Previous Article | Next Article 
Volume 16, Number 17,
Issue of September 1, 1996
pp. 5415-5424
Copyright ©1996 Society for Neuroscience
Stoichiometry of a Recombinant GABAA Receptor
Yongchang Chang,
Ruoping Wang,
Sonal Barot, and
David S. Weiss
Neurobiology Research Center, Department of Physiology and
Biophysics, University of Alabama at Birmingham, Birmingham, Alabama
35294
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES
ABSTRACT
GABA is the main inhibitory neurotransmitter in the mammalian
brain. The postsynaptic GABAA receptor/pore complex is
presumed to be a pentamer typically composed of a combination of
,
, and
subunits, although the stoichiometry remains
controversial. We probed the stoichiometry of the GABAA
receptor by site-directed mutagenesis of a conserved leucine (to
serine) in the putative second membrane-spanning domain of the rat
1(
L263S),
2(
L259S), and
2(
L274S) subunit isoforms.
Coexpression of wild-type and mutant subunits of each class (e.g.,
and
L263S), along with their wild-type counterparts (e.g.,
and
), in Xenopus laevis oocytes resulted in mixed
populations of receptors with distinct GABA sensitivities. This is
consistent with the interpretation that the leucine mutation increased
the GABA sensitivity in proportion to the number of incorporated mutant
subunits. The apparent number of incorporated subunits for each class
(
,
, and
) could then be determined from the number of
components comprising the compound GABA dose-response relationships.
Using this approach, we conclude that the recombinant
1
2
2
GABAA receptor is a pentamer composed of two
subunits,
two
subunits, and one
subunit.
Key words:
GABA;
stoichiometry;
receptor;
ion channel;
mutagenesis
INTRODUCTION
The release of GABA from presynaptic nerve
terminals in the CNS inhibits the postsynaptic neuron by gating a
chloride-selective ion pore that is an integral component of the
receptor complex. Four different classes of GABAA receptor
subunits have thus far been identified in the mammalian brain:
,
,
, and
(Schofield et al., 1987
; Khrestchatisky et al., 1989
;
Lolait et al., 1989
; Shivers et al., 1989
; Ymer et al., 1989
; Harvey et
al., 1993
). In addition, multiple isoforms have been isolated for each
of the
,
, and
subunit classes. Exogenous expression of
various combinations of subunits suggests that GABAA
receptors must contain
,
, and
subunits to reconstitute the
major features of native GABAA receptors (Levitan et al.,
1988
; Pritchett et al., 1989
; Malherbe et al., 1990
; Sigel et al.,
1990
; Verdoorn et al., 1990
). Although structure-function studies of
recombinant receptors have elucidated subunit domains and residues
critical for activation and modulation (Pritchett and Seeburg, 1991
;
Sigel et al., 1992
; Wieland et al., 1992
; Amin and Weiss, 1993
), the
stoichiometry of the GABAA receptor has been elusive.
Mutation of a conserved leucine in the putative second
membrane-spanning domain of homomeric neuronal nicotinic acetylcholine
(nACh) and serotonin type 3 (5-HT3) receptors induced an
increase in agonist sensitivity (Revah et al., 1991
; Yakel et al.,
1993
). (nACh and 5-HT3 receptors are members of the same
ligand-operated ion channel superfamily as the GABAA
receptor.) More recent studies of heteromeric nACh receptors determined
that each subunit of the receptor complex carrying this leucine
mutation contributed an
10-fold increase in ACh sensitivity (Filatov
and White, 1995
; Labarca et al., 1995
).
We have used this leucine mutation to infer the number of each subunit
type that comprises recombinant
1
2
2 GABA receptors expressed
in Xenopus oocytes. Mutation of this leucine to serine in
either the rat
1,
2, or
2 GABA subunit isoforms increased the
sensitivity to GABA. Coexpression of wild-type and mutant subunits of
each class (e.g.,
and
-mutant), along with their wild-type
counterparts (e.g.,
and
), resulted in mixed populations of
receptors with distinct GABA sensitivities. We were then able to infer
the number of each subunit type from the number of components
comprising these compound GABA dose-response relationships. Using this
approach, we conclude that the
1
2
2 GABA receptor is a pentamer
composed of two
subunits, two
subunits, and one
subunit.
MATERIALS AND METHODS
Site-directed mutagenesis and in vitro
transcription. The cDNAs were isolated by the PCR (Saiki et al.,
1988
) as described previously (Amin et al., 1994
). The cDNAs were
cloned into the pALTER vector for oligonucleotide-mediated
site-directed mutagenesis using Altered Sites (Promega, Madison WI).
The oligonucleotides (complementary to the sense strand) used to make
the leucine to serine substitutions were
1(L263S): 5
-CAA GGT TGT
CAT GGT ACT AAC GGT CGT CAC TCC-3
;
2(L259S): 5
-GAT TGT
GGT CAT CGT ACT GAC AGT TGT AAT TCC-3
; and
2(L274S):
5
-GAG AGT GGT CAT CGT ACT GAC AGT CGT GAT TCC-3
.
The mismatched base pairs are indicated in bold. Successful
mutagenesis was confirmed by sequencing (Sanger et al., 1977
). cDNAs
were linearized with SspI, which leaves a several hundred
base pair tail that may increase cRNA stability in the oocyte. cRNA was
transcribed from the linearized cDNAs by standard in vitro
transcription procedures. Methods used to match the cRNA concentrations
of the three subunits have been described previously (Amin and Weiss,
1996
).
Oocyte injection. Xenopus laevis (Xenopus I, Ann
Arbor, MI) were anesthetized by hypothermia, and ovarian lobes were
surgically removed from the frog and placed in a solution that
consisted of (in mM): 82.5 NaCl, 2.5 KCl, 10 HEPES, 2 CaCl2, 1 MgCl2, and 10 Na2HPO4, 50 U/ml penicillin, and 50 µg/ml
streptomycin, pH 7.5. Oocytes were dispersed in this same solution
minus CaCl2 plus 0.3% Collagenase A (Boehringer Mannheim,
Indianapolis, IN). After isolation, the oocytes were rinsed thoroughly.
Stage VI oocytes were separated and maintained overnight at
18°C.
Micropipettes for injecting cRNA were fabricated on a Sutter P87
horizontal puller, and the tips were cut off with microscissors. The
wild-type and mutant cRNAs were then mixed at the desired ratios and
diluted with diethyl pyrocarbonate-treated water. Except for the
experiments presented in Figure 5, the
:
:
cRNA injection ratio
was always 1:1:1. The cRNA mixture was then drawn up into the
micropipette and injected with the Nanoject injector (Drummond
Scientific, Broomall, PA).
Fig. 5.
The stoichiometry does not depend on the 

