recombinant receptor studies have indicated that _6x2 receptors, in contrast to _1x2 receptors, exhibit a high affinity [³H]Ro 15-4513 binding that could not be inhibited by diazepam (Lüddens et al., 1990; Sieghart, 1995). Other studies have indicated that in GABA_A receptors containing ₆ and ₁ (Khan et al., 1996) or ₁ and ₃ subunits (Araujo et al., 1996), each one of the subunits expressed its characteristic benzodiazepine pharmacology. Because 32% of ₆ receptors are composed of _6x2, whereas 37% are composed of _16x2 subunits, these two receptor subtypes are responsible for 46.4% and 53.6% of all [³H]Ro 15-4513 binding sites precipitated by ₆(1-15) antibodies, respectively. Assuming that _6x2 receptors contain two ₆ subunits (Im et al., 1995), these two receptor subtypes contain a total of 73% ₆ and 27% ₁ subunits. The present observation that 23 ± 2% of [³H]Ro 15-4513 binding precipitated by ₆(1-15) antibodies could be inhibited by diazepam is supported by a recent study (Khan et al., 1996) and is in agreement with the conclusion that each one of the subunits expresses its characteristic benzodiazepine pharmacology.

Further fractionation of the 30% ₆ receptors containing  subunits using an ₁ subunit-specific column indicated that 15 ± 3% of all ₆ receptors were composed of _16x and 14 ± 2% of _6x receptors. Although the existence of _16x receptors in cerebellum has been implicated previously (Pollard et al., 1995), their abundancy was not determined.

Subunit composition of ₆ receptors

When ₁-, ₂-, and ₃-specific immunoaffinity columns were used to eliminate GABA_A receptors from cerebellar extracts in parallel experiments, it was demonstrated that the total percentage of ₆ receptors removed was 118%. In the absence of a significant cross-reactivity of the ₁, ₂, or ₃ subunit-specific antibodies, these data suggested the colocalization of different  subunits in 18% of the ₆ receptors. This conclusion is supported by recent evidence indicating the colocalization of two different  subunits in native receptors (Li and De Blas, 1997). The proportion of ₆ receptors containing homogeneous  subunits was then determined by measuring ₆ receptors remaining in the extract after the removal of the other two  subunits. The results obtained indicated that 10, 51, or 21% of all ₆ receptors contained homogeneous ₁, ₂, or ₃ subunits, respectively. Because of the variability of binding data, a reliable estimation of the  subunit composition of the remaining 18% of ₆ receptors was not possible. The observation that ₁ and ₂ as well as ₃ subunits are co-purifying with ₆ and ₂ (Fig. 3C) or ₆ and  subunits (Fig. 4C), however, indicates that the _6x2 or _6x receptor subtypes might exist in up to six isoforms containing different  subunit combinations (homogeneous ₁, ₂, or ₃ subunits, ₁₂, ₁₃, or ₂₃). The same might be true for receptors consisting of _16x2 or _16x subunits. Whether all of the resulting 24 ₆ receptors with different subunit composition actually exist cannot be answered by this study.

Subunit stoichiometry of native ₆ receptors

The present results, in agreement with studies investigating other receptors, indicate that native ₆ receptors can contain two different  (Sieghart, 1995) or two different  subunits (Li and De Blas, 1997), and in addition contain either a ₂ or a subunit. Overall, these results suggest a subunit stoichiometry of two , two , and one  (or one ) subunit for native ₆ receptors. This is in agreement with studies investigating the subunit stoichiometry of ₆₂₂ (Im et al., 1995) or of other recombinant receptors (Chang et al., 1996; Tretter et al., 1997). The method of subtractive purification of GABA_A receptors developed in the present study can be used to investigate whether all native ₆ receptors exhibit this stoichiometry or whether other stoichiometries also exist (Backus et al., 1993). In addition, this method can also be applied to the investigation of other hetero-oligomeric receptors.

