©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Quantitative Characterization of 6 and 16 Subunit-containing Native -Aminobutyric Acid Receptors of Adult Rat Cerebellum Demonstrates Two Subunits per Receptor Oligomer (*)

(Received for publication, May 23, 1995; and in revised form, July 7, 1995)

Simon Pollard Christopher L. Thompson F. Anne Stephenson (§)

From the Department of Pharmaceutical Chemistry, School of Pharmacy, University of London, 29/39 Brunswick Square, London WC1N 1AX, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

-Aminobutyric acid(A) (GABA(A)) receptors were purified from adult rat cerebella by anti-alpha6(1-16 Cys) antibody affinity chromatography. Immunoblots of the alpha6 subunit-containing receptors showed the copurification of the alpha1, beta2/3, 2, but not alpha2 and alpha3 GABA(A) receptor polypeptides. Further fractionation of this receptor subpopulation by anti-GABA(A) receptor subunit alpha6(1-16 Cys) and anti-alpha1(413-429) antibody affinity columns in series substantiated the coassociation of the alpha1 and alpha6 polypeptides. The percentage of coexistence of the two subunits was determined by quantitative immunoblotting, which found that 41 ± 12% of alpha6 subunit immunoreactivity is associated with the alpha1 subunit. The ratios of the alpha1:alpha6 subunits in the double purified receptor preparations was found to be 1:1, thus determining directly for the first time subunit ratios within native GABA(A) receptors. The benzodiazepine pharmacology of the alpha1alpha6 subunit-containing receptors was shown to be predominantly benzodiazepine-insensitive by quantitative immunoprecipitation assays. These results are the first direct quantitative studies of subunit ratios within a population of native GABA(A) receptors.


INTRODUCTION

The GABA(A)(^1)receptors of mammalian brain are fast-acting ligand-gated chloride ion channels. Multiple genes encoding GABA(A) receptor subunits have been identified by molecular cloning. These are classified on the basis of their respective amino acid sequence similarities into five subunit types. Thus known mammalian GABA(A) receptor subunits are the alpha1-alpha6, beta1-beta3, 1-3, , and 1-2, comprising 15 identified to date (for review, see McDonald and Olsen(1994)). Different combinations of these subunits are thought to assemble, probably in pentameric combinations, in vivo to form functional GABA(A) receptors. The polypeptide complement of any one native GABA(A) receptor has not been elucidated. However, the biophysical and pharmacological properties of cloned receptors suggest that most probably consist of an alphabeta combination (Olsen and McDonald, 1994; Stephenson, 1995). A subunit stoichiometry (alpha)(2)(beta)(1)()(2) was found for a defined expressed GABA(A) receptor (Backus et al., 1993). For native receptors, a five-fold axis of symmetry was revealed by negative stain electron microscopy thus providing the first evidence for a pentameric quaternary structure in vivo (Nayeem et al., 1994). Furthermore, Khan et al.(1994) recently deduced by immunoprecipitation studies that a GABA(A) receptor subpopulation in the cerebellum consisted of alpha1alpha62(S)2(L)beta2/3 subunits where 2(S) and 2(L) are splice variants of the 2 subunit.

We have adopted the approach of the determination of native GABA(A) receptor subunit complements by the purification of subsets of receptors by immunoaffinity chromatography using subunit-specific antibodies (e.g. Duggan et al. (1991) and Pollard et al.(1993)). We have focused primarily on the alpha subunit complements of native receptors. We found, in agreement with several other groups, that the majority of native GABA(A) receptors contain a single alpha subunit variant (e.g. Duggan et al.(1991), McKernan et al.(1991), and Benke et al.(1991)). However, we identified, by the use of different specificity antibody affinity columns in series, minor receptor populations that were heterogeneous with respect to their alpha subunit complement e.g. alpha1alpha2, alpha1alpha3, and alpha2alpha3 subunit-containing receptors (Duggan et al., 1991; Pollard et al., 1993). Thus, we proposed the assembly of at least two alpha subunits per receptor oligomer. Several other groups have since reported the copurification or coimmunoprecipitation of other alpha subunit variants (e.g. Mertens et al.(1993) and Kern and Sieghart(1994); summarized by Stephenson(1995)).