cRNA injection ratio. The
m
and


m cRNA combinations were injected into oocytes at
ratios of 4:1:1 and 1:1:4, respectively. The GABA dose-response
relationships were then fit by the Hill equation and compared with
those determined with 1:1:1 cRNA injections. The EC50
values of the GABA dose-response relationships for
m
cRNA ratios of 1:1:1 and 4:1:1 were (mean ± SD) 0.30 ± 0.13 µM (n = 9)
and 0.30 ± 0.04 µM (n = 5),
respectively. The EC50 values of the GABA dose-response
relationships for 

m cRNA ratios of 1:1:1 and 1:1:4
were 0.99 ± 0.23 µM (n = 4) and
0.98 ± 0.062 µM (n = 4),
respectively. These data indicate that at least at these cRNA ratios,
the number of
and
subunits in the GABA receptor is fixed.
[View Larger Version of this Image (13K GIF file)]
Electrophysiological recording. One to three days after
injection, oocytes were placed on a 300 µm nylon mesh suspended in a
small volume chamber (<100 µl). The chamber and perfusion system,
which allows up to 18 different solutions to be introduced to an
individual oocyte, has been described previously (Amin et al., 1994
).
The oocyte was perfused continuously with a solution that consisted of
(in mM): 92.5 NaCl, 2.5 KCl, 10 HEPES, 2 CaCl2,
and 1 MgCl2, pH 7.5, and briefly switched to the test
solution containing GABA.
Recording microelectrodes were fabricated with a P87 Sutter horizontal
puller and filled with 3 M KCl. They had resistances of
1-3 M
. Standard two-electrode voltage-clamp techniques were used to
record currents in response to the application of GABA. In all cases,
the membrane potential was clamped to
70 mV. Data were played out on
a chart recorder during the experiment and recorded on tape for
off-line analysis.
Peak current amplitudes were measured directly from the chart record or
from the computer screen. To quantify the agonist sensitivity, each
dose-response relationship was fitted with the following equation
using a nonlinear least-squares method:
where x is the number of fitted components and can
vary from 1 to 3, I is the peak current at a given
concentration of GABA (A), Imax is
the maximum current, EC50 is the concentration of GABA
yielding a current half the maximum, and n is the Hill
coefficient.
RESULTS
Mutation of the conserved leucine in
1,
2, or
2 increases
the GABA sensitivity
The isoforms
1,
2, and
2 are widely distributed in the
brain (Benke et al., 1991
; Wisden et al., 1992
; Ruano et al., 1994
), so
we selected the
1
2
2 combination for this particular study.
Given the remarkable sequence homology between isoforms within a class,
the conclusions we reach will likely apply to recombinant GABA
receptors composed of other
,
, and
isoforms. In this
manuscript, we designate the rat
1,
2, and
2 subunit isoforms
as simply
,
, and
.
A conserved leucine in the putative second membrane-spanning domain
(TM2) was mutated to serine in the
(
L263S),
(
L259S), and
(
L274S) subunits. (Henceforth, the subunits with the leucine
mutation will be designated
m,
m, and
m). cRNA was in vitro transcribed for both
mutant and wild-type
,
, and
subunits, mixed in the
combinations 