	FOOTNOTES

Received Nov. 6, 1997; revised Jan. 20, 1998; accepted Jan. 21, 1998.

This work was supported by Grants P9003-Med and P9828-Med of the Austrian Science Foundation and by the European Commission Biotechnology Programme, Project ERBBIO4CT960585. The participation of W. Kern in the initial experiments of this study is gratefully acknowledged.

Correspondence should be addressed to Dr. Werner Sieghart, Section of Biochemical Psychiatry, University Clinic for Psychiatry, Währinger Gürtel 18-20, A-1090 Vienna, Austria.

	REFERENCES

Top Abstract Introduction Materials & Methods Results Discussion References

• Araujo F, Tan S, Ruano D, Schoemaker H, Benavides J, Vitorica J (1996) Molecular and pharmacological characterization of native cortical -aminobutyric acid_A receptors containing both 1 and 3 subunits. J Biol Chem 271:27902-27911[Abstract/Free Full Text].
• Backus KH, Arigoni M, Drescher U, Scheurer L, Malherbe P, Möhler H, Benson JA (1993) Stoichiometry of a recombinant GABA_A receptor deduced from mutation-induced rectification. NeuroReport 5:285-288[Web of Science][Medline].
• Chang Y, Wang R, Barot S, Weiss DS (1996) Stoichiometry of a recombinant GABA_A receptor. J Neurosci 16:5415-5424[Abstract/Free Full Text].
• Chen CA, Okayama H (1988) Calcium phosphate-mediated gene transfer: a highly efficient transfection system for stably transforming cells with plasmid DNA. Biotechniques 6:632-638[Web of Science][Medline].
• Davies PA, Hanna MC, Hales TG, Kirkness EF (1997) Insensitivity to anaesthetic agents conferred by a class of GABA_A receptor subunit. Nature 385:820-823[Medline].
• Ebert V, Scholze P, Sieghart W (1996) Extensive heterogeneity of recombinant -aminobutyric acid_A receptors expressed in 432-transfected human embryonic kidney 293 cells. Neuropharmacology 35:1323-1330[Web of Science][Medline].
• Fuchs K, Sieghart W (1989) Evidence for the existence of several different - and -subunits of the GABA/benzodiazepine receptor complex from rat brain. Neurosci Lett 97:329-333[Web of Science][Medline].
• Fuchs K, Zezula J, Slany A, Sieghart W (1995) Endogenous [³H]flunitrazepam binding in human embryonic kidney cell line 293. Eur J Pharmacol 289:87-95[Web of Science][Medline].
• Im WB, Pregenzer JF, Binder JA, Dillon GH, Alberts GL (1995) Chloride channel expression with the tandem construct of 6-2 GABA_A receptor subunit requires a monomeric subunit of 6 or 2. J Biol Chem 270:26063-26066[Abstract/Free Full Text].
• Jones A, Korpi ER, McKernan RM, Pelz R, Nusser Z, Mäkelä R, Mellor JR, Pollard S, Bahn S, Stephenson FA, Randall AD, Sieghart W, Somogyi P, Smith AJH, Wisden W (1997) Ligand-gated ion channel subunit partnerships: GABA_A receptor 6 subunit gene inactivation inhibits  subunit expression. J Neurosci 17:1350-1362[Abstract/Free Full Text].
• Khan ZU, Gutierrez A, De Blas AL (1994) The subunit composition of a GABA_A/benzodiazepine receptor from rat cerebellum. J Neurochem 63:371-374[Web of Science][Medline].
• Khan ZU, Gutierrez A, De Blas AL (1996) The 1 and 6 subunits can coexist in the same cerebellar GABA_A receptor maintaining their individual benzodiazepine-binding specificities. J Neurochem 66:685-691[Web of Science][Medline].
• Laurie DJ, Seeburg PH, Wisden W (1992) The distribution of 13 GABA_A receptor subunit mRNAs in the rat brain. II. Olfactory bulb and cerebellum. J Neurosci 12:1063-1076[Abstract].
• Li M, De Blas AL (1997) Coexistence of two  subunit isoforms in the same -aminobutyric acid type A receptor. J Biol Chem 272:16564-16569[Abstract/Free Full Text].
• Lüddens H, Pritchett DB, Köhler M, Killisch I, Keinänen K, Monyer H, Sprengel R, Seeburg PH (1990) Cerebellar GABA_A receptor selective for a behavioural alcohol antagonist. Nature 346:648-651[Medline].
• Mossier B, Tögel M, Fuchs K, Sieghart W (1994) Immunoaffinity purification of -aminobutyric acid_A (GABA_A) receptors containing ₁-subunits. Evidence for the presence of a single type of -subunit in GABA_A receptors. J Biol Chem 269:25777-25782[Abstract/Free Full Text].