We have recently concentrated efforts on the GABA(A) receptor alpha6 subunit-containing receptors. This is because this subunit is uniquely expressed in adult rat brain in a single cell type, the cerebellar granule cell (Luddens et al., 1990; Thompson et al., 1992). Furthermore, cloned alpha6betaxx (and also alpha4betaxx) receptors have a distinct benzodiazepine pharmacology in that they have high affinity for the partial inverse agonist Ro 15-4513 but very low affinity for the classical allosteric benzodiazepine regulators such as diazepam (Luddens et al., 1990). This pharmacological profile corresponds to the previously described diazepam-insensitive site (abbreviated in this paper to the benzodiazepine-insensitive site, BZ-IS) (Sieghart et al., 1991). We previously reported the purification of calf cerebellar alpha6 subunit-containing GABA(A) receptors, but low yields in the isolation procedure precluded their detailed characterization (Pollard et al., 1993). The isolation efficiency has been improved by using adult rat cerebellum as starting material, thus permitting further analysis, including for the first time quantitative measurements of immunoreactivity. We report these results in this paper.


EXPERIMENTAL PROCEDURES

Materials

[N-methyl-^3H]Flunitrazepam (85 Ci/mmol), rabbit immunoglobulin, horseradish-linked whole antibody, ECL Western blotting system, Hyperfilm-ECL, and the biotinylation kit were from Amersham International (Aylesbury, Bucks, United Kingdom (UK)). [^3H]Ro 15-4513 (24.3 Ci/mmol) was from Du Pont (UK) Ltd. (Stevenage, Hertfordshire, UK). Flunitrazepam and Ro 15-4513 were from Research Biochemicals Inc. (Natick, MA). Peptides, anti-peptide antibodies, and characterization of their specificities were as described by Thompson et al.(1992) and Pollard et al.(1993); details of the anti-GABA(A) receptor subunit antibodies are given below. The monoclonal antibody bd-17, which recognizes both GABA(A) receptor beta2 and beta3 subunits (Ewert et al., 1990), was from Boehringer Mannheim (Lewes, East Sussex, UK). All other materials were from commercial sources.

Methods

Production of GABA(A)Receptor Subunit Antibodies

The peptides (320-337 Cys), DYRKKRKAKVKVTKPRAEC, and (2-12), PHHGARAMNDIC, were coupled via the C-terminal cysteine to keyhole limpet hemeocyanin. Polyclonal antibodies were raised in rabbits and were affinity-purified by the respective peptide affinity resins all as described previously (Stephenson and Duggan, 1991). Both specificity anti- subunit antibodies recognized a M(r), 57,000 ± 500 immunoreactive band in addition to a protein with M(r) 64,000. The higher molecular weight species has been observed with other cysteine-coupled peptides with completely different amino acid sequences; it is a nonspecific, non- subunit protein.

Preparation of Membrane-bound, Detergent-solubilized, and Detergent-treated Membrane Fractions from Rat and Calf Brain

Membranes and Na deoxycholate extracts of rat and calf cerebellum were prepared as described previously (Duggan and Stephenson, 1990). The pellet obtained after Na deoxycholate solubilization was rehomogenized with 10 mM HEPES, pH 7.4, containing 1 mM EDTA, 1 mM benzamidine, centrifuged at 20,000 g for 30 min at 4 °C, and the pellet retained. This was then resuspended as above and termed the detergent-treated membranes.

Immunoaffinity Purification of GABA(A) Receptors

Immunoaffinity purification of GABA(A) receptors from Na deoxycholate extracts of adult rat cerebellum was carried out using a rabbit anti-alpha6(1-16 Cys) Fab antibody fragment affinity column, a sheep anti-alpha1(413-429) whole antibody affinity column, or the anti-alpha6(1-16 Cys) Fab and anti-alpha1(413-429) antibody affinity columns in series all exactly as described previously, except that in the double immunoaffinity column purification experiments, O.05% (w/v) bovine serum albumin was added to the single GABA(A) receptor alpha6 subunit-purified material (Duggan et al., 1991; Pollard et al., 1993). Purified receptor subpopulations were analyzed by both immunoblotting and radioligand binding assays.

Polyacrylamide Gel Electrophoresis and Immunoblotting

Immunoblotting was carried out as before using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions in 10% polyacrylamide slab gels, the chloroform/methanol method for protein precipitation, and the ECL Western blotting system for detection (Duggan et al., 1992). Immunoblots were quantified by densitometry using the Molecular Dynamics personal densitometer.

Radioligand Binding and Immunoprecipitation Assays

Radioligand binding and immunoprecipitation assays were both carried out as described previously using the polyethyleneimine assay for the measurement of specific ligand binding activities (Stephenson and Duggan, 1990). For the measurement of [^3H]Ro 15-4513 binding activity, total binding was determined using 10 µM Ro 15-4513 to define nonspecific binding. For the measurement of benzodiazepine-sensitive (BZ-S) [^3H]Ro 15-4513 binding activity, 10 µM flunitrazepam was used in the binding assay. BZ-IS [^3H]Ro 15-4513 binding activity was thus calculated as the difference between total and the benzodiazepine-sensitive (BZ-S) binding activity.


