(Received for publication, August 16, 1995; and in revised form, October 16, 1995)
From the
The ability of differing subunit combinations of
-aminobutyric acid type A (GABA
) receptors produced
from murine
1,
2, and
2L subunits to form functional
cell surface receptors was analyzed in both A293 cells and Xenopus oocytes using a combination of molecular, electrophysiological,
biochemical, and morphological approaches. The results revealed that
GABA
receptor assembly occurred within the endoplasmic
reticulum and involved the interaction with the chaperone molecules
immunoglobulin heavy chain binding protein and calnexin. Despite all
three subunits possessing the ability to oligomerize with each other,
only
1
2 and
1
2
2L subunit combinations could
produce functional surface expression in a process that was not
dependent on N-linked glycosylation. Single subunits and the
1
2L and
2
2L combinations were retained within the
endoplasmic reticulum. These results suggest that receptor assembly
occurs by defined pathways, which may serve to limit the diversity of
GABA
receptors that exist on the surface of neurons.
-Aminobutyric acid type A (GABA
) (
)receptors are believed to be the major sites of fast
synaptic inhibition in the brain and are also the sites of action for
many psychoactive drugs including the benzodiazepines and barbiturates
(Olsen and Tobin, 1990). Molecular cloning has revealed a number of
GABA
receptor subunits that can be divided by sequence
homology into subunit classes with multiple members:
(1, 2, 3, 4, 5, 6) ,
(1, 2, 3, 4) ,
(1, 2, 3) , and
(1) ,
creating considerable potential for structural diversity. Additional
diversity of receptor structure is generated by alternative splicing of
some of these subunit mRNAs (Burt and Kamatchi, 1991). In situ hybridization and immunocytochemical methodologies suggest a large
temporal and spatial diversity of receptor structure in the brain with
many neuron types often expressing multiple receptor subunits (Wisden
and Seeburg 1992; Fritschy et al., 1992). It is believed that
GABA
receptors are pentameric in their final assembled
plasma membrane form (Nayeem et al., 1994); however, the
precise subunit composition and stoichiometry of a single population of
native GABA
receptors remains unknown.
Accordingly, the
expression of cDNA clones has been used to examine the minimal subunit
composition required to produce functional GABA receptors,
determined by electrophysiological methodologies. Expression of unitary
subunits has produced conflicting results; some subunits expressed
alone appear to be able to produce GABA-gated ion channels (Blair et al., 1988; Pritchett et al., 1988) or channels
that are sensitive to inhibition by picrotoxin, a GABA
receptor channel blocker (Sigel et al., 1989), whereas
other studies demonstrate that some single subunits do not produce
functional receptors (Sigel et al., 1990; Angelotti and
Macdonald, 1993; Krishek et al., 1994). Expression of some
binary subunit combinations have also produced conflicting data. For
example, GABA-gated channels have been reported upon co-expression of
either
1
2 or
2
2 subunits (Verdoorn et al.,
1990; Draguhn et al., 1990). In contrast, the failure of
co-expressed
1
2 and
1
2 subunits to produce
functional GABA
receptors has also been reported (Sigel et al., 1990; Krishek et al., 1994; Angelotti and
Macdonald, 1993). There is, however, general agreement that
co-expression of
and
subunits is sufficient for the
production of GABA-gated chloride currents, and the co-expression of
and
with either the
2 or
3 subunits produce
GABA
receptors that are sensitive to modulation by
benzodiazepines (Pritchett et al., 1988, 1989; Burt and
Kamatchi, 1991)
To further investigate these observations and
attempt to seek an explanation for the failure of certain subunit
combinations to produce functional GABA receptors, we have
examined the assembly and surface expression of homomeric and
heteromeric GABA
receptors using biochemical,
immunological, and electrophysiological methodologies. We have studied
the assembly of GABA
receptors of varying subunit
composition produced from
1,
2, and
2L subunits
expressed in both Xenopus oocytes and transiently transfected
A293 cells. From in situ hybridization and immunochemical
analyses these subunits are co-localized in many adult brain regions
and comprise up to 30% of all benzodiazepine-sensitive GABA
receptors in the adult brain (Benke et al., 1994;
Fritschy et al., 1992).