• Nayeem N, Green TP, Martin IL, Barnard EA (1994) Quarternary structure of the native GABA_A receptor determined by electron microscope image analysis. J Neurochem 62:815-818[Web of Science][Medline].
• Nusser Z, Sieghart W, Benke D, Fritschy JM, Somogyi P (1996) Differential synaptic localization of two major -aminobutyric acid type A receptor  subunits on hippocampal pyramidal cells. Proc Natl Acad Sci USA 93:11939-11944[Abstract/Free Full Text].
• Ogurusu T, Shingai R (1996) Cloning of a putative -aminobutyric acid (GABA) receptor subunit 3 cDNA. Biochim Biophys Acta 1305:15-18[Medline].
• Persohn E, Malherbe P, Richards JG (1992) Comparative molecular neuroanatomy of cloned GABA_A receptor subunits in the rat CNS. J Comp Neurol 326:193-216[Web of Science][Medline].
• Pollard S, Duggan MJ, Stephenson FA (1993) Further evidence for the existence of  subunit heterogeneity within discrete -aminobutyric acid_A receptor subpopulations. J Biol Chem 268:3753-3757[Abstract/Free Full Text].
• Pollard S, Thompson CL, Stephenson FA (1995) Quantitative characterization of 6 and 16 subunit-containing native -aminobutyric acid_A receptors of adult rat cerebellum demonstrates two  subunits per receptor oligomer. J Biol Chem 270:21285-21290[Abstract/Free Full Text].
• Quirk K, Gillard NP, Ragan CI, Whiting PJ, McKernan RM (1994) Model of subunit composition of -aminobutyric acid A receptor subtypes expressed in rat cerebellum with respect to their  and / subunits. J Biol Chem 269:16020-16028[Abstract/Free Full Text].
• Quirk K, Whiting PJ, Ragan CI, McKernan RM (1995) Characterisation of -subunit containing GABA_A receptors from rat brain. Eur J Pharmacol 290:175-181[Web of Science][Medline].
• Sieghart W (1995) Structure and pharmacology of -aminobutyric acid_A receptor subtypes. Pharmacol Rev 47:181-234[Web of Science][Medline].
• Sieghart W, Item C, Buchstaller A, Fuchs K, Höger H, Adamiker D (1993) Evidence for the existence of differential O-glycosylated 5-subunits of the -aminobutyric acid_A receptor in the rat brain. J Neurochem 60:93-98[Web of Science][Medline].
• Slany A, Zezula J, Tretter V, Sieghart W (1995) Rat 3 subunits expressed in human embryonic kidney 293 cells form high affinity [³⁵S]t-butylbicyclophosphorothionate binding sites modulated by several allosteric ligands of -aminobutyric acid type A receptors. Mol Pharmacol 48:385-391[Abstract].
• Tögel M, Mossier B, Fuchs K, Sieghart W (1994) -Aminobutyric acid_A receptors displaying association of ₃-subunits with _2/3 and different  subunits exhibit unique pharmacological properties. J Biol Chem 269:12993-12998[Abstract/Free Full Text].
• Tretter V, Ehya N, Fuchs K, Sieghart W (1997) Stoichiometry and assembly of a recombinant GABA_A receptor subtype. J Neurosci 17:2728-2737[Abstract/Free Full Text].
• Varecka L, Wu CH, Rotter A, Frostholm A (1994) GABA_A/benzodiazepine receptor 6 subunit mRNA in granule cells of the cerebellar cortex and cochlear nuclei: expression in developing and mutant mice. J Comp Neurol 339:341-352[Web of Science][Medline].
• Wessel D, Flügge UI (1984) A method for the quantitative recovery of proteins in dilute solution in the presence of detergent and lipids. Anal Biochem 138:141-143[Web of Science][Medline].
• Wisden W, Laurie DJ, Monyer H, Seeburg PH (1992) The distribution of 13 GABA_A receptor subunit mRNAs in the rat brain. I. Telencephalon, diencephalon, mesencephalon. J Neurosci 12:1040-1062[Abstract].
• Zezula J, Sieghart W (1991) Isolation of type I and type II GABA_A-benzodiazepine receptors by immunoaffinity chromatography. FEBS Lett 284:15-18[Web of Science][Medline].
• Zezula J, Fuchs K, Sieghart W (1991) Separation of 1, 2 and 3 subunits of the GABA_A-benzodiazepine receptor complex by immunoaffinity chromatography. Brain Res 563:325-328[Web of Science][Medline].
• Zezula J, Slany A, Sieghart W (1996) Interaction of allosteric ligands with GABA_A receptors containing one, two, or three different subunits. Eur J Pharmacol 301:207-214[Web of Science][Medline].