RESULTS

We have previously reported the purification of alpha6 subunit-containing GABA(A) receptors from calf cerebellum by anti-alpha6(1-16 Cys) Fab antibody affinity chromatography (Pollard et al., 1993). In that study we reported the copurification of the alpha6 and alpha1 subunit immunoreactivities. However, because of the low yield in the isolation procedure, we were unable to characterize in detail the coassociation of alpha6 and alpha1 subunit immunoreactivities by the use of different specificity antibody columns in series, as has been employed for other GABA(A) receptor subpopulations (cf. Duggan et al., 1991; Pollard et al., 1993). Furthermore, there were no detectable radioligand binding activities in the purified receptor preparations, thus precluding the characterization of the pharmacological properties of native GABA(A) receptor alpha6 subunit-containing receptors. An investigation of the efficiency of the purification procedure showed that for the standard conditions of receptor solubilization using 0.5% (w/v) Na deoxycholate and 150 mM KCl, the majority of alpha6 subunit immunoreactivity remained in the calf cerebellar detergent-treated membranes (Fig. 1). This was in contrast to the efficiency of solubilization under the same conditions from adult rat cerebellum, where at least 50% of the alpha6 subunit immunoreactivity was found in the solubilized preparation (Fig. 1). The comparative results for the extraction of BZ-S and BZ-IS [^3H]Ro 15-4513 binding sites from rat cerebellum are shown in Table 1. There was no appreciable difference in the efficiency of detergent solubilization between the two pharmacological classes of receptor. Furthermore, the percentage efficiency of solubilization of binding sites agreed with that found for alpha6 subunit immunoreactivity ( Table 1and Fig. 1). This is in contrast to Uusi-Oukari(1992), who showed that BZ-S sites were preferentially solubilized from pig cerebellum. Although this differential sensitivity to detergent extraction appears to be species-dependent, it is important also to make the point that it may reflect differences between receptor subtypes in their subcellular compartmentalization and/or association with cytoskeletal elements particularly at less accessible sites such as synapses. For the results herein and indeed for all other papers addressing native GABA(A) receptor subunit complements by biochemical approaches, the assumption is made that the solubilized preparation is representative of the entire functional, alpha6 subunit-containing GABA(A) receptor population.


Figure 1: Solubilization of GABA(A) receptor alpha6 immunoreactivity from adult rat and calf cerebellum. Membranes, Na deoxycholate extract, and Na deoxycholate-treated membranes were all prepared from adult rat and calf cerebellum. Samples (25 µg of protein/gel lane) were precipitated and analyzed by immunoblotting using affinity-purified anti-alpha6(1-16 Cys) antibodies (2.5 µg, final concentration) all as described under ``Experimental Procedures.'' Lane1, benzodiazepine affinity-purified GABA(A) receptor from calf cortex; lanes2-4, calf cerebellum; lanes 5-7, rat cerebellum. Lanes2 and 5, detergent-treated membranes; lanes 3 and 6, Na deoxycholate-solubilized membranes; lanes4 and 7, membranes. The positions of prestained protein standards (kDa 10) are on the left; alpha1 and alpha6 refer to the antibodies used for immunoblotting.





Purification and Characterization of alpha6 Subunit-containing GABA(A)Receptors from Adult Rat Cerebellum

The alternative use of rat cerebellum as the source of alpha6 subunit-containing GABA(A) receptors with the increased efficiency of solubilization permitted their detailed biochemical characterization. Table 2shows the [^3H]Ro 15-4513 binding results of a typical purification of GABA(A) receptors from adult rat cerebellum by anti-alpha6(1-16 Cys) Fab immunoaffinity chromatography. The pH 11.5 elution profile of the total [^3H]Ro 15-4513 binding activity from the anti-alpha6 subunit antibody column was concomitant with the M(r) 57,000 alpha6 immunoreactive band (results not shown, but see also Fig. 3). For total [^3H]Ro 15-4513 binding activity, it can be seen that 18% of the sites applied are retained by the anti-alpha6(1-16 Cys) Fab antibody column. There is a recovery of 4% of the total [^3H]Ro 15-4513 sites following pH 11.5 elution. This efficiency is the same order of magnitude found using different specificity GABA(A) receptor antibody columns, e.g. for alpha1 and alpha2 (Duggan et al., 1991). When the pharmacology of the total [^3H]Ro 15-4513 binding activity bound to the anti-alpha6(1-16 Cys) Fab antibody column was subfractionated into the BZ-S and BZ-IS sites, typical values found were 75% of the BZ-IS sites compared to 7% of the BZ-S sites (Table 2). Mean values obtained for the retention of BZ-IS and BZ-S were 74 ± 3% and 2 ± 5%, respectively (n = 4). From Table 3, it can be seen that the percentage of alpha6 subunit immunoreactivity (79 ± 7%) and BZ-IS sites retained by the anti-alpha6(1-16 Cys) Fab antibody affinity column were the same.