,
m
, 
m
,
and 

m, and injected into Xenopus laevis
oocytes. Figure 1A shows examples of
GABA-activated currents from each of the four combinations of subunits.
Note that much lower concentrations of GABA were required to activate
the GABA receptors containing either an
m,
m, or
m subunit. Figure 1B
shows average GABA dose-response relationships for all four subunit
combinations and illustrates the leftward shifts in the GABA dose-
response relationships induced by the leucine to serine
substitutions. The continuous lines are the best fits of the Hill
equation to the data. The EC50 values (concentration of
GABA required for half-maximal activation) determined from the fits
were (mean ± SD) 

, 45.8 ± 3.6 µM;
m
, 0.30 ± 0.13 µM;

m
, 0.035 ± 0.004 µM; and


m, 0.99 ± 0.23 µM.
Fig. 1.
Mutation of the conserved leucine in TM2 of the
1(L263S),
2(L259S), and
2(L274S) subunit increases the GABA
sensitivity of the GABAA receptor. A,
GABA-activated currents from oocytes expressing 

,
m
, 
m
, and


m subunit combinations. The subscript
``m'' indicates that the conserved leucine in TM2 was mutated
to serine. GABA was bath-applied at the indicated concentrations. Note
the increase in GABA sensitivity induced by the mutation in each
subunit. Calibration: 100 sec; 350, 65, 35, and 500 nA for the four
rows of traces, respectively. B, Average GABA
dose-response relationships for each of the four combination of
subunits (mean ± SEM). The continuous lines are the
best fit of the Hill equation to the data points. The
EC50 values and Hill coefficients (mean ± SD) for the
fits are 

: 45.8 ± 3.6 µM, 1.59 ± 0.09 (n = 5);
m
: 0.30 ± 0.050 µM, 0.85 ± 0.10 (n = 9);

m
: 0.035 ± 0.004 µM, 1.12 ± 0.04 (n = 3); and 

m:
0.99 ± 0.23 µM, 1.78 ± 0.36 (n = 4).
[View Larger Version of this Image (19K GIF file)]
Studies of heteromeric nACh receptors, in which the stoichiometry has
been established (Reynolds and Karlin, 1978
; Lindstrom et al., 1979
;
Raftery et al., 1980
), concluded that each subunit of the receptor
complex carrying this leucine mutation contributed an
10-fold
increase in ACh sensitivity (Filatov and White, 1995
; Labarca et al.,
1995
). For example, if two subunits in the nACh receptor complex are
mutated, an
100-fold increase in ACh sensitivity was observed. In
this study, we are attempting to work in the other direction; that is,
infer the number of each subunit type comprising the GABAA
receptor from the shift in GABA sensitivity induced by the mutations.
The data presented in Figure 1 demonstrate a 153-, 1308-, and 46-fold
decrease in the EC50 with
m,
m, and
m, respectively. Because the
effects of the mutations may not be equivalent for
,
, and
subunits, we could not ascertain directly the stoichiometry from these
observed shifts in GABA sensitivity. Similarly, the shift in ACh
sensitivity for the nACh receptor with one mutant subunit varied from
13-fold to 49-fold depending on which particular subunit (
,
,
, or
) carried the mutation (Labarca et al., 1995
). We now
describe a strategy to ascertain the stoichiometry that is not
compromised by differences in the magnitude of the shifts
induced by
m,
m, and
m.
Coexpression of wild-type and mutant subunits
Predictions
Assume there is only one
subunit in the GABAA
receptor complex. Coexpression of both
and
m
subunits along with wild-type
and
subunits would result in GABA
dose-response relationships having two components (Fig.
2A): one component from activation
of the
m
receptors and one component from
activation of the 