---

Copyright © 1998 Society for Neuroscience  0270-6474/98/1872449-09$05.00/0

This article has been cited by other articles:

				K. H. Kaur, R. Baur, and E. Sigel Unanticipated Structural and Functional Properties of {delta}-Subunit-containing GABAA Receptors J. Biol. Chem., March 20, 2009; 284(12): 7889 - 7896. [Abstract] [Full Text] [PDF]

				R. W. Olsen and W. Sieghart International Union of Pharmacology. LXX. Subtypes of {gamma}-Aminobutyric AcidA Receptors: Classification on the Basis of Subunit Composition, Pharmacology, and Function. Update Pharmacol. Rev., September 1, 2008; 60(3): 243 - 260. [Abstract] [Full Text] [PDF]

				C. F. Valenzuela, R. A. Radcliffe, P. Botta, and M. Mameli Response to Comment on "Ethanol Sensitivity of GABAergic Currents in Cerebellar Granule Neurons Is Not Increased by a Single Amino Acid Change (R100Q) in the {alpha}6 GABAA Receptor Subunit" J. Pharmacol. Exp. Ther., January 1, 2008; 324(1): 401 - 403. [Full Text] [PDF]

				H. L. Payne, W. M. Connelly, J. H. Ives, R. Lehner, B. Furtmuller, W. Sieghart, P. Tiwari, J. M. Lucocq, G. Lees, and C. L. Thompson GABAA {alpha}6-Containing Receptors Are Selectively Compromised in Cerebellar Granule Cells of the Ataxic Mouse, Stargazer J. Biol. Chem., October 5, 2007; 282(40): 29130 - 29143. [Abstract] [Full Text] [PDF]

				N. Zhang, W. Wei, I. Mody, and C. R. Houser Altered Localization of GABAA Receptor Subunits on Dentate Granule Cell Dendrites Influences Tonic and Phasic Inhibition in a Mouse Model of Epilepsy J. Neurosci., July 11, 2007; 27(28): 7520 - 7531. [Abstract] [Full Text] [PDF]

				S. H. Hadley and J. Amin Rat {alpha}6beta2{delta} GABAA receptors exhibit two distinct and separable agonist affinities J. Physiol., June 15, 2007; 581(3): 1001 - 1018. [Abstract] [Full Text] [PDF]