Figure 3: Purification of GABA(A) receptors from adult rat cerebellum by anti-alpha6(1-16 Cys) Fab and anti-alpha1(413-429) antibody affinity columns in series. GABA(A) receptors were purified by anti-alpha6(1-16 Cys) Fab affinity chromatography. Receptor-containing fractions were pooled and applied to an anti-alpha1(413-429) antibody column. The filtrate was retained, the column washed, then eluted at pH 11.5, and all fractions analyzed by immunoblotting as described under ``Experimental Procedures.'' A quantitative analysis of the immunoblots is given in Table 3. A, immunoblotting with alpha6; B, immunoblotting with alpha1 affinity-purified antibodies. The gel lay-out is the same for both: lane1, Na deoxycholate extract of adult rat cerebellum; lane2, anti-alpha6(1-16 Cys) Fab antibody post-column filtrate; lane3, alpha6(1-16 Cys) immunoaffinity-purified receptors; lane4, anti-alpha1(413-419) antibody post-column filtrate; lanes 5-9, pH 11.5 eluted fractions 1-5 from the anti-alpha1 413-429 antibody affinity column. The positions of prestained protein standards (kDa 10) are shown on the left. A and B show the results from a single representative purification, whereas C shows the densitometric profile for alpha1 () and alpha6 (up triangle, filled) immunoreactivities eluted by pH 11.5 from the anti-alpha1(413-429) antibody affinity column from n = 3 preparations. The results are expressed as a percentage of the peak fraction immunoreactivity for each antibody specificity and are the mean ± S.D.





In the pH 11.5 eluted fractions, again, BZ-S and BZ-IS [^3H]Ro 15-4513 binding activity were both assayed. The results were difficult to quantify because of the low level of total [^3H]Ro 15-4513 binding activity, but it was observed that flunitrazepam displaced a significant proportion of the total binding activity (see below). There was no significant retention of specific [^3H]flunitrazepam binding sites by the anti-alpha6(1-16 Cys) antibody column. In agreement, [^3H]flunitrazepam binding to the purified receptors was not detectable (n = 2, results not shown).

To determine the other GABA(A) receptor polypeptides that copurified with the alpha6 subunit-containing GABA(A) receptors, the pH 11.5 fractions 2-4 were pooled and analyzed by immunoblotting. Fig. 2shows the results, where it can be seen that alpha1, beta2/3, 2, and subunit immunoreactivities were found. As for the bovine preparations, there was no detectable alpha2 or alpha3 subunit immunoreactivities. Note that we were unable to use the anti-beta3(379-393) antibody (Pollard et al., 1991); this was raised to the bovine beta3(379-393) sequence. The homologous rat sequence has three amino acid differences, and the rat beta3 subunit is not recognized by the bovine antibody. The beta subunits were thus identified by the monoclonal antibody bd-17, which recognizes both rat beta2 and beta3 subunits.


Figure 2: Demonstration by immunoblotting of the coassociation of GABA(A) receptor alpha1, beta2/3, 2, and with the alpha6 subunit. GABA(A) receptors were purified from adult rat cerebellum by anti-alpha6(1-16 Cys) Fab affinity chromatography. The alpha6 subunit-containing fractions were pooled and analyzed by immunoblotting for reactivity with alpha1 (M(r) 51,000), beta2/3 (M(r) 59,000 and 57,000), 2 (M(r) 49,000), (M(r) 57,000 ± 500), and alpha6 antibodies as described under ``Experimental Procedures.'' Lanes 1, 5, and 8, Na deoxycholate-solubilized rat cerebellum; lanes 2, 4, 6, 7, 9, and 10, anti-alpha6(1-16 Cys) Fab affinity-purified GABA(A) receptors; lane3, benzodiazepine affinity-purified receptor. 1 and 2, immunoblotting with anti-(2-12 Cys) and anti-(320-337 Cys) antibodies, respectively. The positions of prestained protein standards (kDa 10) are shown on the left.