receptors. Furthermore, on the basis of the
data presented in Figure 1, the GABA EC50 values of these
two components would be
0.30 and
46 µM,
respectively. Alternatively, if the GABA receptor complex contained two
subunits, the GABA dose-response relationships resulting from
coexpression of
m,
,
, and
subunits would have
three components (Fig. 2B): one component from receptors in
which both
subunits are wild type (EC50
46
µM), one component from receptors in which both
subunits are mutant (EC50
0.30 µM), and
one component with an intermediate GABA sensitivity from receptors with
one wild-type and one mutant
subunit. If mutation of the two
subunits has an equivalent effect, the shift in sensitivity contributed
by each
m subunit would be the square root of the shift
in EC50 observed when both
subunits are mutant
(153-fold; Fig. 1B). Thus, each
m subunit
would contribute a 12.4-fold increase in GABA sensitivity predicting an
intermediate component with an EC50 of 3.7 µM. (Similar logic could be applied to arrive at the four
components that would result from 
m
coexpression, assuming three
subunits in the receptor complex.)
Fig. 2.
The number of components comprising the GABA
dose-response relationship with 
m
coexpression
depends on the number of
subunits in the GABA receptor complex.
A, With one
subunit in the GABA receptor complex,
the GABA dose-response relationship from oocytes coexpressing
,
m,
, and
subunits would be composed of two
components (continuous line): one component from
activation of 

receptors and one component from activation of
m
receptors (dashed lines, scaled
to facilitate comparison with the continuous line). The
EC50 values of these two components would be
0.30 and
45.8 µM (indicated on the abscissa) as demonstrated by
the data in Figure 1. The mutant
subunit is shown
shaded. B, With two
subunits in the
GABA receptor complex, the GABA dose-response relationship from
oocytes coexpressing
,
m,
, and
subunits would
be composed of three components (continuous line): one
component from activation of 

receptors, one component from
activation of
m
receptors, and an intermediate
component from activation of receptors containing both an
and an
m subunit (dashed lines). The
EC50 values of the first and third components would be
0.30 and
45.8 µM, and the predicted
EC50 of the intermediate component, assuming the two
m subunits contribute equally to the shift, would be 3.7 µM. A similar logic could be applied to


m
and 


m coexpression to
determine the number of
and
subunits, respectively.
[View Larger Version of this Image (18K GIF file)]

m
coexpression
Figure 3A shows the results from
experiments in which
m,
,
, and
subunits were
all coexpressed in the same oocyte. The resulting dose-response
relationship was well described by the sum of three Hill equations
(continuous line). The EC50 of the first component,
0.26 ± 0.05 µM, corresponds to that determined with
m
coexpression (0.30 ± 0.13 µM), and the EC50 of the third component,
36.3 ± 8.1 µM, corresponds to that of the 

receptor (45.8 ± 3.6 µM). The presence of an
intermediate component (EC50 = 2.2 ± 0.1 µM) indicates that the GABA receptor must contain more
than one
subunit (Fig. 2B). The EC50 of this
intermediate component is in excellent agreement with the predicted
intermediate EC50 of 3.7 µM (Fig.
2B). Our interpretation of this intermediate component is
that it represents the activation of receptors containing both an
and an
m subunit. This interpretation is supported by
the experiment shown in the inset of Figure 3A. Oocytes were
injected with 

cRNA; 5 d later, the same oocytes were
injected with
m
cRNA. Previous studies demonstrate
that the cRNA for glycine subunits (Kuhse et al., 1993
) or GABA
subunits (Amin and Weiss, 1996
) injected into Xenopus
oocytes is degraded within 2-3 d. Therefore, after the second
injection, the oocytes should contain only 

and
m
receptors, and no intermediate component
(attributable to 
m
receptors) should be
observed. This was indeed the case, because the dose-response
relationship in these delayed-injection experiments was described by
the sum of two Hill equations (continuous line) with EC50
values of (mean ± SD) 0.42 ± 0.17 µM and
30.0 ± 7.5 µM (n = 3).
Fig. 3.
GABA dose-response relationships from
coexpression of 
m
, 

m
,
and 


m subunits. A, cRNA encoding
for
,
m,
, and
subunits was coinjected into
oocytes. The dose-response relationship for GABA (mean ± SEM,
n = 4) was well described by the sum of three Hill
equations (continuous line), suggesting, in terms of
GABA sensitivity, three receptor subtypes. The EC50 values
of the three components were 0.26 ± 0.05, 2.2 ± 0.1, and
36.3 ± 8.1 µM (
-to-
m cRNA
injection ratio = 1:3). The dashed lines
representing the first component and the combination of the first and
second component are shown to delineate the individual components. The
three components suggest that there are two
subunits in the
GABAA receptor complex. The inset is a GABA
dose-response relationship from an oocyte expressing primarily


