The effect of subunit composition of rat brain GABAA receptors on channel function (Sigel, et al., 1990)

Neuron,
Vol. 5, 703-711, November, 1990, Copyright
0 1990 by Cell Press
The Effect of Subunit Composition
of Rat Brain GABA* Receptors
on Channel Function
Erwin Sigel: Roland Baur: Gerhard Trube,+
Hanns MGhler,* and Pari Malherbet
*Department of Pharmacology
University of Bern
Bern
Switzerland
+Pharmaceutical Research
F. Hoffmann-La Roche Ltd.
Base1
Switzerland
*Department of Pharmacology
University of Zurich
Zurich
Switzerland
Summary
Different combinations of cloned rat brain subunit isoforms of the GABAA receptor channel were expressed
in Xenopus oocytes. The voltage-clamp technique was
then used to measure properties of the CABA-induced
membrane currents and to study the effects of various
modulators of the CABAA receptor channel (diazepam,
DMCM, pentobarbital, and picrotoxin). This approach
was used to obtain information on the minimal structural requirements for several functional properties of
the ion channel. The combination a5B2y2 was identified as the minimal requirement reproducing consensus
properties of the vertebrate CABAA receptor channel,
including cooperativity of CABAdependent channel
gating with a K, in the range of 10 PM, modulation by
various drugs acting at the benzodiazepine binding site,
picrotoxin sensitivity, and barbiturate effects.
Introduction
Biochemical isolation of a GABAJbenzodiazepine
receptor complex from bovine brain, followed by analysis using gel electrophoresis under denaturing conditions, indicated the existence of two subunits, which
were named a and p (Sigel et al., 1983; Sigel and Barnard, 1984). Primary peptide sequences derived from
the purified material enabled molecular cloning of
two subunits from bovine brain (Schofield et al., 1987).
Subsequent functional expression in Xenopus oocytes led to ion channels, claimed to have many of the
properties known for the native GABAA receptor channel, in particular benzodiazepine sensitivity of the
GABA-induced ion current (Schofield et al., 1987). It
soon became clear that bovine brain contains additional subunit isoforms (Levitan et al., 1988a; Ymer et
al., 1989a) and that expressed a-8 subunit combinations lack benzodiazepine responsiveness in the low
micromolar concentration range and display noncooperative GABA gating of the channel (Levitan et al.,
198813; Malherbe et al., 1989a). The functional defi-
ciencies are not simply due to the inability of the
Xenopus oocyte to assemble GABA* receptor channels properly, since expression of mRNA derived from
whole brain leads to expression of channels exhibiting the same pharmacological properties (Parker et
al., 1986; Sigel and Baur, 1988) as measured in vertebrate neurons (Yakushiji et al., 1989; Akaike et al.,
1990).
Subsequent cDNA cloning revealed a bewildering
number of distinct subunit isoforms (reviewed by
Schofield, 1989; Olsen and Tobin, 1990). The molecular cloning of multiple subunit isoforms from bovine,
human, and rat brain (Schofield et al., 1989; Pritchett
et al., 1989b; Ymer et al., 1989a, 1989b; Lolait et al.,
1989; Shivers et al., 1989; Khrestchatisky et al., 1989;
Malherbe et al., 1990a, 1990b, 199Oc; Pritchett and Seeburg, 1990) strongly supports the notion of GABAA
receptor heterogeneity (reviewed by Sieghart, 1989).
Although it is clear from biochemical work that not
more than four or five subunits may be accommodated to form a channel complex (Sigel et al., 1983),
it is not clear, so far, which subunits form a functional
ion channel in situ. A precise description of the functional properties of the channels expressed from
different subunit combinations is clearly needed.
In the a-j3 combinations, which lead to expression
of large GABA-gated currents, the respective isoform
of a subunit has been reported to affect the affinity
for GABA gating (Levitan et al., 1988a; Malherbe et al.,
1990a) and the pharmacological properties (Pritchett
et al., 1989a) of the channel. In the case of human
receptor subunits, combined transfection into 293
cells of the al and PI subunit together with the ~2
subunit led to full sensitivity to benzodiazepine receptor ligands (Pritchett et al., 1989b), whereas for the
same rat subunit combination expressed in the Xenopus oocyte, only a weak sensitivity was found (Malherbe et al., 1990b). On the other hand, it has been
impossible to express a channel displaying cooperativity to GABA gating. Furthermore, most functional
studies performed so far with cloned subunit isoforms lack detailed analysis of the channel properties.
Very often drugs were applied at a single concentration only. The results of these studies are very difficult
to interpret, since, for example, the concentration
dependence of drugs acting at the benzodiazepine
binding site is biphasic (Sigel and Baur, 1988; Yakushiji
et al., 1989) and may vary for channels formed by
different subunit combinations.