Quantification of the Percentage Coassociation of the alpha1 and alpha6 GABA(A)Receptor Subunits in Adult Rat Cerebellum

The coassociation of the alpha1 and alpha6 subunits was investigated further and quantitatively by using different immunoaffinity columns in series. Thus GABA(A) receptors were first purified by anti-alpha6(1-16 Cys) Fab antibody affinity chromatography. The receptor-containing fractions were pooled and applied to a sheep anti-alpha1(413-429) whole antibody affinity column. The affinity column filtrate and the pH 11.5 eluted fractions were analyzed for radioligand binding activity and immunoreactivity. No [^3H]Ro 15-4513 binding was detected in the eluted fractions, but alpha6 and alpha1 GABA(A) receptor subunit immunoreactivities were found and these were quantified by densitometry. Fig. 3shows representative results for these experiments. On application of the alpha6 subunit-containing receptors to the anti-alpha1(413-429) antibody column, it can be seen that all the alpha1 subunit immunoreactivity is retained in contrast to the M(r) 57,000 alpha6 subunit, which was detectable in the anti-alpha1(413-429) antibody affinity column filtrate. Both immunoreactivities were found in the pH 11.5 eluted fractions. Fig. 3(A and B) shows the elution profile from a single experiment. Fig. 3C summarizes the results obtained from three separate purifications. It demonstrates the coelution of the alpha1 and alpha6 subunit immunoreactivities. The results for the quantification of the immunoblots are summarized in Table 3. For these experiments, equal volumes were applied for each sample thus permitting direct comparisons following densitometry. It was found that 79 ± 7% of the applied alpha6 subunit immunoreactivity was retained initially by the anti-alpha6 subunit antibody column. On application to the second different specificity antibody column, 41 ± 12% alpha6 subunit immunoreactivity was retarded (n = 2).

To determine the ratios of the alpha1:alpha6 subunits in the double purified receptor preparations, two sets of experiments were carried out. First, the primary alpha1 and alpha6 affinity-purified antibody dose dependences were determined, for a fixed antigen concentration, in immunoblots of double immunoaffinity-purified receptors (Fig. 4A). Second, using the antibody concentration at saturation (Fig. 4A), the alpha6alpha1 subunit-containing antigen was varied and the resultant immunoreactive bands quantified (Fig. 4B). The alpha1:alpha6 subunit ratio was 0.95 ± 0.1, which was the mean value for each antigen concentration and for n = 2 preparations. In immunoprecipitation assays (see below), it was found that the anti-alpha6(1-16 Cys) antibody did not pellet all the alpha6 subunit immunoreactivity even when used at saturation. Although immunoblotting and immunoprecipitation are two different experimental paradigms, further investigation was required to ensure that the alpha6 subunit was not being underestimated by using an antibody with low avidity. Thus, two immunoblots were carried out in parallel. In the first a single incubation with a saturating concentration of primary anti-alpha6(1-16 Cys) antibody was used. For the second immunoblot, this was processed as for the first except that the initial primary antibody was aspirated and the immunoblot then incubated with a fresh primary anti-alpha6(1-16 Cys) antibody at the same saturating concentration (Fig. 4A). Both immunoblots were then quantified by densitometry but no differences in the amount of alpha6 subunit immunoreactivity were found (results not shown).


Figure 4: Determination of the alpha1:alpha6 subunit ratio in alpha6alpha1 double immunopurified GABA(A) receptors from adult rat cerebellum. GABA(A) receptors were purified from adult rat cerebellum by anti-alpha6(1-16 Cys) and alpha1(413-429) antibody affinity columns in series as described under ``Experimental Procedures.'' A shows the results for an immunoblot where a fixed amount of alpha6alpha1 subunit-containing receptors as antigen (70 µl) was used with increasing concentrations of affinity-purified anti-alpha1(324-341) (up triangle, filled) or anti-alpha6(1-16 Cys) (black square) GABA(A) receptor subunit antibody. B shows an immunoblot where the antigen concentration was varied using a fixed concentration of affinity-purified antibody, which gave saturation (Fig. 5A) for immunoblotting.




Figure 5: Immunoprecipitation of [^3H]Ro 15-4513 binding sites from cerebellar detergent extracts and GABA(A) receptors purified by anti-alpha1(413-429) antibody affinity chromatography. Immunoprecipitation was carried out from Na deoxycholate extracts of rat cerebellum and GABA(A) receptor anti-alpha1(413-429) antibody immunoaffinity-purified preparations as described under ``Experimental Procedures'' using affinity-purified antibodies at a concentration of 75 µg/ml. The results are expressed as the percentage of [^3H]Ro 15-4513 binding sites immunoprecipitated with respect to the total activity in the initial immunoprecipitation incubation mixture. The values are the means ± S.D. for n = 7 independent determinations for immunoprecipitation from soluble cerebella extracts and n = 2 for imunoprecipitation from anti-alpha1(413-429) immunopurified receptors. A, total; B, BZ-IS; C, BZ-S [^3H]Ro 15-4513 binding activity, respectively. Ig, nonimmune protein A-purified Ig; alpha1, anti-alpha1(413-429); alpha6, anti-alpha6(1-16 Cys) antibodies, respectively.