and
m
receptors. This was achieved by
injecting 

cRNA, waiting 5 d, and then injecting
m
cRNA. This ensures that
and
m
cRNAs do not coexist to any appreciable extent in the oocyte. In this
case there was no intermediate component, and the GABA dose-response
relationship was described by the sum of two Hill equations with
EC50 values of 0.34 and 33.4 µM (indicated by
arrows). The EC50 values from three such
experiments (mean ± SD) were 0.42 ± 0.17 and 30.0 ± 7.5 µM for the first and second components, respectively.
B, cRNA encoding for
,
,
m, and
subunits was coinjected into oocytes. The dose-response relationship
for GABA (mean ± SEM, n = 3) was also well
described by the sum of three Hill equations (continuous
line). The EC50 values of the three components were
0.025 ± 0.01, 0.94 ± 0.07, and 39.2 ± 7.9 µM (
-to-
m cRNA injection ratio = 1:1). The three components suggest that there are two
subunits in
the GABAA receptor complex. C, cRNA encoding
for
,
,
, and
m subunits was coinjected into
oocytes. The dose-response relationship for GABA (mean ± SEM,
n = 3) was described by the sum of two Hill
equations (continuous line) with EC50 values
of 1.09 ± 0.12 and 40.9 ± 4.8 µM
(
-to-
m cRNA injection ratio = 1:1). The
dashed line represents the first component. Two
components suggest that there is one
subunit in the
GABAA receptor complex.
[View Larger Version of this Image (19K GIF file)]


m
coexpression
Figure 3B presents the average GABA
dose-response relationship from oocytes coexpressing
and
m subunits along with wild-type
and
subunits.
The continuous line in Figure 3B is the best fit of the sum
of three Hill equations. The EC50 of the first component,
0.025 ± 0.01 µM, corresponds to that determined
with 
m
coexpression (0.035 ± 0.004 µM), and the EC50 of the third component,
39.2 ± 7.9 µM, corresponds to that of the 

receptor (45.8 ± 3.6 µM). Again, the presence of an
intermediate component (EC50 = 0.94 ± 0.07 µM) indicates that the GABA receptor contains more than
one
subunit (Fig. 2B). Assuming two
subunits and the
shift observed in Figure 1B for 
m
coexpression (1308-fold), each
m subunit would
contribute a 36.2-fold increase in GABA sensitivity, predicting an
intermediate EC50 of 1.27 µM. This is in
excellent agreement with the observed intermediate EC50 of
0.94 ± 0.07 µM.



m coexpression
The data presented thus far (Fig. 3A,B) suggest the
presence of two
subunits and two
subunits. If, by analogy with
other members of this receptor-operated super-family, we assume that
the GABAA receptor is a pentameric complex (Langosch et
al., 1988
; Anand et al., 1991
; Cooper et al., 1991
; Unwin, 1993
; Nayeem
et al., 1994
; Macdonald and Olsen, 1994
), we can assume there is only
one
subunit. This inferred presence of one
subunit is confirmed
by the 


m coexpression studies presented in
Figure 3C. In contrast to that of 
m
and 

m
coexpression, the



m dose-response relationships were described
by the sum of only two Hill equations (continuous line). Furthermore,
the EC50 of the first component, 1.09 ± 0.12 µM, corresponds to that determined with


m coexpression (0.99 ± 0.23), and the
EC50 of the second component, 40.9 ± 4.8 µM, corresponds to that determined for 

receptors (45.8 ± 3.6 µM). Therefore, the two
components in Figure 3C represent the activation of
receptors containing either a
or
m subunit, and the
GABA receptor complex contains only one
subunit.
Varying the ratio of wild-type and mutant subunits
We next considered the possibility that there are multiple
indistinguishable intermediate components in the dose-response
relationships of Figure 3. To address this issue, the ratio of
wild-type and mutant cRNA (e.g.,
and
m) coinjected
with their wild-type counterparts (e.g.,
and
) was varied. For
example, if the intermediate component of the GABA dose-response
relationship in Figure 3A consists of more than one
component, then varying the
-to-
m cRNA ratio should
vary the relative fractions of the different receptor combinations
underlying the intermediate components and shift the apparent
EC50 of the intermediate component. A similar argument
would apply for changes in the
-to-
m and
-to-
m cRNA ratio.
At all cRNA injection ratios tested, the GABA dose-response
relationships consisted of three components for

m
and 

m
coexpression
and two components for 


m coexpression. Figure
4 presents plots of the EC50 values versus
the fraction of the wild-type component for each of the three
coexpression experiments. The fraction of the wild-type component is
the amplitude of the wild-type component divided by the total amplitude
and is determined from the fits to the individual compound
dose-response relationships. The EC50 values and component
fractions are presented in Table 1. The relative
fraction of the components varied in a manner that would be expected
for the different cRNA injection ratios; that is, the
amplitude of the wild-type component increased with an increase in the
ratio of
-to-
m cRNA. Figure 4A is a plot
of the EC50 values of the three components versus the
fraction of the wild-type component for 
m
coexpression. The dashed lines represent the mean EC50
determined from expression of 