In this study we demonstrate that
GABA receptor assembly occurs in the endoplasmic reticulum
(ER), where interactions with the molecular chaperones immunoglobulin
heavy chain binding protein (BiP) and calnexin were detected. Access to
the cell surface was, however, limited to receptors composed of
1
2 and
1
2
2L subunits. Single subunits and the
binary combinations of
1
2L and
2
2L, although
capable of oligomerization, were retained within the ER, presumably via
interactions with BiP and calnexin. Receptor assembly and transport to
the cell surface was not dependent on N-linked glycosylation,
although its efficiency was enhanced. These results suggest that
GABA
receptor assembly occurs by defined mechanisms, which
may serve to regulate the diversity of GABA
receptors
expressed on the surface of neurons.
Reimmunoprecipitation was performed on immunoprecipitates in reducing sample buffer. These were diluted 20-fold in lysis buffer containing 2% Triton X-100 prior to the second immunoprecipitation with either 9E10 supernatant, anti-calnexin, or anti-BiP as described above.
To aid biochemical and morphological analyses, GABA receptor subunits were tagged using the epitopes of 9E10 or FLAG.
These epitopes were added to the
1,
2, and
2L subunits
by site-directed mutagenesis between amino acids 4 and 5 of the mature
subunits to create
1
,
2
, and
2L
. The functional effects of these additions were
tested by electrophysiological analysis in A293 cells and Xenopus oocytes. Receptors incorporating 9E10-tagged subunits produced
GABA-activated responses, which were indistinguishable from receptors
comprised of wild-type subunits (Draguhn et al., 1990; Smart et al., 1991; Angelotti and Macdonald, 1993) with regard to
zinc insensitivity and benzodiazepine modulation (Fig. 1).
Similar results were obtained with subunits containing the FLAG
epitope. (
)Therefore, the addition of these small epitopes
to the extreme N-terminal domains of GABA
receptor subunits
appears to be ``functionally silent.''
Figure 1:
Functional properties of
recombinant GABA receptors incorporating the 9E10 epitope
tag expressed in A293 cells. A, GABA equilibrium concentration
response curves for
1
2 (
) and
1
2
(
) constructs (n = 3-5 cells). The curves were fitted using a
nonlinear least squares Marquadt routine with the following logistic
state function: I/I
= 1/{1
+ (EC
/[A]
)},
where I represents the GABA-activated current at a given
concentration of GABA ([A]), and I
is
the maximum current induced by a saturating concentration of GABA.
EC
describes the concentration of GABA inducing a
half-maximal response, and n is the Hill coefficient. The
determined EC
values and Hill coefficients for the
1
2 are 0.77 ± 0.07 µM and 1.45 ±
0.22, and for the
1
2
, the
values are 0.82 ± 0.03 µM and 1.66 ± 0.12. B, whole cell recordings of GABA-activated currents after
rapid application of GABA to A293 cells at -50 mV expressing
1
2
constructs. The solid
lines indicate the duration of the GABA application. W represents the recovery times (min) for the GABA-induced responses
following exposure to 1 µM flurazepam or 10
µM Zn
. C, concentration
response curves for the recombinant GABA
receptors
1
2
2L (
) and
1
2
2L
(
) expressed in A293 cells. The EC
values and Hill
coefficients were 4.61 ± 0.83 µM and 1.38 ±
0.32 and 4.8 ± 0.63 µM and 0.98 ± 0.1,
respectively. D, examples of GABA-induced membrane currents
recorded from
1
2
2L
receptors after application of 1 µM flurazepam or 50 µM Zn
. Note the
enhanced response to GABA after flurazepam and the reduced inhibition
by Zn
in the
2L subunit containing GABA
receptors. The calibration in D applies to the upper and
lower series of responses.
The subcellular
distribution of these tagged GABA receptors expressed in
A293 cells was determined by immunofluorescence of the FLAG epitope
followed by confocal microscopy. Expression of
1
,
2
, or
2L
alone revealed an
ER-like staining pattern similar to that for horseradish peroxidase
containing the C-terminal ER retention signal Lys-Asp-Glu-Leu
(horseradish peroxidase-KDEL) (Fig. 2A), which is
almost exclusively localized to the ER (Connolly et al.,
1994). To further investigate the localization of these GABA
receptor subunits, immunofluorescence was performed on cells in
which the peroxidase reaction had been performed using diaminobenzidine
as the substrate. This results in the production of a dark insoluble
precipitate, which cross-links all proteins that co-localize with
horseradish peroxidase (Courtoy et al., 1984; Ajioka and
Kaplan, 1987). The reaction product should therefore prevent the
detection of fluorescence (DAB quenching) if the candidate protein
shares the same intracellular localization as horseradish peroxidase.