Immunoblots of alpha6alpha1 double immunoaffinity-purified receptors showed the coassociation of beta2/3 and subunits (n = 1; results not shown), but attempts to show the localization of the 2 subunit have so far been negative.

The Benzodiazepine Pharmacology of alpha1alpha6 Subunit-containing GABA(A)Receptors

Since binding activity was not detectable in the alpha6:alpha1 double immunopurified GABA(A) receptors, the benzodiazepine pharmacology of the alpha1alpha6 subunit-containing receptors was investigated by quantitative immunoprecipitation using affinity-purified antibody at saturating concentrations. The results are summarized in Fig. 5. First, the alpha6 subunit antibody immunoprecipitated 23 ± 5% (n = 9) of the total [^3H]Ro 15-4513 binding sites from solubilized rat cerebellar membranes. This value is in agreement with the percentage of total [^3H]Ro 15-4153 binding sites retained by the anti-alpha6(1-16 Cys) antibody affinity column (Table 2). It corresponded to 54 ± 7% of the BZ-IS sites but no significant immunoprecipitation of the BZ-S sites was found (values were 5 ± 4%; see also Table 2), again in agreement with results obtained from anti-alpha6(1-16 Cys) Fab affinity chromatography (Table 2). In contrast, the GABA(A) receptor alpha1 subunit immunoprecipitated 72 ± 6% of total [^3H]Ro 15-4513 sites. Significant immunoprecipitation of both BZ-S (77 ± 6%) and BZ-IS (64 ± 10%) sites was found (Fig. 5). These values agree with results obtained using anti-alpha1(413-429) immunoaffinity chromatography from adult rat cerebellum where 81 ± 5% of total, 85 ± 10% BZ-S, and 71 ± 10% BZ-IS [^3H]Ro 15-4513 binding sites were retained (n = 3; results not shown).

The same immunoprecipitation assays were carried out on GABA(A) receptors purified from adult rat cerebellum by anti-alpha1(413-429) antibody affinity chromatography. Here, the alpha1 antibody immunoprecipitated close to 100% of total, BZ-S, and BZ-IS [^3H]Ro 15-4513 sites as should be the case for an alpha1 subunit-purified preparation. However, the alpha6 subunit antibody immunoprecipitated a maximum of 30 ± 5% of total [^3H]Ro 15-4513 sites (i.e. alpha1alpha6 subunit containing). When these binding sites were subfractionated into the BZ-IS and BZ-S sites, the values were not significant for the BZ-S but 47 ± 7% compared to a predicted 100% for the BZ-IS sites. Increasing the antibody concentration did not effect the percentage of [^3H]Ro 15-4513 binding sites precipitated, but it was found that at these high antibody concentrations, alpha6 subunit immunoreactivity was still present in the supernatant. Thus, the inability to immunoprecipitate all the BZ-IS [^3H]Ro 15-4513 sites may be attributed to the low avidity for the antibody. This has been encountered before for immunoprecipitations with the anti-2(1-15 Cys) antibody where the problem was overcome by sequential immunoprecipitations with fresh batches of antibody (Duggan et al., 1992). This was not possible here because of the low levels of binding activity. Significantly, when the pharmacology of the immunoprecipitated pellet was determined directly instead of as a percentage of the total starting activity, 91 ± 10% (n = 2) of the [^3H]Ro 15-4513 binding was BZ-IS.


DISCUSSION

In this paper, we have described the purification of alpha6 subunit-containing GABA(A) receptors with the retention of their [^3H]Ro 15-4513 radioligand binding activities. We have substantiated the coexistence of the alpha1 and alpha6 subunit in single receptor oligomers and, in addition, we have quantified their percentage coassociation. In double immunoaffinity-purified receptors, the alpha1:alpha6 subunit ratio was 1:1 and the benzodiazepine pharmacology of this subset of receptors was BZ-IS. Thus the use of the alpha6 and alpha1 immunoaffinity columns in series not only proved the coexistence of these two gene products in one receptor (41% of the alpha6 subunit receptor population, Table 3) but also showed that in the rat cerebellum, either single variant alpha1 or alpha6 subunit-containing receptors exist. These results are in agreement with the emerging pattern from several groups. That is, that at least for the GABA(A) receptor alpha subunits, different isoforms do partially coexist within the same receptor molecule (summarized by Stephenson, 1995). With specific reference to the alpha1 and alpha6 subunits, the findings herein are in agreement with the localization of alpha1 and alpha6 GABA(A) receptor subunit-like immunoreactivities at the electron microscopic level where synapses in the cerebellum were found containing either alpha1 or alpha6 or both alpha1 and alpha6 polypeptides (Nusser et al., 1995). Moreover, Mathews et al.(1994) coexpressed alpha1, alpha6, beta2, and 2 polypeptides in mammalian cells and showed that the resultant pharmacological properties were distinct from both alpha1beta12 and alpha6beta12 receptors and best explained by an alpha1alpha6beta22 hybrid receptor. Quirk et al.(1994), however, found no evidence for coassociation of alpha1 and alpha6 subunits but this may be explained by low avidity antibodies. Similarly, Korpi and Luddens(1993) found no evidence for the coassociation of all four subunits following transient expression in mammalian cells.