and
m
(Fig. 1B). The EC50 values of the first and
third components do not depend on the relative proportions of the
different receptor combinations consistent with the interpretation that
they represent the activation of receptors containing all mutant and
all wild-type
subunits, respectively. The continuous line is from a
linear regression to the EC50 of the intermediate component
and demonstrates a very slight, but significant
(p < 0.05), negative dependence on the fraction
of the wild-type component. That is, the EC50 decreased
slightly with increasing availability of the wild-type
subunit. If
the intermediate component was the combination of more than one
subcomponent, the expected relationship should be in the opposite
direction than that observed; that is, the EC50 should
increase with an increase in the fraction of the wild-type component.
The slight decrease in the EC50 with increasing fraction of
the wild-type component may reflect differences in the ability to
extract the EC50 values from these compound dose-response
relationships as the amplitudes of the components vary. Figure
4B,C are similar plots of the EC50 values as a
function of the fraction of the wild-type component with


m
and 


m coexpression.
In both cases, the EC50 values of all components appeared
independent of the component amplitudes (Table 1). The amplitudes of
the components in these wild-type and mutant coexpression experiments
did not correspond in all cases to that predicted by the binomial
hypothesis. Possible explanations for this discrepancy will be
considered in the Discussion.
Fig. 4.
The EC50 values of the components with

m
, 

m
, and



m coexpression do not depend on the relative
amplitudes of the components. A, Plot of the
EC50 values of the three components as a function of the
fraction of the wild-type component for 
m
coexpression. The fraction of the wild-type component is the amplitude
of the wild-type component divided by the total amplitude and is
determined from the fits to the individual compound dose-response
relationships. In these experiments, GABA dose-response relationships
were constructed from oocytes in which the
-to-
m cRNA
injection ratio was varied (1:3, 1:1, and 3:1) to shift the relative
amplitudes of the different components. The total amount of
and
m cRNA remained fixed with respect to
and
. For
all
-to-
m ratios tested, the GABA dose-response
relationships were described by the sum of three Hill equations. The
dashed lines represent the mean EC50 values
determined from 

and
m
coexpression (Fig.
1). The continuous line is a linear regression to the
EC50 of the intermediate component and demonstrates a
slight but significant (p < 0.05) negative
dependence that is in the opposite direction from that predicted if
there were multiple, indistinguishable, intermediate components (see
text). These data indicate three components and hence two
subunits
in the GABA receptor complex. B, Similar plot as in
A, but for 

m
coexpression at
different ratios of
to
m (1:1 and 3:1). The
dashed lines represent the mean EC50 values
determined from 

and 
m
coexpression (Fig.
1). These data indicate three components and hence two
subunits in
the GABA receptor complex. C, Similar plot as in
A, but for 


m coexpression at
different ratios of
to
m (1:3 and 1:1). These data
indicate two components and hence one
subunit in the GABA receptor
complex.
[View Larger Version of this Image (18K GIF file)]
Nevertheless, the data presented in Figure 4 suggest that in terms of
GABA sensitivity, there are three receptor types with

m
and 

m
coexpression
and two receptor types with 


m coexpression,
further supporting a pentameric GABAA receptor composed of
two
subunits, two
subunits, and one
subunit.
The stoichiometry does not depend on the relative availability of
,
, and
subunits
Finally, we considered whether the stoichiometry of the GABA
receptor is fixed or can vary, depending on the relative abundance of
the different subunit types. To address this issue, we injected
m,
, and
cRNA at a ratio of 4:1:1 or
,
,
and
m cRNA at a ratio of 1:1:4. If the increased
availability of
or
subunits can increase the number of
or
subunits incorporated into the GABA receptor, the EC50
values should shift further to the left than that observed with
equivalent cRNA injection ratios (Fig. 1B). The results from
these experiments are presented in Figure 5. For both
cases, the EC50 values of the GABA dose-response
relationships with nonequivalent injection ratios were
indistinguishable from those determined with equivalent cRNA injection
ratios. These data indicate that at least at these cRNA ratios, the
number of
and
subunits in the GABA receptor is fixed. By
default, the number of
subunits should be fixed as well.
DISCUSSION
Summary, assumptions, and limitations
By taking advantage of a mutation in the putative second
membrane-spanning domain of the GABAA receptor subunits
that increased the GABA sensitivity in relative proportion to the
number of subunits carrying the mutation, we were able to infer that
recombinant
1
2
2 receptors are pentamers composed of two
subunits, two
subunits, and one
subunit. We have also
demonstrated that at least over a fourfold ratio of injected cRNA
concentrations, the stoichiometry appears to be fixed. Behe et al.
(1995)
used a similar strategy of wild-type and mutant subunit
coexpression to determine the number of incorporated NR1 subunits in
recombinant N-methyl-D-aspartate (NMDA)
receptors, although their approach differs from the present study in
that they relied on mutation-induced alterations in the single-channel
conductance.
Our conclusions are tied strongly to the assumption that the mutations
do not alter the normal stoichiometry of the
1
2
2
GABAA receptor. Although we have no direct evidence, it
seems unlikely that a point mutation in a residue that is presumed to
face the interior of the pore (Revah et al., 1991
; Unwin, 1995
; Xu and
Akabas, 1996
) would so drastically affect subunit-subunit interactions
as to induce an atypical stoichiometry. We have also assumed that the
different possible subunit arrangements of the mutant and wild-type
subunits within the pentameric complex are functionally equivalent.
To uncover any possible hidden components in the compound
dose-response relationships of Figure 3, we varied the
wild-type-to-mutant cRNA ratio for each of the subunits (e.g.,
,
m) and coexpressed this mixture along with their
wild-type counterparts (e.g.,
,
). Although the relative
amplitudes of the components depended on the wild-type-to-mutant cRNA
ratio, the observed number of components and their EC50
values were essentially independent of the cRNA ratio. This supports
the conclusion that the dose-response relationships in Figure
3A-C are composed of only three, three, and two components,
respectively.
We do not believe that the shifts in EC50 in Figure 1, and
the additional components in Figure 2, result from the expression of
subpopulations of GABA receptors with different GABA sensitivities
(e.g., 
, 
, etc.) for the following reasons. (1) Expression
of
,
, or
subunits alone, as well as
/
coexpression,
does not yield functional GABA receptors in oocytes. (2) 
receptors have an EC50 of 30.1 ± 8.4 µM, which is similar to that of 