				I. Sarto-Jackson, R. Furtmueller, M. Ernst, S. Huck, and W. Sieghart Spontaneous Cross-link of Mutated {alpha}1 Subunits during GABAA Receptor Assembly J. Biol. Chem., February 16, 2007; 282(7): 4354 - 4363. [Abstract] [Full Text] [PDF]

				D. Chandra, F. Jia, J. Liang, Z. Peng, A. Suryanarayanan, D. F. Werner, I. Spigelman, C. R. Houser, R. W. Olsen, N. L. Harrison, et al. GABAA receptor {alpha}4 subunits mediate extrasynaptic inhibition in thalamus and dentate gyrus and the action of gaboxadol PNAS, October 10, 2006; 103(41): 15230 - 15235. [Abstract] [Full Text] [PDF]

				V. Santhakumar, H. J. Hanchar, M. Wallner, R. W. Olsen, and T. S. Otis Contributions of the GABAA receptor alpha6 subunit to phasic and tonic inhibition revealed by a naturally occurring polymorphism in the alpha6 gene. J. Neurosci., March 22, 2006; 26(12): 3357 - 3364. [Abstract] [Full Text] [PDF]

				S. i Storustovu and B. Ebert Pharmacological Characterization of Agonists at {delta}-Containing GABAA Receptors: Functional Selectivity for Extrasynaptic Receptors Is Dependent on the Absence of {gamma}2 J. Pharmacol. Exp. Ther., March 1, 2006; 316(3): 1351 - 1359. [Abstract] [Full Text] [PDF]

				E. M. Petrini, I. Marchionni, P. Zacchi, W. Sieghart, and E. Cherubini Clustering of Extrasynaptic GABAA Receptors Modulates Tonic Inhibition in Cultured Hippocampal Neurons J. Biol. Chem., October 29, 2004; 279(44): 45833 - 45843. [Abstract] [Full Text] [PDF]

				D. Benke, P. Fakitsas, C. Roggenmoser, C. Michel, U. Rudolph, and H. Mohler Analysis of the Presence and Abundance of GABAA Receptors Containing Two Different Types of {alpha} Subunits in Murine Brain Using Point-mutated {alpha} Subunits J. Biol. Chem., October 15, 2004; 279(42): 43654 - 43660. [Abstract] [Full Text] [PDF]

				Z. Peng, C. S. Huang, B. M. Stell, I. Mody, and C. R. Houser Altered Expression of the {delta} Subunit of the GABAA Receptor in a Mouse Model of Temporal Lobe Epilepsy J. Neurosci., September 29, 2004; 24(39): 8629 - 8639. [Abstract] [Full Text] [PDF]

				F. Minier and E. Sigel Positioning of the {alpha}-subunit isoforms confers a functional signature to {gamma}-aminobutyric acid type A receptors PNAS, May 18, 2004; 101(20): 7769 - 7774. [Abstract] [Full Text] [PDF]

				W. Wei, N. Zhang, Z. Peng, C. R. Houser, and I. Mody Perisynaptic Localization of {delta} Subunit-Containing GABAA Receptors and Their Activation by GABA Spillover in the Mouse Dentate Gyrus J. Neurosci., November 19, 2003; 23(33): 10650 - 10661. [Abstract] [Full Text] [PDF]

				I. Spigelman, Z. Li, J. Liang, E. Cagetti, S. Samzadeh, R. M. Mihalek, G. E. Homanics, and R. W. Olsen Reduced Inhibition and Sensitivity to Neurosteroids in Hippocampus of Mice Lacking the GABAA Receptor {delta} Subunit J Neurophysiol, August 1, 2003; 90(2): 903 - 910. [Abstract] [Full Text] [PDF]

				A. C. Engblom, F. F. Johansen, and U. Kristiansen Actions and Interactions of Extracellular Potassium and Kainate on Expression of 13 gamma -Aminobutyric Acid Type A Receptor Subunits in Cultured Mouse Cerebellar Granule Neurons J. Biol. Chem., May 2, 2003; 278(19): 16543 - 16550. [Abstract] [Full Text] [PDF]