For the non-alpha subunits coassociated with the alpha6 polypeptide, the results reported here are in agreement with previous reports, where the coassociation of alpha6 with 2 (Khan et al., 1994; Quirk et al., 1994), (Quirk et al., 1994), and beta2/3 (Khan et al., 1994) was found. But Quirk et al.(1994) identified alpha62 and alpha6 as two distinct populations, where the latter did not bind [^3H]Ro 15-4513. In the alpha1alpha6 double immunoaffinity-purified receptors, we were unable to detect the GABA(A) receptor 2 subunit by immunoblotting. Negative results here are difficult to interpret definitively because they may be explained by both the low levels of purified receptor and antibody avidity, a particular problem with the anti-2 subunit antibody used (cf. Stephenson et al., 1990; Duggan et al., 1992). But, it may also be that the 2 subunit is associated with single alpha6 variant receptors. Consequently, the (alpha1alpha6beta2/3) receptor identified here may be similar to (alpha6) receptors described by Quirk et al. (1994), which do not bind [^3H]Ro 15-4513. Further analysis of the anti-alpha1(413-429) post-column filtrate should clarify this.

The direct determination of the number of alpha subunits (a 1:1 ratio for alpha1:alpha6, therefore predicting two per receptor) is the first for native GABA(A) receptors. It is in agreement with the 1:1 ratio predictions for native receptors where the coexistence of two but not three different alpha subunits were detectable (Duggan et al., 1991), the inferred subunit complement of native cerebellar receptors, alpha1alpha6beta2/32(L)2(S) (Khan et al., 1994), and the subunit complement of an (alpha1)(2)(beta1)(1)(2)(2) cloned receptor (Backus et al., 1993). The quantification described in this paper is not ideal because antibody molecules are bivalent and the antibodies used are polyclonal albeit to a restricted epitope. Thus it is a possibility that the number of antibodies bound per subunit may not be stoichiometric. However, this is unlikely because 1) steric hindrance would reduce the probability of two antibody molecules binding to different epitopes within the restricted 16-amino acid peptide sequence, and 2) the binding of one antibody molecule to two subunits would have an equal probability for the alpha1 and alpha6 subunits following reduction and denaturation in SDS-PAGE.

The study of cloned, single alpha GABA(A) receptors showed that the benzodiazepine subpharmacology was dependent on the type of alpha subunit (Luddens and Wisden, 1991). The alpha6 subunit-containing cloned receptors show BZ-IS pharmacology in contrast to alpha1 receptors, which are BZ-S (Luddens et al., 1990). A single point mutation in the alpha6 subunit, alpha6R100H, results in a mutant receptor with high affinity for the classical benzodiazepines (Wieland et al., 1992). From the results reported herein, for (alpha1alpha6) receptors, the alpha6 subunit dominates the pharmacology yielding BZ-IS pharmacology. This would be in agreement with Mathews et al.(1994).

Conclusions

In conclusion, we have demonstrated a direct correlation between the BZ-IS, [^3H]Ro 15-4513 binding site pharmacology and native, alpha6 subunit-containing GABA(A) receptors. Furthermore, quantitative results showed that 41% of all the alpha6 subunit-containing receptors coexist with an alpha1 subunit and that in these receptors, the alpha6:alpha1 ratio was 1:1 and they displayed BZ-IS pharmacology. The functional significance of the extensive GABA(A) receptor heterogeneity remains to be solved. However, the fact that we find all combinations of association of these two polypeptides in percentages that preclude the random association of actively transcribed genes suggests that there may be an active sorting/assembly mechanism to form functional receptor subtypes.


FOOTNOTES

*
This work was supported by the Medical Research Council (United Kingdom). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 44-171-753-5877; Fax: 44-171-278-1939.

(^1)
The abbreviations used are: GABA(A), -aminobutyric acid(A); BZ-IS, benzodiazepine-insensitive; BZ-S, benzodiazepine-sensitive; ECL, enhanced chemiluminesence; PAGE, polyacrylamide gel electrophoresis.