GABA receptors
(45.8 ± 3.6 µM). (3) 
GABA receptors have an
EC50 of 4.2 ± 0.9 µM (Amin et al.,
1994
), which is less GABA-sensitive than any of the nonwild-type
components (Figs. 2 and 3; Table 1). The observation that the
EC50 values do not shift with changes in the
wild-type-to-mutant coinjection ratios (Fig. 4; Table 1) further
strengthens the conclusion that subpopulations of GABA receptors cannot
account for the shifts in GABA sensitivity.
The amplitudes of the components for the different cRNA injection
ratios deviated somewhat from that predicted by the binomial
distribution. For example, at a wild-type-to-mutant cRNA ratio of 1:1
(Table 1), the fractional amplitudes of the wild-type components were
0.72 ± 0.07 (n = 3), 0.27 ± 0.07 (n = 4), and 0.72 ± 0.12 (n = 3)
for 
m
, 

m
, and



m, respectively. Assuming that the percentages
of the different receptor combinations were to follow a binomial
distribution, the fraction of wild-type receptors should be 0.25 for

m
and 

m
coexpression
and 0.50 for 


m coexpression. Several factors
could account for this discrepancy. First, the mutations may affect
subunit assembly. If the mutant subunit does not assemble as
efficiently as that of the wild type, the amplitudes of the components
would deviate from those predicted by the binomial distribution.
Second, if the mutation altered the maximum percent time open or the
single-channel conductance, the relative amplitudes of the components
would be altered accordingly. In fact, the mutations do affect the Hill
coefficients slightly (see Fig. 1 legend), suggesting an alteration in
the gating kinetics. Third, the determination of the amplitudes of the
components may be influenced by receptor desensitization. At high
agonist concentrations, desensitization would diminish the relative
contribution of the receptors in the more GABA-sensitive components,
thus depressing the apparent amplitude of the wild-type component.
Fourth, in the case of
m
and

m
coexpression, we observed a picrotoxin-sensitive
current in the absence of GABA that was not observed in oocytes
expressing 

m or 

receptors (data not
shown). This may represent the spontaneous opening of GABA receptors
with two mutant subunits incorporated. If so, this would alter the
relative contributions of the components in the compound dose-response
relationship with 
m
and