In this paperwe have focused on the functional role
of six rat brain GABAA receptor subunit isoforms, al,
a3, a5 (this subunit has also been named a4 [Khrestchatisky et al., 1989]), pl, 82, and ~2. These subunits
were functionally expressed in different combinations in order to explore the contribution of the different subunit isoforms to the properties of GABA-gated
receptor channel. The GABA concentration eliciting
NWVXl
704
half-maximal current (K,) and the Hill coefficient for
GABA gating, along with the effect of drugs acting at
the benzodiazepine binding site and at the barbiturate site, were investigated. The study led to the assignment of some of the functional properties to
defined subunit isoforms. In addition, a minimal composition of a5B2y2 has been found to display the behavior known for the GABAA receptor channel in situ.
Results
Six cloned rat GABAA receptor subunit isoforms were
available for this study. In principle, there exist 63
different subunit combinations, whose detailed functional analysis clearly exceeded our working capacity.
Therefore, we decided first to analyze functional expression of the subunits individually and then to proceed with pairs and triples of subunit combinations,
with the aim to define minimal structural requirements for the individual functional parameters.
Homomeric Channels and Suppression
of Their Formation
As reported for some of the subunits earlier (Sigel et
al., 1989), oocytes injected with RNA coding for a single subunit showed either little response to the application of 100 PM GABA (Table 1) or no response at all.
This is in agreement with the report by Khrestchatisky
et al. (1989), but in contrast to that by Blair et al. (1988),
who reported single-channel recordings from expressed individual subunits. The Bl subunit resulted
in the formation of an anion-selective channel that
was open in the absence of GABA, but closed in response to the channel blocker picrotoxin (Sigel et al.,
1989). It is interesting to note that coinjection of
stoichiometric amounts of all the available a subunit
RNAs, but not of the ~2 subunit RNA, together with
the Bl RNA suppressed the formation of this nongated anion channel (Table 1).
GABA-Dependent Gating of the Channel:
Affinity and Cooperativity
Subunit combinations consisting of two subunit isoforms displayed different affinity for GABA-dependent gatingof the channel. a5 in combination with Bl
led to formation of channels displaying a higher affinity toward GABA compared with aIs1 and a3Bl and
led to some cooperativity in the opening of the channel by GABA (Malherbe et al., 1990a). It is also interesting to note that a subunits are not strictly needed in
order to observe GABA-activated currents, since B2y2
was sufficient to induce these currents (Table 1). However, from the combinations studied, it seems that the
current amplitudes are much smaller in the absence
of a subunits than in their presence. In the dual combinations studied, no functional channels were observed in the absence of a B subunit.
Coexpression of y2 together with alB1 or alB2 induced in each case a Ffold increase in the K, for
channel gating by GABA. Replacement of Bl by B2, in
the subunit combinations alB1 and alBly2, led in
both cases to a 13-fold increase in K,. Thus, a, 8, and
y subunits affect the affinity of GABA gating. None of
the dual and triple subunit combinations mentioned
so far displayed cooperativity for GABA-dependent
gating of the channel, with the possible exception of
a5Bl.
Subsequently, all six subunit isoforms available to
us were coexpressed, although biochemical size determinations exclude that hexameric subunit isoforms
form a single-channel complex (Sigel et al., 1983). To
our surprise, an apparently homogeneous population
of ion channels was expressed and displayed cooperative gating by GABA. This encouraged us to try other
combinations with a smaller number of subunits. Expression of five different subunits led to results that
were difficult to interpret. This was due to K, shifts
toward lower values, which were observed for some
combinations during the expression period. The values shown in Table 1 were obtained at least 4 days after injection of the RNAs. Except for the combination
that lacked a5, all combinations showed strong cooperativity for GABA gating, again pointing to a role
of this subunit isoform in cooperativity. Omission of
a5 also significantly increased the K, for GABA. In
line with the postulated role of y2 in mediating the effect of diazepam (see below), the combination lacking
y2 completely lost modulation by diazepam. Subsequently, we returned to combinations with a smaller
number of subunits. We found that combining ~2, the
subunit conferring benzodiazepine sensitivity, with
a5, the subunit probably responsible for cooperativity and high affinity for GABA-dependent channel gating, and 82 led to the formation of an ion channel with
the desired properties (Table 1; a5B2y2). Addition of
either al or a3 to a5B2y2 had little effect on the propertiesof the resulting channel. We subsequently found
that neither the ~2 nor a B subunit was required for
cooperativity of GABA gating (Table 1, u3a5y2 and
ala582) and that a B subunit was not required for diazepam sensitivity (a3a5y2). However, in the absence
of a B subunit, the expressed current amplitudes were
small.