				I. Sarto, T. Klausberger, N. Ehya, B. Mayer, K. Fuchs, and W. Sieghart A Novel Site on gamma 3 Subunits Important for Assembly of GABAA Receptors J. Biol. Chem., August 16, 2002; 277(34): 30656 - 30664. [Abstract] [Full Text] [PDF]

				S. B. Christie, C. P. Miralles, and A. L. De Blas GABAergic Innervation Organizes Synaptic and Extrasynaptic GABAA Receptor Clustering in Cultured Hippocampal Neurons J. Neurosci., February 1, 2002; 22(3): 684 - 697. [Abstract] [Full Text] [PDF]

				H. Mohler, J. M. Fritschy, and U. Rudolph A New Benzodiazepine Pharmacology J. Pharmacol. Exp. Ther., January 1, 2002; 300(1): 2 - 8. [Abstract] [Full Text] [PDF]

				Z. Nusser, L. M. Kay, G. Laurent, G. E. Homanics, and I. Mody Disruption of GABAA Receptors on GABAergic Interneurons Leads to Increased Oscillatory Power in the Olfactory Bulb Network J Neurophysiol, December 1, 2001; 86(6): 2823 - 2833. [Abstract] [Full Text] [PDF]

				T. Klausberger, I. Sarto, N. Ehya, K. Fuchs, R. Furtmuller, B. Mayer, S. Huck, and W. Sieghart Alternate Use of Distinct Intersubunit Contacts Controls GABAA Receptor Assembly and Stoichiometry J. Neurosci., December 1, 2001; 21(23): 9124 - 9133. [Abstract] [Full Text] [PDF]

				C. Sur, K. A. Wafford, D. S. Reynolds, K. L. Hadingham, F. Bromidge, A. Macaulay, N. Collinson, G. O'Meara, O. Howell, R. Newman, et al. Loss of the Major GABAA Receptor Subtype in the Brain Is Not Lethal in Mice J. Neurosci., May 15, 2001; 21(10): 3409 - 3418. [Abstract] [Full Text] [PDF]

				T. Klausberger, K. Fuchs, B. Mayer, N. Ehya, and W. Sieghart GABAA Receptor Assembly. IDENTIFICATION AND STRUCTURE OF gamma 2 SEQUENCES FORMING THE INTERSUBUNIT CONTACTS WITH alpha 1 AND beta 3 SUBUNITS J. Biol. Chem., March 17, 2000; 275(12): 8921 - 8928. [Abstract] [Full Text] [PDF]

				E. Louiset, R. McKernan, W. Sieghart, and H. Vaudry Subunit Composition and Pharmacological Characterization of {gamma}-Aminobutyric Acid Type A Receptors in Frog Pituitary Melanotrophs Endocrinology, March 1, 2000; 141(3): 1083 - 1092. [Abstract] [Full Text] [PDF]

				E. I. Tietz, X. J. Zeng, S. Chen, S. M. Lilly, H. C. Rosenberg, and P. Kometiani Antagonist-Induced Reversal of Functional and Structural Measures of Hippocampal Benzodiazepine Tolerance J. Pharmacol. Exp. Ther., December 1, 1999; 291(3): 932 - 942. [Abstract] [Full Text]

				Z. Nusser, W. Sieghart, and I. Mody Differential regulation of synaptic GABAA receptors by cAMP-dependent protein kinase in mouse cerebellar and olfactory bulb neurones J. Physiol., December 1, 1999; 521(2): 421 - 435. [Abstract] [Full Text] [PDF]

				R. M. Mihalek, P. K. Banerjee, E. R. Korpi, J. J. Quinlan, L. L. Firestone, Z.-P. Mi, C. Lagenaur, V. Tretter, W. Sieghart, S. G. Anagnostaras, et al. Attenuated sensitivity to neuroactive steroids in gamma -aminobutyrate type A receptor delta subunit knockout mice PNAS, October 26, 1999; 96(22): 12905 - 12910. [Abstract] [Full Text] [PDF]