Figure 2:
Surface expression and intracellular
localization of GABA receptor unitary, binary, and tertiary
subunit combinations. A293 cells were analyzed for GABA
receptor/horseradish peroxidase-KDEL expression 1 day after
transfection. A, co-immunofluorescence of individual
GABA
receptor subunits (via FLAG epitope) and horseradish
peroxidase-KDEL was performed on permeabilized cells (using
anti-horseradish peroxidase antiserum). B, DAB quenching in
cells co-expressing horseradish peroxidase-KDEL and
1
2 (
),
1
2L (
), or
2
2L (
), followed by
immunofluorescence for the FLAG tag. Areas were selected to illustrate
immunofluorescence versus DAB reaction product. C,
GABA
receptor detection, via FLAG tag, on the surface of
nonpermeabilized cells expressing
1
2 (
),
2
1 (
), or
1
2
2L
(
). The scale bar represents 10
microns.
To confirm this, we examined the ability to detect endogenous markers in the presence of DAB reaction product produced from horseradish peroxidase-KDEL. After completion of the DAB reaction, under conditions identical to those shown in Fig. 2B, fluorescence was performed on horseradish peroxidase-KDEL-expressing cells using either antibodies against BiP and calnexin (localized to the ER; Fig. 3A) or fluorescent-labeled wheat germ agglutinin and lens culinaris lectins (markers for the Golgi apparatus; Fig. 3B). In cells expressing horseradish peroxidase-KDEL, no fluorescence was detected using antibodies to the ER markers, BiP, and calnexin, although fluorescence was detected in untransfected cells. In contrast, high levels of fluorescence for the Golgi markers, wheat germ agglutinin, and lens culinaris were observed in horseradish peroxidase-KDEL-transfected cells. These results confirm DAB quenching as a valid protocol for the detection of components within the same compartment.
Figure 3:
Localization of ER and Golgi markers using
DAB quenching from horseradish peroxidase-KDEL. A293 cells were
transfected with the horseradish peroxidase-KDEL cDNA expression
construct. 12-18 h after transfection, the cells were fixed and
exposed to 0.02% HO
, 0.15% diaminobenzidine,
and 0.1 M imidazole for 1 h at 37 °C. The cells were then
processed for immunohistochemical analysis using antisera directed
against either ER or Golgi markers. A, ER markers: BiP (Clone
5A5) and calnexin (AF8). B, Golgi markers detected by
rhodamine-labeled wheat germ agglutinin (WGA) and fluorescein
isothiocyanate-labeled lens culinaris lectin (LC). The left-hand panels are phase images, whereas the right-hand panels represent fluorescent images recorded from the same field.
The scale bar represents 10
microns.
Using this methodology we analyzed all
three binary subunit combinations 1
2,
1
2L, and
2
2L (Fig. 2B). Immunofluorescence was rare
(approximately 0.01%) in
1
2L- or
2
2L-transfected
cells and was never coincident with cells exhibiting DAB staining,
suggesting co-localization of these binary subunit combinations with
horseradish peroxidase-KDEL. Similar results were also seen with single
subunits utilizing DAB quenching.
These results are
consistent with the restriction to an ER-like staining pattern observed
for these combinations in the absence of horseradish peroxidase-KDEL (Fig. 2B;
and
)
and their failure to reach the cell surface, as determined in
nonpermeabilized cells.
Together these results demonstrate
that single subunits and the
1
2L and
2
2L binary
subunit combinations are restricted to the ER.