A typical example of the determination of the affinity and cooperativity of GABA-dependent gating is
shown in Figure 1. An oocyte was exposed to increasing concentrations of GABA, and the log of the current amplitude elicited versus the log of the GABA
concentration was plotted. The limiting slopes from
at least three experiments performed with three different oocytes were averaged to obtain the Hill coefficient, n (Table 1). At the end of the experiment, the oocytes were exposed to 10 mM GABA in order to obtain
an estimate of the maximal current amplitude, and
the K, was estimated. In the above experiments emphasis was put on the lower GABA concentration
range. Full dose-response curves (Figures 2a and 2b)
were also evaluated in order to obtain K, and n for
some subunit combinations. In the experiments shown
in Figures 1 and 2, oocytes were injected with the
Functional Architecture of CABAA Receptor Channels
705
Table 1. Functional
Subunits
injected
Chick Braina
Properties
of Different
lsoform Combinations
after Expression
in the Xenopus
CABA
Picrotoxin
Pentobarbital
I,,,
I
I (%I,,,; 1 mM)
(nA)
- 2500
al
a3
n
n
n
n
n
- 3
(10/2)
(8/2)
(6/2)
(8/2)
(10/2)
(6/2)
W/2
P2Y2
>3000
>3000
>3000
- 890
n
n
n
- 16
- 155
(25/6)
(25/4)
(26/4)
(7/2)
(13/2)
(7/2)
(6/2)
(9/l)
(7/2)
aluSe
ala5y2
a3a5y2
alply2e
alpZy2
a3ply2
a3P2y2
a5ply2
a5P2y2
>2000
n
- 470
>3000
>3000
>3000
>2000
>3000
>3000
(7/l)
(7/2)
(8/2)
(34/6)
(12/4)
(3/l)
(4/l)
(3/l)
(23/3)
ala3P2y2
ala5P2y2
a3a5!32y2
>3000 (8/l)
>3000 (7/l)
>3000 (7/l)
ala3a5plp2s
ala3a5fily2s
ala3a5P2y2s
ala3plp2y2s
ala5Blp2y2s
a3a5blp2y2s
>3000
>3000
>3000
>3000
>3000
>3000
ala3a5plp2y2
>3000 (1012)
;;15b
P2
Y2
alplc
a3DlC
a5PlC
aID
aly2
a3y2
a5y2
Subunit
K, (cLM)
H”
21
1.7
13
17
1.1
139
*7
* 11
* 0.7
* 18
(411)
(3/W
(5/l)
(311)
64
f 11
(311)
18
*3
147
74
985
240
487
*
+
f
f
f
17
14
*7
*3
145
(3/l )
(4/l)
(3/l)
(3/l)
(5/l)
(4/l)
39
13
352
136
42
0.9
1.0
1.2
1.1
n
*
f
*
*
0.1
0.0
0.1
0.1
(3/l)
(4/Z)
(5/2)
(3/I)
(3/l)’
(3/l)
1.7
1.0
1.0
1.2
1.3
1.6
1.7
(3/l)
(7/2)
* 75
(3/l)
*I
*5
(311)
(311)
9
44
20
* 22
(l/V
(3/l)
(l/l)h
13 f 4
n
n
n
408
n
n
(10/2)
(3/I)
(3/l)
(4716)
(6/I)
(3/I)
28
5
10
n
n
(712)
(712)
(912)
(6/I)
(13/2)
*
f
*
*
*
+
*
0.1
0.0
0.1
0.0
0.0
0.0
0.1
*
f
*
*
*
*
88
6
+I
(l/W
cwh
17
*7
(3/l)
62
f 28
(5/2)
1.6 f 0.2
-2+5
VI
-1
(6)
f IO
44 f 13
-2*4
15 * 6
30 f 10
198 * 49
(5)
4 * 16
(3)
176 * 61
(4)
(13)
(IO)
(3)
(3)
(3)
(4)
32 f 8
(7)
(3)
20 (6/2)
n (6/2)
98 f 15
65 f 16
n (l/l)
29 f 9
15 *
108 *
23 *
299 *
31 *
136*41
16 f a
34 f 14
12 f 3
355 * 163 (4)
191 * 26 (3)
16Oi26
(3)
1.9 * 0.1
1.7 f 0.0
2.0 f 0.0
1.7
1.4
1.4
1.2
1.5
1.5
Stimulation by
Diazepam (o/o;1 PM)
130
132 f 13
196 (9/l)
n (6/I)
1.6 f 0.0
21
34
(nA; 10 PM)
Oocyte
0.1
0.1
0.1
0.1
0.1
0.1
-5
57
150
113
f
*
_t
*
7
37
25
50
14
5
13
25
21
(3)
(3)
(3)
(3)
35 f 14
(3)
(3)
36 f 18
(4)
a+3
n
6r2
In the body of the table, n represents <3 nA; a dash indicates not determined.
Data are given as mean * SD (number of oocytes
and number of different batches used). The value of K, was for each case confirmed in additional batches of oocytes by determining
the ratio of the currents elicited by two different GABA concentrations.