m
coexpression. And finally, there may be
slight errors in estimating the relative wild-type and mutant cRNA
concentrations from the agarose gels. Although these factors could
influence the estimation of the parameters in these compound
dose-response relationships, it is not apparent how they would alter
the general conclusions from this study, which rely more heavily on a
detection of the number of components. Moreover, our conclusions are
strengthened by the excellent agreement between the observed and
predicted EC50 values of the components in Figures 3 and
4.
Comparison with other studies
Our studies are consistent with evidence indicating that some
native (Duggan et al., 1991
; Luddens et al., 1991
; Pollard et al.,
1995
) and recombinant (Verdoorn, 1994
) GABA receptors can contain
multiple subtypes of the
subunit. In addition, Im et al. (1995)
,
using tandem constructs of
6-
2 GABA subunits, concluded that
6
2
2 GABA receptors contain two
subunits, two
subunits,
and one
subunit.
Our results are in contrast to those of Benke et al. (1994)
, which
infer that only one
subunit is present in the native GABA receptor.
This conclusion was reached from the observation that
[3H]flumazenil or [3H]muscimol binding to
native GABA receptors immunoprecipitated from brain by
1,
2, or
3 antiserum sum to ~100% of the total binding. If neurons
dominantly express one isoform of the
subunit, however, the results
of Benke et al. (1994)
would be obtained regardless of the number of
subunits in the GABA receptor complex. In fact, immunolocalization
studies indicate some segregation of the
subunit isoforms to
different brain regions (Benke et al., 1994
).
Quirk et al. (1994)
observed that the sum of the percentages of
[3H]muscimol binding sites immunoprecipitated by
2 and
3 subunit antisera was slightly greater than the
[3H]muscimol binding observed with
2 and
3 antisera
in combination, although as the authors report, the difference was
within the limit of error of the measurement. Khan et al. (1994)
made a
similar observation using antibodies raised to
2S and
2L
subunits. In both cases, the data were used to conclude that a minor
fraction of the GABA receptors can contain more than one type of
subunit. Furthermore, GABA receptors obtained by
3
immunoprecipitation were labeled in a Western blot probed with
2
antisera, again suggesting that
2 and
3 subunits can coexist in
the same GABA receptor complex. This conclusion seems to contradict our
findings that the
1
2
2 GABA receptor contains a single copy of
the
2 subunit. It should be kept in mind, however, that
immunoprecipitation studies investigate total GABA or benzodiazepine
binding to isolated membranes. This approach may include (1) a
subpopulation of nonfunctional GABA receptors having an aberrant
stoichiometry (our approach is limited to functional GABAA
receptors) or (2) a subpopulation of 
or 
GABA receptors
that may contain multiple
subunits. It is also possible that GABA
receptors may have a stoichiometry that depends on the expression
system (neurons, transfected mammalian cells, oocytes) or the
particular
,
, and
subunit isoforms.
Finally, Backus et al. (1993)
examined the stoichiometry of rat
3
2
2 receptors in transfected human embryonic kidney cells
(HEK293) and concluded that the possible subunit stoichiometries for
this receptor were 2
1
2
, 2
2
1
, or 1
2
2
, of
which 2
1
2
was favored. This study examined the degree of
outward rectification induced by charged substitutions in homologous
positions of the putative pore-forming domain of the
3,
2, and
2 subunit isoforms. As stated by the authors, the conclusions
assumed that the degree of the effect of changing the charge on the
outward rectification is the same for all the subunits. Given that the
amino acid at one of the two mutated positions was not conserved across
the three subunits (arginine, tyrosine, or lysine), this assumption may
not hold true. Perhaps both conservative and nonconservative
substitutions (in terms of charge) at these positions, along with
wild-type and mutant coexpression of each subunit type, could be used
to test this assumption.
Arrangement of the subunits
The question of the precise manner in which the two
subunits,
two
subunits, and one
subunit are arranged within the
pentameric structure is still unanswered. Some insight may be provided
by structure/function studies of the GABAA receptor. For
example, site-directed mutagenesis has previously identified a residue
on the
subunit and two domains on the
subunit that seem to form
part of the GABA binding site (Sigel et al., 1992
; Amin and Weiss,
1993
). If, as for the nACh receptor, the agonist binding sites are at
subunit interfaces (Kurosaki et al., 1987
; Blount and Merlie, 1989
;
Pedersen and Cohen, 1990
; Czajkowski and Karlin, 1991
; Sine and
Claudio, 1991
; Sine, 1993
), at least one of the two GABA binding sites
may be at the
-
subunit interface (Smith and Olsen, 1995). A
detailed single-channel kinetic analysis (Sine and Steinbach, 1987
;
Colquhoun and Ogden, 1988
; Weiss and Magleby, 1989
; Twyman et al.,
1990
; Newland et al., 1991
) may provide information on the equivalence
of the two GABA binding sites and help place the second binding site at
either the other
-
interface or at a
-
or
-
interface.
Although the potential subunit interactions underlying activation and
modulation of GABAA receptors are yet to be elucidated,
knowledge of the stoichiometry will facilitate the incorporation of the
wealth of structural and functional information into a cohesive model
of the GABAA receptor/pore complex.
FOOTNOTES
Received May 1, 1996; revised June 14, 1996; accepted June 18, 1996.
This research was supported by National Institutes of Health Grants
AA09212 and NS35291 to D.S.W.
Correspondence should be addressed to David S. Weiss, Neurobiology
Research Center, University of Alabama at Birmingham, 1719 Sixth Avenue
South, CIRC 410, Birmingham, AL 35294-0021.
REFERENCES
-
Amin J,
Weiss DS
(1993)
GABAA receptor needs
two homologous domains of the
subunit for activation by GABA, but
not by pento