In contrast, the
1
2 combination was capable of leaving the ER as determined by
the co-existence of immunofluorescence and DAB staining within the same
cells, consistent with the apparent surface staining (Fig. 2B,
) and confirmed in
nonpermeabilized cells (Fig. 2C,
and
). Only when all three subunits were
co-expressed could the
2L subunit be detected on the cell surface (Fig. 2C,
). These results
correlated with electrophysiological recordings made from A293 cells
expressing all subunit combinations. Consistent with the results from
the immunofluorescence experiments, only cells expressing either
1
2 or
1
2
2L receptor subunits exhibited
resolvable GABA-gated currents (Table 1). Similar results were
obtained from expression studies performed using Xenopus oocytes up to 72 h after injection. Thus it appears that only the
combinations
1
2 and
1
2
2L can access the cell
surface. The accumulation of all nonsurface receptor combinations
(
,
,
,
, and
) in the ER strongly
suggests that receptor assembly occurs in this intracellular
compartment.
Figure 4:
Immunoprecipitation and glycosylation of
GABA receptor
1,
2, and
2L subunits from
transfected A293 cells. A293 cells expressing
1
(
),
2
(
),
2L
(
), or untransfected (C)
were [
S]methionine-labeled in the
absence(-) or presence (+) of 5 µg/ml tunicamycin
(present 2 h prior to and during labeling). Receptor subunits were then
immunoprecipitated using 9E10 antibody coupled to protein G-Sepharose.
Immune complexes were then separated by SDS-polyacrylamide gel
electrophoresis using 8% gels. The molecular masses of marker proteins
(Bio-Rad) are indicated on the right. A co-immunoprecipitating
band migrating at approximately 75 kDa (*) was consistently
observed.
The 2
subunit
exhibits apparent molecular masses of 53 and 56 kDa plus a weak band at
50 kDa (Fig. 4,
, -). Again, this subunit
contains two consensus sequences for N-linked glycosylation at
positions 8 and 80. Tunicamycin treatment produced a major band at 50
kDa (Fig. 4,
, +), as predicted by cDNA
cloning (Kamatchi et al., 1995). Thus, the
2 forms are
also consistent with the presence of zero, one, and two sites of N-linked glycosylation. The
2L
subunit
migrated as a broad band at approximately 42 kDa (Fig. 4,
, -) and, following tunicamycin treatment, as a
broad band of around 30 kDa (Fig. 4,
, +);
this shift is consistent with the predicted presence of three consensus
sites for N-linked glycosylation at positions 13, 90, and 208.
The apparent molecular masses of both glycosylated and unglycosylated
forms of
2L are much smaller than its 48-kDa predicted molecular
mass derived from cDNA cloning (Pritchett et al., 1989). This
may be due to proteolysis, a common observation for this subunit (Moss et al., 1992; Haddingham et al., 1992) and may
explain the appearance of a smear rather than discreet bands.
Consistent with
the surface expression of the 1
2 subunit combination, it was
found that
2 co-immunoprecipitates both the 48-kDa unglycosylated
and the 50-kDa partially glycosylated but not the 52-kDa fully
glycosylated forms of the
1 subunit (Fig. 5A, lane 3). The reciprocal co-immunoprecipitation was not so
clear and may be complicated by the co-migration of both the
1 and
2 bands at 50 kDa (Fig. 5A, lane 4).
Despite being transport incompetent, the
1 and
2L subunits
could also co-immunoprecipitate each other (Fig. 5B, lanes 3 and 4). As seen with the
2 subunit, the
2L also binds exclusively to the two lower forms of
1 (Fig. 5B, lane 3). Thus, neither
2 nor
2L show detectable oligomerization with the 52-kDa fully
glycosylated form of the
1 subunit, even though it is the major
species present. Similarly, the
2 and
2L subunits are also
capable of oligomerizing despite their transport incompetence. In this
case,
2L binds predominantly to the nonglycosylated 50-kDa form of
2 (Fig. 5C, lane 3). It also appears that
both
1 and
2 bind preferentially to lower molecular mass
2L (Fig. 5, B, lane 4, and C, lane 4, respectively) compared to the major species present
when immunoprecipitated directly (Fig. 5, B, lane
2, and C, lane 2). These preferences for subunit
binding occur in the presence of the nonbinding forms, which exist
within 5 min of [
S]methionine labeling, a period
in which oligomerization has occurred.
Similar patterns of
subunit oligomerization were seen under nonreducing conditions,
suggesting that they did not result from intermolecular disulfide
bridges, as well as after solubilization in a range of other detergents
including CHAPS (1%) and digitonin (1%).