This represents a sensitive measure for K, shifts. The Hill
coefficient (H,) was determined from the limiting slope of log/log plots. The currents expressed from some subunit combinations
containing PI were affected by culturing of the oocytes in the presence of actinomycin D. For details see text.
a Part of the data are from Sigel and Baur (1988).
b Part of the data are from Sigel et al. (1989); additionally, it has now been found that the oocyte may contribute a factor necessary
for biosynthesis or function of the nongated channel.
c Part of the data are from Malherbe et al. (1990a).
d In two additional oocytes, a K, in the submicromolar
range was found.
e Part of the data are from Malherbe et al. (1990b).
‘A second component with K, < 1 PM was evident. The maximal current of this component was <I% of the dominant component.
g Data on K, and H, are from the late expression phase.
h A K, about 3- to Cfold higher was found 2-3 days after injection with mRNA.
’A second component with K, < 1 PM was evident even in the late phase of expression (see also text).
subunit
combinations
alp2y2 and a5P2y2, respective-
ly. Fitting of the entire dose-response
coefficients
almost
identical
curves gave Hill
to those
is interesting
tions that resulted
sitization
of the
to note
current
elicited
and extent of desensitization
combina-
showed
by GABA.
depended
combination
Effect of Actinomycin D
that all subunit
in GABA responses
but not on the subunit
expressed.
determined
from the above type of experiments.
It
concentration,
desen-
The
rate
on the GABA
Recently
it has been found
endogenously
subunit
transcribes
of the nicotinic
nel (Buller
and White,
that the Xenopus
an mRNA
acetylcholine
coding
oocyte
for an a
receptor
1990). This subunit
chan-
is able to
Neuron
706
Table 2. Effect of Actinomycin
D
Relative Expression (% of Control)
Subunit Combination
loAsA
fP,CrOtDXl”
81
alB1
a5Bl
P2Y2
a5Bly2
aSB2y2
-7
-6
-5
-4
-3
log GABA (M)
-2
Figurel. Determination
of the Hill Coefficients
for CABADependent
Channel
Gating of the Subunit Combinations
alB2y2 and a5P2y2
Oocytes were injected with cDNA-derived mRNAs and cultured.
Follicular cell layers were removed, and the newly expressed ion
currents were characterized as indicated in Experimental Procedures. Current amplitudes were standardized to the response
elicited by 10 mM GABA in order to correct for the variation in
current expression in different oocytes. Each point represents
the mean * SD of at least three oocytes. The limiting slope of
the log/log plot was drawn by hand. Closed circles, u5B2~2; open
circles, alB2y2.
combine with subunits of the same receptor produced from exogenous mRNA, giving rise to false interpretation of the ability of exogenous subunits to
produce functional channels. To exclude such an endogenous influence of the oocyte on our results, we
cultured oocytes injected with RNA coding for various subunit combinations in the presence of the inhibitor of transcription, actinomycin D. The results
obtained are summarized in Table2. Expression of the
nongated anion channel produced by PI was strongly
inhibited in the presence of actinomycin D. When PI
was expressed in combination with al or a5, the maximal current amplitude elicited by GABA was somewhat inhibited, whereas the affinity of the CABA gat-
55
a7
95
146
172
+
k
*
f
*
12 (611)
45 (4/i)
24(4/l)
40 (4/l)
44 (10/2)
17 * 4 (1012)
103 f 10 (311)
73 ir 34 (311)
-
Following injection, oocytes were cultured in the presence of
50 ug/ml actinomycin D or in its absence. The average current
expressed in treated oocytes was calculated relative to the average current found in untreated cells. The GABA-induced current
(lo,& was measured at saturating CABA concentration. lplcrotuxln
is the current component inhibited by IO uM picrotoxin. Data
are given as mean * propagated SEM (number
number of different batches of oocytes).
of oocytes and
ingwas unaltered (data not shown). Interestingly, little
effect was observed on the nongated current amplitude in the latter cases. These findings probably indicate that the oocyte contributes an endogenous protein factor that is essential for the biosynthesis of
the PI subunit. The current amplitude expressed by
the subunit combination p2y2 was not affected, and
the ones expressed by a50272 and a5@ly2 were even
stimulated, again without effect on the affinity of the
GABA gating. This stimulation of channel expression
is very likely a consequence of diminshed competition of endogenous mRNA for the translational machinery in the oocyte (Richter et al., 1983).
Modulation by Drugs Acting at the
Benzodiazepine Binding Site
Diazepam is known to shift the GABA dose-response
curve to lower GABA concentrations. To provide similar conditions for the different subunit combinations,
we determined diazepam effects using GABA concentrations that produced about 10% of the respective
Figure 2. GABA Dose-Response
Curves
for the Subunit Combinations alB2y2 and
a5B2y2
Experiments were carried out as indicated
in Figurel. (a) Current traces recorded
from an oocyte expressing a5B2y2. Different concentrations of CABA were applied
for the times indicated by the bar. The concentrations are given (micromolar) above
12.5
25
50
the bar. The horizontal calibration bar is 1
min for GABA concentrations <I3 uM and
30 s for higher concentrations. The vertical
calibration bar is 20 nA for CABA applications of <I uM and 60,100,500, and 1000 nA
for the GABA applications of 1.6, 3.1, 6.3,
and >I0 uM, respectively. (b) Individual
log GABA (MI
dose-response
curves were fitted using
the equation given in Experimental Procedures. Curves obtained from different oocytes were standardized to the fitted Imaxand refitted using the same equation. Each point represents the mean and SD of at least three oocytes. Closed circles, u5B2~2; open circles,
al B2y2.