REFERENCES

  1. Backus, K. H., Arigoni, M., Drescher, U., Scheurer, L., Malherbe, P., Mohler, H., and Benson, J. A. (1993) NeuroReport 5,285-288 [Medline] [Order article via Infotrieve]
  2. Benke, D., Mertens, S., Trceziak, A., Gillessen, D., and Mohler, H. (1991) J. Biol. Chem. 266,4478-4483 [Abstract/Free Full Text]
  3. Duggan, M. J., Pollard, S., and Stephenson, F. A. (1991) J. Biol. Chem. 266,24778-24784 [Abstract/Free Full Text]
  4. Duggan, M. J., Pollard, S., and Stephenson, F. A. (1992) J. Neurochem. 58,72-77 [Medline] [Order article via Infotrieve]
  5. Ewert, M., Shivers, B. D., Luddens, H., Mohler, H., and Seeburg, P. H. (1990) J. Cell Biol. 110,2043-2048 [Abstract]
  6. Kern, W., and Sieghart, W. (1994) J. Neurochem. 62,764-769 [Medline] [Order article via Infotrieve]
  7. Khan, Z. U., Gutierrez, A., and De Blas, A. L. (1994) J. Neurochem. 63,371-374 [Medline] [Order article via Infotrieve]
  8. Korpi, E. R., and Luddens, H. (1993) Mol. Pharmacol. 44,87-92 [Abstract]
  9. Luddens, H., and Wisden, W. (1991) Trends Pharmacol. Sci. 12,49-51 [Medline] [Order article via Infotrieve]
  10. Luddens, H., Pritchett, D. B., Kohler, M., Killisch, I., Keinanen, K., Monyer, H., Sprengel, R., and Seeburg, P. H. (1990) Nature 346,648-651 [CrossRef][Medline] [Order article via Infotrieve]
  11. MacDonald, R. L., and Olsen, R. W. (1994) Annu. Rev. Neurosci. 17,569-602 [CrossRef][Medline] [Order article via Infotrieve]
  12. Mathews, G. C., Bolos-Sy, A. M., Holland, K. D., Isenberg, K. E., Covey, D. F., Ferrendelli, J. A., and Rothman, S. M. (1994) Neuron 13,149-158 [CrossRef][Medline] [Order article via Infotrieve]
  13. McKernan, R. M., Quirk, K., Prince, R., Cox, P. A., Gillard, N. P., Ragan, I. C., and Whiting, P. J. (1991) Neuron 7,667-676 [Medline] [Order article via Infotrieve]
  14. Mertens, S., Benke, D., and Mohler, H. (1993) J. Biol. Chem. 268,5965-5973 [Abstract/Free Full Text]
  15. Nayeem, N., Green, T. P., Martin, I. L., and Barnard, E. A. (1994) J. Neurochem. 62,815-818 [Medline] [Order article via Infotrieve]
  16. Nusser, Z., Pollard, S., Thompson, C. L., Stephenson, F. A., Sieghart, W., and Somogyi, P. (1995) Brain Res. Assoc. Abstr. 12,114
  17. Pollard, S., Duggan, M. J., and Stephenson, F. A. (1991) FEBS Lett. 295,81-83 [CrossRef][Medline] [Order article via Infotrieve]
  18. Pollard, S., Duggan, M. J., and Stephenson, F. A. (1993) J. Biol. Chem. 268,3753-3757 [Abstract/Free Full Text]
  19. Quirk, K., Gillard, N. P., Ragan, C. I., Whiting, P. J., and McKernan, R. M. (1994) J. Biol. Chem. 269,16020-16028 [Abstract/Free Full Text]
  20. Sieghart, W., Eichinger, A., Richards, J. G., and Mohler, H. (1987) J. Neurochem. 48,46-52 [Medline] [Order article via Infotrieve]
  21. Stephenson, F. A. (1995) Biochem. J. 310,1-9 [Medline] [Order article via Infotrieve]
  22. Stephenson, F. A., and Duggan, M. J. (1991) in Molecular Neurobiology: A Practical Approach (Wheal, H., and Chad, J., eds) pp. 183-204, IRL Press, Oxford
  23. Stephenson, F. A., Duggan, M. J., and Pollard, S. (1990) J. Biol. Chem. 265,21160-21165 [Abstract/Free Full Text]
  24. Thompson, C. L., Bodewitz, G., Stephenson, F. A., and Turner, J. D. (1992) Neurosci. Lett. 144,53-56 [CrossRef][Medline] [Order article via Infotrieve]
  25. Uusi-Oukari, M. (1992) J. Neurochem. 59,568-573 [Medline] [Order article via Infotrieve]
  26. Wieland, H. A., Luddens, H., and Seeburg, P. H. (1992) J. Biol. Chem. 267,1426-1429 [Abstract/Free Full Text]

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