To test the
oligomerization of the triple subunit combination
1
2
2L
immunoprecipitation utilizing
2L
subunit was
performed. These results demonstrated association of the
2 and
1 subunits with the
2L subunit as expected.
Figure 5:
Oligomerization of GABA receptor subunits expressed in A293 cells. A293 cells expressing
single or binary combinations of GABA
receptor subunits
were labeled with [
S]methionine, cells were
lysed, and GABA
receptor subunits were purified using 9E10
antibody coupled to protein G-Sepharose. GABA
receptor
subunits were then separated by SDS-polyacrylamide gel electrophoresis
using 8% gels. Control cells show the presence of a contaminating band
at approximately 40 kDa, which is sometimes observed regardless of the
antibody used (arrowhead). A, oligomerization of
1 and
2 subunits: lane 1,
1
; lane 2,
2
; lane 3,
2
1; lane 4,
1
2. B, oligomerization of
1 and
2L subunits: lane 1,
1
; lane
2,
2L
; lane 3,
2L
1; lane 4,
1
2L. C, oligomerization of
2 and
2L subunits: lane 1,
2
; lane
2,
2L
; lane 3,
2L
2; lane 4,
2
2L.
Taken together with the immunolocalization studies, these
experiments detailing the oligomerization of receptor subunits
demonstrate that GABA receptor assembly is primarily
localized to the ER.
Figure 6:
Effect of tunicamycin on GABA
receptor subunit oligomerization. A293 cells transfected with
1
(lane 1);
2
(lane 2);
1
,
2, and
2L (lane 3);
1
and
2L (lane 4);
or control untransfected cells were labeled with
[
S]methionine, immunoprecipitated using 9E10
antibody coupled to protein G-Sepharose, and resolved by
SDS-polyacrylamide gel electrophoresis using 8% gels. Cells were
treated with 5 µg/µl tunicamycin for 2 h prior to and during
labeling with [
S]methionine. C represents immune precipitations from control untreated cells. The
presence of a contaminating band of 40 kDa, which is sometimes observed
regardless of antibody used, is indicated
(*).
Although the
inhibition of N-linked glycosylation did not affect subunit
oligomerization, N-linked glycosylation may be important for
subsequent maturation. Therefore, we examined cell surface receptor
expression in the presence of tunicamycin. This treatment caused other
effects, most notably a reduction in cell number, reduced transfection
efficiency, and changes in morphology. However, cell surface expression
was not prevented, as determined by immunofluorescence for receptors
consisting of 1
2
2L subunits (Fig. 7A)
but not for intracellular markers such as BiP, although the efficiency
was significantly reduced from 95 ± 7.5% in the absence to 37
± 15.52% in the presence of tunicamycin (Fig. 7B). This is consistent with the report of the
1 subunit (lacking N-linked glycosylation sites by
site-directed mutagenesis) in the presence of
1 and
2, which
produced functional GABA
receptors with pharmacological
properties similar to those observed for the wild-type receptor but
with reduced levels (Buller et al., 1994).
Figure 7:
Effect
of tunicamycin treatment on cell surface expression. A,
immunofluorescence for the 1
subunit in the
presence (+) or the absence(-) of TX100 was performed on
A293 cells transfected with
1
2
2L
(-tunicamycin). In a duplicate experiment, the cells were treated
with tunicamycin constantly post-transfection, as well as 2 h
pretransfection (+tunicamycin). B, quantitation of the
frequency of surface (-TX100) fluorescence as a percentage of the
frequency of total (+TX100) fluorescence. In the absence of
tunicamycin 38.23 ± 3.03% (n = 519) were
positive in the absence of TX100, with 40.3 ± 7.84% (n = 445) in the presence of TX100. In the presence of
tunicamycin 6.05 ± 2.53% (n = 843) were positive
in the absence of TX100, with 16.3 ± 7.2% (n =
336) in the presence of TX100 (where n = total number
of cells counted).
Figure 8:
Interaction of GABA receptor
subunits with the ER chaperones: calnexin and BiP. A, cells
transfected with the GABA
1
subunit
were labeled with [
S]methionine and then
immunoprecipitated using 9E10 antibody coupled to protein G-Sepharose.