Functional Architecture of CABAA Receptor Channels
707
diazepam
diozepam
v:
II
r
a5492 y2
a5191 y2
J
-I
200 nA
40s
:
500 nA
40s
L
Figure 3. Diazepam Stimulation of the CABA Current Expressed
in Oocytes Injected with the a5P2y2 or the a5ply2 Subunit Combination
An oocyte injected with either the a5p2y2 or the a5ply2 combination was exposed first to 5 PM CABA and subsequently to the
same concentration of GABA in combination with 1 PM diazepam. The CABA concentration
was chosen to produce about
10% of the maximal CABA current amplitude. The bar indicates
the time period of drug application.
maximal current response. As shown in Table 1, GABA
currents induced in oocytes injected with the combination P2y2 could be greatly enhanced by 1 PM diazepam. Thus, an a subunit was not needed for such a
J
Ge9
I
-8
-7
-6
Diazepam
Figure4.
azepam
Concentration
Dependence
-5
(Ml
of the Response
to Di-
For a number different subunit combinations, experiments of
the type shown in Figure 3 were carried out for a wide range of
diazepam concentrations. Each point represents the mean f SD
of at least three oocytes. Data for the subunit combination
alply
are taken from Malherbe et al. (199Ob). Closed circles,
a.Sp2y2; open circles, alb2y2; open triangles, ulfHy2; open
squares, fi2y2; closed triangles, a5Bly2.
stimulation. Coexpression of this subunit combination with al even reduced stimulation by diazepam
(Table 1). Diazepam effects on some subunit combinations were measured over a wide concentration
range of this drug (Figure 4). All subunit combinations
studied showed a similar concentration dependence,
which was characterized by an optimum at 0.3-1.0 WM
diazepam. Higher concentrations resulted in less stimulation of the GABA current. Replacement in any subunit combination of /32 by PI, or omission of ~2, resulted in a drastic loss of diazepam stimulation (Figures
3 and 4; Table 1).
Figure 5 shows results on the concentration dependence of effects of the negative allosteric modulator
of the GABA receptor channel, DMCM (methyl-6,7dimethoxy4ethyl-&carboline-3-carboxylate).
It is interesting to note that all subunit combinations tested
show the same degree of responsiveness to the negative allosteric modulator DMCM as to the positive allosteric modulator diazepam. Thus, it appears that the
response to 1 pM diazepam can be used as a diagnostic tool for the presence of a functionally normal benzodiazepine binding site. The subunit combinations
studied here responded in a way qualitatively similar
to that of the receptor expressed from total chick
brain mRNA (Sigel and Baur, 1988), except that the
transition from inhibition of the GABA response by
DMCM to activation took place at lower DMCM concentrations. The precise transition concentration depended on the subunit combination expressed (Figure 5). The fact that a transition occurs with the
cloned subunit combinations is itself interesting. It
demonstrates that this effect observed for receptors
expressed from total tissue mRNA is not a consequence of the expression of several channels showing
a different response to DMCM.
Direct Gating of the Channel by Pentobarbital
The GABA receptor channel expressed from rat and
chick brain mRNA (Parker et al., 1986; Table 1) and in
frog sensory neurons (Akaike et al., -1990)can be activated by pentobarbital in the absence of GABA. We
have observed that the current elicited by 1 mM pentobarbital in oocytes expressing chick brain mRNA
displays the same reversal potential as the current
elicited by GABA in the same oocyte (data not shown).
However, the maximal currents observed with pentobarbital do not exceed about 15% of the maximal
current amplitude that can be elicited by high concentrations of GABA (Parker et al., 1986; Akaike et al.,
1990; Table 1). An interesting feature of the alply
subunit combination is that pentobarbital can fully
activate the resulting channel with a Hill coefficient of
2 (Malherbe et al., 199Ob). Similar findings have previously been reported by Levitan et al. (1988b) using bovine subunits. We have extended these observations
to the study of several more rat subunit combinations.
The results are shown in Table 1. In the case of the
alp1
subunit combination, 1 mM pentobarbital resulted in even larger currents than IO mM CABA.
NWrOn
708
i
1,
-7
-// -9
I
-8
-6
DMCili
Figure 5. Concentration
DMCM
Dependence
Figure 6. Concentration
toxin
-5
(Ml
of
the
Response
to
For a number different subunit combinations, experiments of
the type shown in Figure 3 were carried out for a wide range of
DMCM concentrations. Each point represents the mean f SD
of at least three oocytes. At higher DMCM concentrations, the
inhibition reverted to a stimulation of the GABA current response. This switch to diazepam-like
responses occurred at
different DMCM concentrations for the individual subunit combinations. Data for the subunit combination alPly’ are taken
from Malherbe et al. (1990b). Closed circles, aSp2y2; open circles, alp2y2; open triangles, alply2; open squares, p2y2; closed
triangles, a5!3ly2.