				F. Araujo, D. Ruano, and J. Vitorica Native gamma -Aminobutyric Acid Type A Receptors from Rat Hippocampus, Containing Both alpha 1 and alpha 5 Subunits, Exhibit a Single Benzodiazepine Binding Site with alpha 5 Pharmacological Properties J. Pharmacol. Exp. Ther., September 1, 1999; 290(3): 989 - 997. [Abstract] [Full Text]

				T. P. Bonnert, R. M. McKernan, S. Farrar, B. le Bourdelles, R. P. Heavens, D. W. Smith, L. Hewson, M. R. Rigby, D. J. S. Sirinathsinghji, N. Brown, et al. theta , a novel gamma -aminobutyric acid type A receptor subunit PNAS, August 17, 1999; 96(17): 9891 - 9896. [Abstract] [Full Text] [PDF]

				K. Kannenberg, M. T. Schaerer, K. Fuchs, W. Sieghart, and E. Sigel A Novel Serine Kinase with Specificity for beta 3-Subunits Is Tightly Associated with GABAA Receptors J. Biol. Chem., July 23, 1999; 274(30): 21257 - 21264. [Abstract] [Full Text] [PDF]

				E. Bencsits, V. Ebert, V. Tretter, and W. Sieghart A Significant Part of Native gamma -Aminobutyric AcidA Receptors Containing alpha 4 Subunits Do Not Contain gamma or delta Subunits J. Biol. Chem., July 9, 1999; 274(28): 19613 - 19616. [Abstract] [Full Text] [PDF]

				W. Bonigk, J. Bradley, F. Muller, F. Sesti, I. Boekhoff, G. V. Ronnett, U. B. Kaupp, and S. Frings The Native Rat Olfactory Cyclic Nucleotide-Gated Channel Is Composed of Three Distinct Subunits J. Neurosci., July 1, 1999; 19(13): 5332 - 5347. [Abstract] [Full Text] [PDF]

				E. A. Barnard, P. Skolnick, R. W. Olsen, H. Mohler, W. Sieghart, G. Biggio, C. Braestrup, A. N. Bateson, and S. Z. Langer International Union of Pharmacology. XV. Subtypes of gamma -Aminobutyric AcidA Receptors: Classification on the Basis of Subunit Structure and Receptor Function Pharmacol. Rev., June 1, 1998; 50(2): 291 - 314. [Abstract] [Full Text] [PDF]

				N. J. Brandon, P. Delmas, J. T. Kittler, B. J. McDonald, W. Sieghart, D. A. Brown, T. G. Smart, and S. J. Moss GABAA Receptor Phosphorylation and Functional Modulation in Cortical Neurons by a Protein Kinase C-dependent Pathway J. Biol. Chem., December 1, 2000; 275(49): 38856 - 38862. [Abstract] [Full Text] [PDF]

				T. Klausberger, N. Ehya, K. Fuchs, T. Fuchs, V. Ebert, I. Sarto, and W. Sieghart Detection and Binding Properties of GABAA Receptor Assembly Intermediates J. Biol. Chem., May 4, 2001; 276(19): 16024 - 16032. [Abstract] [Full Text] [PDF]

				V. Tretter, B. Hauer, Z. Nusser, R. M. Mihalek, H. Hoger, G. E. Homanics, P. Somogyi, and W. Sieghart Targeted Disruption of the GABAA Receptor delta Subunit Gene Leads to an Up-regulation of gamma 2 Subunit-containing Receptors in Cerebellar Granule Cells J. Biol. Chem., March 23, 2001; 276(13): 10532 - 10538. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit an eLetter Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (107) Citing Articles via Google Scholar Google Scholar Articles by Jechlinger, M. Articles by Sieghart, W. Search for Related Content PubMed PubMed Citation Articles by Jechlinger, M. Articles by Sieghart, W.

Home  |   Search  |   Archive  |   Subscribe  |   Contact  |   Help