The precipitated material was then reprecipitated with antibodies
against BiP (lane 1), calnexin (AF8) (lane 2), or
9E10 (lane 3). B, A293 cells expressing
1
(lane 1),
2
(lane 2), or
2L
(lane 3)
and untransfected cells (lane 4) were immunoprecipitated using
either anti-calnexin antibody (AF8) or by 9E10 antibody (9E10). Migration of the 90-kDa calnexin (AF8) and
the 75-kDa BiP bands as well as the mature GABA
receptor
subunits are identified.
This interaction with BiP and calnexin is
not unique to the 1
subunit, because the
1
,
2
, and
2L
subunits can also be co-immunoprecipitated by anti-calnexin
antibody (Fig. 8B) with no apparent preference for
different forms. In all three cases, a protein migrating at the same
molecular mass as BiP is also co-immunoprecipitated, suggesting some
overlap in the binding abilities of BiP and calnexin. This overlap
appears to occur with some endogenous proteins in A293 cells as
evidenced by the untransfected control lane (Fig. 8B),
in which anti-calnexin antibody co-immunoprecipitated a band coincident
with BiP. When these apparent complexes were immunoprecipitated by
9E10, calnexin is only weakly observed, consistent with its low
turnover rate and subsequently low [
S]methionine
incorporation (Hammond et al., 1994). Upon expression of all
three, subunits BiP is still immunoprecipitated (Moss et al.,
1995). However, whether this is due to interactions occurring between
unitary, binary, or tertiary subunit complexes under these conditions
is difficult to ascertain.
To date, 15 different GABA receptor cDNAs have
been isolated from a variety of vertebrates (Burt and Kamatchi, 1991).
Many of these subunits exhibit differing patterns of both spatial and
developmental expression in the CNS, with many neurons often expressing
multiple numbers of receptor subunits (Wisden and Seeburg, 1992). A
major challenge in trying to analyze the diversity of GABA
receptor structure in the brain is determining what processes
control receptor assembly. Regulation of receptor assembly could occur
at numerous stages, including subunit oligomerization or export to the
cell surface. Unfortunately, to date there is little experimental data
on these important questions. To address this, we have examined the
assembly of GABA
receptors of differing subunit
compositions constructed from those of
1,
2 and
2L
subunits expressed in both A293 cells and Xenopus oocytes
using immunological, biochemical, and electrophysiological
methodologies. These subunits are thought to co-localize in many brain
regions and comprise up to 30% of all benzodiazepine-sensitive
GABA
receptors in the adult brain (Fritschy et
al., 1992; Benke et al., 1994). For immunological and
biochemical analyses the 9E10 (Evan et al., 1985) or the FLAG
epitopes were added between amino acids 4 and 5 of each of these
subunits. As demonstrated by electrophysiological analyses, these
additions appeared to be functionally silent.
Using
immunolocalization, epitope tagged 1,
2, or
2L subunits
expressed alone are incapable of leaving the ER, as are the binary
subunit combinations of
1
2L and
2
2L, demonstrated
by co-localization with horseradish peroxidase-KDEL (Connolly et
al., 1994). The only combinations of receptors produced from these
subunits that are capable of exiting the ER and accessing the cell
surface are
1
2 and
1
2
2L. In agreement with
this only the latter two subunit combinations exhibited resolvable
GABA-gated currents when expressed in either A293 cells or Xenopus oocytes.
Previous studies have produced conflicting data on the
expression of single subunits and the combinations of 1
2L and
2
2L, as determined by electrophysiological analysis. Blair et al. (1988) reported the production of GABA-gated channels
on the expression of single
1 or
1 subunits in Xenopus oocytes, and Sigel et al.(1989) reported finding chloride
currents that could be blocked by picrotoxin on the expression of the
rat
1 subunit. Moreover, Verdoorn et al.(1990) and
Draguhn et al.(1990) found robust receptor expression from
1
2 subunits and smaller currents from
2
2L subunits
in A293 cells. In contrast to these results Angelotti and
Macdonald(1993) and Krishek et al.(1994) found no GABA-gated
currents when expressing
1
2 or
1
2 subunits in L929
or A293 cells. These discrepancies could be due to differences in the
expression systems used or differences in the type of
subunit
used in some of these experiments (
1 versus
2).