Inhibition of the Channel by Picrotoxin
It has previously been shown that injection into oocytes of RNA coding for the rat brain PI subunit is sufficient to express an anion channel retaining full sensitivity toward the channel blocker picrotoxin (Sigel et
al., 1989). Thus, this subunit is, with a possible contribution by the oocyte (see above), sufficient to form a
binding site for picrotoxin. We now have tested several subunit combinations for sensitivity toward this
channel blocker. Only small variations in the Ki for
picrotoxin were observed between the subunit combinations investigated (Figure 6). Thus, the binding
site for picrotoxin seems to be relatively nonspecific
in terms of subunit isoform requirements. It is interesting to note that, in spite of the fact that the PI
subunit is sufficient to form a picrotoxin binding site,
it is not an essential component for the formation of
a picrotoxin binding site. Even a subunit combination
containing no fl subunits, a3aSy2, retained sensitivity
toward picrotoxin. In the latter subunit combination
10 VM picrotoxin inhibited 92% + 4% (n = 3) of the
current elicited by 100 PM GABA.
Discussion
We have shown that a wide variety of subunit isoform
‘f\
’
0
A-
-6
-5
Pi crotox i n (M)
Dependence
of the Inhibition
-4
by Picro-
For a number different subunit combinations, experiments of
the type shown in Figure 3 were carried out for a wide range of
picrotoxin concentrations.
Each point represents the mean +
SD of at least three oocytes. Data for the subunit combinations
alPly
are taken from Malherbe et al. (1990b). Closed circles,
tifi2y2; open circles, alB2y2; open triangles, al@lyZ; open
squares, p2y2.
combinations of the rat brain GABAA receptor, expressed in the Xenopus oocyte plasma membrane, result in chloride channels with different functional
characteristics. The properties of the resulting channels vary drastically. Before we compare the channel
properties resulting from the expression of different
subunit combinations, the following limitations deserve to be considered:
-It is not quite clear what the functional properties
of the “normal” GABA receptor channel in vertebrate
brain are. In fact, there are few quantitative data concerning cooperativity of GABA gating, K, for GABA,
and diazepam and pentobarbital effects on GABAgated currents available in the literature. And in those
cases in which such parameters have been measured,
the subunit composition is not known. For convenience, we compare our present results with data previously obtained in our laboratory following expression of crude chick brain mRNA in the Xenopus
oocyte (Sigel and Baur, 1988). These data are very similar to the results obtained in isolated frog sensory
neurons (Yakushiji et al., 1989; Akaike et al., 1990).
With this assumption we do not imply that these
GABA channels display all the properties of all types
of CABA receptor channels existing in vertebrate
brain.
-It should be remembered that the terms a subunit
and B subunit were first used for polypeptide bands
identified after gel electrophoresis under denaturing
conditions (Sigel et al., 1983). At present, it is not
known how all the cloned rat brain subunit isoforms
used here migrate in denaturing gels under the same
Functional Architecture of CABAA Receptor Channels
709
conditions. However, it has been shown that at least
the a3 subunit migrates very much like the original p
subunit (Stephenson et al., 1989). Thus, it is difficult
to relate earlier assignments of binding sites to
subunits, made with the purified isolated protein, to
results with the cloned subunit isoforms used here.
-It should be kept in mind that expression in the
Xenopus oocyte does not automatically assemble a
channel composed of all injected subunits. As mentioned before, molecular weight determinations of
the purified channel complex indicate that it is composed of five or less subunits (Sigel et al., 1983). An
additional subunit or a subunit not fitting into the
context of the coinjected subunits may obstruct proper
assembly of a channel. Therefore, it was very surprising that coinjection of six different subunit isoforms
resulted in an apparently homogeneous population
of ion channels. This indicates that at least under
some circumstances the assembly process selects appropriate subunits to build a functional channel.
-Different receptor channel subunits may interact allosterically and, as a consequence, acquire properties
that cannot be assigned to individual subunits.
-It cannot be excluded that the Xenopus oocyte carries out biosynthesis of the GABA receptor channel in
a slightly different manner than rat neurons. It is also
impossible to exclude contribution by the oocyte of
a subunit synthesized from an endogenous mRNA
stored in the cytoplasm.
Keeping these limitations in mind, we can discuss
the possible contributions made by the different subunit isoforms, with respect to CABAA receptor channel function.
