(Received for publication, May 23, 1995; and in revised form, July 7, 1995)
From the
-Aminobutyric acid
(GABA
) receptors
were purified from adult rat cerebella by anti-
6(1-16 Cys)
antibody affinity chromatography. Immunoblots of the
6
subunit-containing receptors showed the copurification of the
1,
2/3,
2,
but not
2 and
3 GABA
receptor polypeptides. Further fractionation of this receptor
subpopulation by anti-GABA
receptor subunit
6(1-16 Cys) and anti-
1(413-429) antibody affinity
columns in series substantiated the coassociation of the
1 and
6 polypeptides. The percentage of coexistence of the two subunits
was determined by quantitative immunoblotting, which found that 41
± 12% of
6 subunit immunoreactivity is associated with the
1 subunit. The ratios of the
1:
6 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
receptors. The benzodiazepine pharmacology of the
1
6
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
receptors.
The GABA(
)receptors of mammalian brain
are fast-acting ligand-gated chloride ion channels. Multiple genes
encoding GABA
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
receptor subunits are the
1-
6,
1-
3,
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
receptors. The polypeptide complement of
any one native GABA
receptor has not been elucidated.
However, the biophysical and pharmacological properties of cloned
receptors suggest that most probably consist of an
combination (Olsen and McDonald, 1994; Stephenson, 1995). A subunit
stoichiometry (
)
(
)
(
)
was found for a defined expressed GABA
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
receptor subpopulation in the cerebellum
consisted of
1
6
2
2
2/3
subunits where
2
and
2
are splice
variants of the
2 subunit.
We have adopted the approach of the
determination of native GABA 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
subunit complements of native receptors.
We found, in agreement with several other groups, that the majority of
native GABA
receptors contain a single
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
subunit complement e.g.
1
2,
1
3, and
2
3 subunit-containing
receptors (Duggan et al., 1991; Pollard et al.,
1993). Thus, we proposed the assembly of at least two
subunits
per receptor oligomer. Several other groups have since reported the
copurification or coimmunoprecipitation of other
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 receptor
6
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
6
x
x (and
also
4
x
x) 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
6 subunit-containing GABA
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.
We have previously reported the purification of 6
subunit-containing GABA
receptors from calf cerebellum by
anti-
6(1-16 Cys) Fab antibody affinity chromatography
(Pollard et al., 1993). In that study we reported the
copurification of the
6 and
1 subunit immunoreactivities.
However, because of the low yield in the isolation procedure, we were
unable to characterize in detail the coassociation of
6 and
1
subunit immunoreactivities by the use of different specificity antibody
columns in series, as has been employed for other GABA
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
receptor
6 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
6
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
6 subunit
immunoreactivity was found in the solubilized preparation (Fig. 1). The comparative results for the extraction of BZ-S and
BZ-IS [
H]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
6 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
receptor subunit complements by biochemical approaches, the
assumption is made that the solubilized preparation is representative
of the entire functional,
6 subunit-containing GABA
receptor population.
Figure 1:
Solubilization of GABA
receptor
6 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-
6(1-16 Cys) antibodies (2.5 µg, final
concentration) all as described under ``Experimental
Procedures.'' Lane1, benzodiazepine
affinity-purified GABA
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;
1 and
6 refer to the antibodies used for
immunoblotting.
Figure 3:
Purification of GABA receptors
from adult rat cerebellum by anti-
6(1-16 Cys) Fab and
anti-
1(413-429) antibody affinity columns in series.
GABA
receptors were purified by anti-
6(1-16 Cys)
Fab affinity chromatography. Receptor-containing fractions were pooled
and applied to an anti-
1(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
6; B, immunoblotting with
1 affinity-purified
antibodies. The gel lay-out is the same for both: lane1, Na
deoxycholate extract of adult rat
cerebellum; lane2, anti-
6(1-16 Cys) Fab
antibody post-column filtrate; lane3,
6(1-16 Cys) immunoaffinity-purified receptors; lane4, anti-
1(413-419) antibody post-column
filtrate; lanes 5-9, pH 11.5 eluted fractions 1-5
from the anti-
1 413-429 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
1
(
) and
6 (
) immunoreactivities eluted by pH 11.5
from the anti-
1(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
[H]Ro 15-4513 binding activity were both assayed.