Expression of murine
1 subunits alone in both A293 cells and Xenopus oocytes can produce low levels of surface expression, (
)in common with the observations of Blair et
al.(1988) and Sigel et al.(1989). Therefore some of this
variability in expression may be subunit-specific. A second reason for
such discrepancies may result from over-expression. It is possible that
the longer expression periods used in many previous studies
(48-72 h in A293 cells and 2-6 days in oocytes (Blair et al., 1988; Verdoorn et al., 1990) compared with
12-24 h and 2-3 days for 293 cells and Xenopus oocytes, respectively, used in this study) may have resulted in
the escape of normally transport-incompetent receptor complexes through
saturation of the ER retention system, resulting in low levels of
surface expression.
In spite of the apparent inability of the
1
2L and
2
2L subunit combinations to leave the ER,
they were still capable of rapid oligomerization. In fact, all
oligomerization events were not dependent on N-linked
glycosylation and could occur with unglycosylated subunits.
Furthermore, N-linked glycosylation was not required for the
transport of receptor heteroligomers to the cell surface.
Interestingly, the
2 subunit appeared to oligomerize more
efficiently to lower molecular mass forms of the
1 (and possibly
the
2L subunits), and the
2L subunit appeared to oligomerize
more efficiently to lower molecular mass forms of both the
1 and
the
2 subunits. The significance of these interactions is
uncertain; they may represent preferred patterns of subunit
oligomerization if these incompletely processed forms are represented
in cell surface receptors in the presence of glycosylated forms.
Whether these interactions are important in controlling the final
assemblies of GABA
receptors produced warrants further
investigation.
GABA receptor subunits interacted with at
least two ER chaperone proteins, BiP and calnexin, whose function is to
retain misfolded and unassembled proteins. Interactions with BiP are
thought to result from the exposure of hydrophobic domains in
incorrectly folded proteins (Pelham, 1989), whereas calnexin appears to
show specificity for glycoproteins containing partially
``glucose-trimmed'' carbohydrate side chains (Hammond et
al., 1994). Calnexin is thought to hold glycoproteins in the ER
until folding/assembly is complete, thus preventing their aggregation
and/or premature exit from the ER (Hochstenbach et al., 1992;
Ou et al., 1993; Hammond et al., 1994). Presumably
single GABA
receptor subunits and the binary combinations
of
1
2L and
2
2L are retained in the ER via the
interaction with chaperone proteins such as calnexin and BiP.
The
demonstration of the intracellular localization of GABA receptor heteroligomers has important consequences for our
understanding of GABA
receptor structure. To date, the
method of choice for examining the complexity of GABA
receptor structure in the brain has been the use of
subunit-specific antisera to immunoprecipitate receptor complexes from
solubilized brain tissue (e.g. Duggan and Stephenson, 1990;
Mertens et al., 1993; Mckernan et al., 1991; Mossier,
1994). These procedures are complex and often yield contradictory
results (cf. Pollard et al.(1993), Quirk et
al.(1994), and Khan et al.(1994)). It is possible that
some of the interactions seen following immunoprecipitation in these
experiments and thereby proposed to represent cell surface receptor
subunit combinations may in fact represent ER-retained forms of
GABA
receptors. Such complexes as demonstrated in our study
may have differing subunit combinations from GABA
receptors
expressed on the surface of neurons.
Finally, the inability of
GABA receptor heteroligomers consisting of
1
2L
and
2
2L subunits to reach the cell surface suggests that
assembly of GABA
receptors might share similar mechanisms
to those employed by muscle acetylcholine receptors. Assembly of these
receptors is believed to utilize intracellular dimer or trimer
intermediates (Green and Miller, 1995). Further studies will elucidate
whether the ER-retained subunit heteroligomers described in our work
are important intermediates in the production of fully assembled
pentameric GABA
receptors. This may be of significance
because the consensus of opinion (Burt and Kamatchi, 1991; Pritchett et al., 1988, 1989), derived from a combination of molecular,
pharmacological, and electrophysiological methodologies, suggests that in vivo benzodiazepine-responsive GABA
receptors
are composed of
in unknown stoichiometries. The
relevance of these
1
2L and
2
2L intracellular
subunit complexes to the final functional GABA
receptors
produced is currently under investigation.