Functional Contributions by the a
Subunits (al, a3, and a5)
Previously, it has been reported that the type of a
subunit expressed in combination with additional
subunits affects the GABA affinity of the channel gating (Levitan et al., 1988a; Malherbe et al., 1990a) and
the pharmacological properties of the expressed
receptor channel (Pritchett et al., 1989a; Pritchett and
Seeburg, 1990). In our experiments, the presence of at
least one a subunit in the subunit combination expressed in the Xenopus oocyte was required for induction of large GABA-gated ion currents. On the
other hand, a subunits were required neither for the
formation of an ion channel, nor for GABA-dependent
gating of the channel, nor for the modulation of GABAdependent gating of the channels by compounds
acting at the benzodiazepine binding site. Subunit a5
showed some differences compared with the other
two a subunits studied here. In combination with the
[31subunit, a5 displayed the highest affinity for GABA
gating of the channel and induced some cooperativity
of the gating. On the other hand, omission of the
same subunit from the combined expression of all six
subunits resulted in partial loss of cooperativity and
affinity. Therefore, a5 (also called a4 by Khrestchatisky
et al. [1989])could be important for high GABA affinity
and cooperativity of GABA gating of the channel.
Some of the results obtained with five different
subunits are difficult to interpret (Table 1). An example
is the loss of part of the diazepam modulation of the
GABA current by omission of a3, leaving behind
subunits that are in principle competent in this respect. This could be a consequence of the perturbation of proper biosynthesis.
Functional Contributions by the b
Subunits (bl and B2)
The presence of a 0 subunit was not required for the
expression of GABA-gated ion currents displaying cooperativity and diazepam sensitivity. Formation of an
anion-selective, picrotoxin-sensitive channel was observed with the B’l (Sigel et al., 1989), but not with the
82, subunit. Formation of this GABA-independent
channel was suppressed bycoexpression of any of the
a subunits, but not by the ~2 subunit. Replacement of
the P2 by the PI subunit in a given subunit combination usually resulted in a decrease of KJGABA) and
in a strongly increased sensitivity to diazepam. While
introduction into the oocyte of the PI subunit is sufficient to form a picrotoxin binding site, the presence
of a fi subunit is not essential for the observation of
channel block. This is indicated by the fact that GABA
currents observed with the subunit combination
a3a5y2 are inhibited by picrotoxin (Figure 6).
Functional Contributions by the y2 Subunit
It has previously been suggested that the ~2 subunit
is essential for modulation of the GABA channel by
drugs acting at the benzodiazepine binding site (Pritchett et al., 1989b). In our experiments, a small effect of
such drugs could be observed in the absence of 72,
but the presence of this subunit was essential for
large effects. The y2 subunit also affected GABA gating
of the channel. y2 subunit added to subunit combinations al@ or alp2 resulted in a T-fold increase of
K,(GABA).
(alla3a5fl2y2
IS a Candidate Composition
of Native GABAA Receptor Channels
From the data compiled in Table 1, it is clear that the
subunit combination a5fJ2y2 displays consensus prop
erties of the GABA receptor channel. Coexpression
with al or a3 had little effect on the properties of the
resulting channels. In view of the colocalization observed in many regions of the brain of al with p2 and
~2 (Shivers et al., 1989), it could be hypothesized that
al is an integral component of the channel consensus
properties. But this is not evident from our functional
data. Although a5 might confer cooperativity and
high affinity of the channel gating by GABA, p2 may
be important, together with the a subunit, for efficient channel biosynthesis, and ~2 might be responsible for benzodiazepine sensitivity.
Functional Similarity of the Different
Subunit lsoforms
Many functions, e.g., GABA-gated channel formation
and picrotoxin sensitivity, do not absolutelv reauire
Neuron
710
the presence of a specific subunit isoform. Obviously,
the subunit isoforms do not only show a high degree
of structural similarity, but also display a high degree
of functional similarity.
Relevance of the Data to GABAA Channel lsoforms
in the Brain
Probably, there exist in vertebrate brain GABA receptor channels with properties that do not agree with
the consensus properties mentioned above (Alger
and Nicholl, 1982; Osmanovic and Shefner, 1990). A
detailed quantitative description of the GABA gating
of these currents and their modulation by drugs is
lacking. It is, therefore, possible that one of the
subunit isoform combinations described here corresponds to one of these irregular types of channel. At
the moment, howeuer, it is impossible to identify such
a composition. As soon as more quantitative data are
available on such channels, our data could help to
identify a presumptive subunit composition in intact
cells.
Different approaches will have to be combined to
confirm whether the subunit compositions proposed
here, or any other presumptive subunit combination,
really form an ion channel in situ. More detailed localization studies for the individual subunit isoforms at
the single-cell level and, if possible, immunoprecipitation of channel complexes from individual types of
neurons using subunit isoform-specific antibodies
are required. In a complementary approach, quantitative electrophysiological characterization of the GABA
current in the corresponding cells will have to be carried out. Hopefully, each of the subunit combinations
will display a different signature at the single-channel
level, facilitating identification of the different channel isoforms.