The results were difficult to quantify because of the low level of
total [
H]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 [
H]flunitrazepam binding sites by the
anti-
6(1-16 Cys) antibody column. In agreement,
[
H]flunitrazepam binding to the purified
receptors was not detectable (n = 2, results not
shown).
To determine the other GABA receptor
polypeptides that copurified with the
6 subunit-containing
GABA
receptors, the pH 11.5 fractions 2-4 were pooled
and analyzed by immunoblotting. Fig. 2shows the results, where
it can be seen that
1,
2/3,
2, and
subunit
immunoreactivities were found. As for the bovine preparations, there
was no detectable
2 or
3 subunit immunoreactivities. Note
that we were unable to use the anti-
3(379-393) antibody
(Pollard et al., 1991); this was raised to the bovine
3(379-393) sequence. The homologous rat sequence has three
amino acid differences, and the rat
3 subunit is not recognized by
the bovine antibody. The
subunits were thus identified by the
monoclonal antibody bd-17, which recognizes both rat
2 and
3
subunits.
Figure 2:
Demonstration by immunoblotting of the
coassociation of GABA receptor
1,
2/3,
2,
and
with the
6 subunit. GABA
receptors were
purified from adult rat cerebellum by anti-
6(1-16 Cys) Fab
affinity chromatography. The
6 subunit-containing fractions were
pooled and analyzed by immunoblotting for reactivity with
1 (M
51,000),
2/3 (M
59,000 and 57,000),
2 (M
49,000),
(M
57,000 ± 500), and
6 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-
6(1-16 Cys) Fab affinity-purified GABA
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.
To determine the ratios of the
1:
6 subunits in the double purified receptor preparations,
two sets of experiments were carried out. First, the primary
1 and
6 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
6
1 subunit-containing antigen
was varied and the resultant immunoreactive bands quantified (Fig. 4B). The
1:
6 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-
6(1-16 Cys)
antibody did not pellet all the
6 subunit immunoreactivity even
when used at saturation. Although immunoblotting and
immunoprecipitation are two different experimental paradigms, further
investigation was required to ensure that the
6 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-
6(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-
6(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
6 subunit immunoreactivity were found (results not shown).
Figure 4:
Determination of the 1:
6 subunit
ratio in
6
1 double immunopurified GABA
receptors
from adult rat cerebellum. GABA
receptors were purified
from adult rat cerebellum by anti-
6(1-16 Cys) and
1(413-429) antibody affinity columns in series as described
under ``Experimental Procedures.'' A shows the
results for an immunoblot where a fixed amount of
6
1
subunit-containing receptors as antigen (70 µl) was used with
increasing concentrations of affinity-purified
anti-
1(324-341) (
) or anti-
6(1-16 Cys)
(
) GABA
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
[H]Ro 15-4513 binding sites from cerebellar
detergent extracts and GABA
receptors purified by
anti-
1(413-429) antibody affinity chromatography.
Immunoprecipitation was carried out from Na
deoxycholate extracts of rat cerebellum and GABA
receptor anti-
1(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 [
H]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-
1(413-429) immunopurified receptors. A, total; B, BZ-IS; C, BZ-S [
H]Ro 15-4513
binding activity, respectively. Ig, nonimmune protein
A-purified Ig;
1, anti-
1(413-429);
6, anti-
6(1-16 Cys) antibodies,
respectively.
Immunoblots of 6
1 double immunoaffinity-purified receptors
showed the coassociation of
2/3 and
subunits (n = 1; results not shown), but attempts to show the
localization of the
2 subunit have so far been negative.
The same immunoprecipitation
assays were carried out on GABA receptors purified from
adult rat cerebellum by anti-
1(413-429) antibody affinity
chromatography. Here, the
1 antibody immunoprecipitated close to
100% of total, BZ-S, and BZ-IS [
H]Ro 15-4513
sites as should be the case for an
1 subunit-purified preparation.
However, the
6 subunit antibody immunoprecipitated a maximum of 30
± 5% of total [
H]Ro 15-4513 sites (i.e.
1
6 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
[
H]Ro 15-4513 binding sites precipitated, but it
was found that at these high antibody concentrations,
6 subunit
immunoreactivity was still present in the supernatant. Thus, the
inability to immunoprecipitate all the BZ-IS
[
H]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
[
H]Ro 15-4513 binding was BZ-IS.
In this paper, we have described the purification of 6
subunit-containing GABA
receptors with the retention of
their [
H]Ro 15-4513 radioligand binding
activities. We have substantiated the coexistence of the
1 and
6 subunit in single receptor oligomers and, in addition, we have
quantified their percentage coassociation. In double
immunoaffinity-purified receptors, the
1:
6 subunit ratio was
1:1 and the benzodiazepine pharmacology of this subset of receptors was
BZ-IS. Thus the use of the
6 and
1 immunoaffinity columns in
series not only proved the coexistence of these two gene products in
one receptor (41% of the
6 subunit receptor population, Table 3) but also showed that in the rat cerebellum, either
single variant
1 or
6 subunit-containing receptors exist.
These results are in agreement with the emerging pattern from several
groups. That is, that at least for the GABA
receptor
subunits, different isoforms do partially coexist within the same
receptor molecule (summarized by Stephenson, 1995). With specific
reference to the
1 and
6 subunits, the findings herein are in
agreement with the localization of
1 and
6 GABA
receptor subunit-like immunoreactivities at the electron
microscopic level where synapses in the cerebellum were found
containing either
1 or
6 or both
1 and
6
polypeptides (Nusser et al., 1995). Moreover, Mathews et
al.(1994) coexpressed
1,
6,
2, and
2
polypeptides in mammalian cells and showed that the resultant
pharmacological properties were distinct from both
1
1
2
and
6
1
2 receptors and best explained by an
1
6
2
2 hybrid receptor. Quirk et al.(1994),
however, found no evidence for coassociation of
1 and
6
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- subunits coassociated with the
6 polypeptide, the results reported here are in agreement with
previous reports, where the coassociation of
6 with
2 (Khan et al., 1994; Quirk et al., 1994),
(Quirk et al., 1994), and
2/3 (Khan et al., 1994) was
found. But Quirk et al.(1994) identified
6
2 and
6
as two distinct populations, where the latter did not bind
[
H]Ro 15-4513. In the
1
6 double
immunoaffinity-purified receptors, we were unable to detect the
GABA
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
6 variant receptors. Consequently, the
(
1
6
2/3
) receptor identified here may be similar to
(
6
) receptors described by Quirk et al. (1994),
which do not bind [
H]Ro 15-4513. Further analysis
of the anti-
1(413-429) post-column filtrate should clarify
this.
The direct determination of the number of subunits (a
1:1 ratio for
1:
6, therefore predicting two per receptor) is
the first for native GABA
receptors. It is in agreement
with the 1:1 ratio predictions for native receptors where the
coexistence of two but not three different
subunits were
detectable (Duggan et al., 1991), the inferred subunit
complement of native cerebellar receptors,
1
6
2/3
2
2
(Khan et
al., 1994), and the subunit complement of an
(
1)
(
1)
(
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
1 and
6
subunits following reduction and denaturation in SDS-PAGE.
The study
of cloned, single GABA
receptors showed that the
benzodiazepine subpharmacology was dependent on the type of
subunit (Luddens and Wisden, 1991). The
6 subunit-containing
cloned receptors show BZ-IS pharmacology in contrast to
1
receptors, which are BZ-S (Luddens et al., 1990). A single
point mutation in the
6 subunit,
6R100H, results in a mutant
receptor with high affinity for the classical benzodiazepines (Wieland et al., 1992). From the results reported herein, for
(
1
6) receptors, the
6 subunit dominates the pharmacology
yielding BZ-IS pharmacology. This would be in agreement with Mathews et al.(1994).