Conclusions
The functional expression of GABA receptor channel
isoforms in a heterologous expression system is a useful approach to the understanding of the functional
implications of the heterogeneity found at the level of
structure. The present study is also a starting point for
structure-function studies using in vitro mutagenesis.
It will be especially interesting to determine which
domains in the individual a and b subunits are responsible for the large functional differences seen between
closely related subunit isoforms. Hopefully, in the future functional expression of different defined subunit
combinations in surrogate systems will also assist in
the identification of drugs specifically acting on individual GABAA receptor isoforms.
Experimental Procedures
Expression of Cloned Subunit lsoform Combinations
in Xenopus Oocytes
The cloning, in vitro transcription, capping, and polyadenylation
of the rat brain subunit isoforms have been described elsewhere
(Malherbe et al., 1990a, 1990b, 19900. The cRNA combinations
were coprecipitated in ethanol and shipped and stored below
O°C. Isolation of follicles from the frogs, culturing of the follicles,
injection with RNA, and removal of the follicular cell layers from
the oocytes were all performed as described earlier (Sigel, 1987).
Follicles were injected with about 50 nl of the capped transcripts.
This solution contained the transcripts coding for each the different subunits at a concentration of 100-200 nM, to allow injection of stoichiometric amounts. The follicular cell layers were
removed from the oocytes 1 or 2 days prior to the electrophysiological experiments. When mentioned, the oocytes were cultured in medium containing 50 pg/ml actinomycin D.
Electrophysiological Experiments
All electrophysiological
measurements
were carried out on
denuded oocytes. Oocytes were placed in a 04ml bath on a nylon grid, and the bath was perfused throughout the experiment
at 6 ml/min with 90 mM NaCI, 1 mM KCI, 1 mM MgCl>, 1 mM
CaCI,, 5 mM HEPES-NaOH
(pH i!4). The perfusion medium
could be switched to one supplied with CABA alone or in combination with other drugs. All experiments were carried out at
room temperature
(20°-240C). For the current measurements,
oocytes were impaled with two microelectrodes and the membrane potential was voltage-clamped at -80 mV.
Data on the gating properties of the different expressed GABA
channels were obtained by using the following two methods.
-CABA dose-response
curves obtained from CABA applications over a wide range of concentrations
were fitted using
a nonlinear least-squares method (Gauss-Newton-Marquardt).
The equation used was I(c) = Imaxc”/(c” + K,“), where c is the
GABA concentration, I the current elicited, K, is the GABA concentration eliciting half-maximal current (% I,,,), and n the Hill
coefficient. At the end of each experiment, the response elicited
by IO mM GABA was additionally determined to verify that data
points in the saturating region had been collected. This determination is not shown in Figure 2. Verifying saturation for the
subunit combination alp2y2 was not possible because of the
high K, of the channel.
-Alternatively, Hill coefficients wereobtained
from the limiting
slope of the log/log plot of the dose-response obtained with low
CABA concentrations. In the latter case, 10 mM CABA was applied at the end of the experiments to allow an estimate of lmdX
and K,.
In some cases both types of analysis were performed with the
same subunit combination.
No difference was found in the
resulting values of n and K,. With either method the same oocyte was exposed to increasing concentrations
of CABA. Between two applications, the oocyte was perfused with standard
medium for 3-15 min (depending on the GABA concentration
previously used) to ensure full recovery from desensitization.
Precautions were taken to minimize the effect of desensitization when current amplitudes were determined.
For the measurement of dose-response curves over a wide GABA concentration range, the perfusion solution was applied through a glass
capillary with an inner diameter of 1.35 mm, the mouth of which
was placed about 04 mm from the surface of the oocyte. This allowed fast changes in agonist concentration around the oocyte.
The rate of change was 70% in less than 0.5 s, as determined by
switching the perfusion medium to a sodium-free medium and
measuring the amplitude of sodium currents expressed in oocytes injected with mRNA isolated from chick forebrain. Current
amplitudes were always back-extrapolated to the time when the
current had reached 50% of the maximal amplitude.
Drug Effects
Diazepam was diluted into standard medium from preparations
used for intravenous application (Valium, 17.6 mM in H,O,
propylene glycol, ethanol, sodium-benzoate,
benzyl alcohol,
benzoic acid); DMCM was obtained from Research Biochemicals (Konstanz, Germany) and diluted from an ethanol solution.
The final concentration of ethanol in the experiment was 0.06%.
Neither of the drug carriers altered the CABA response in control experiments. Pentobarbital was dissolved in standard medium. A fresh solution was prepared daily.
Diazepam effects were determined as follows. Dose-response
curves were used to determine the GABA concentration that
Functional Architecture of CABAA Receptor Channels
711
elicited about 10% of the maximal current response. Control applications of CABA at this concentration were made. The GABA
solution always contained the largest amount of drug carrier
used in the corresponding experiment. This control application
was followed by application of the same GABA concentration,
but including diazepam. DMCM effects on the CABA current
were determined in a similar way. No significant difference was
observed between dose-response curves obtained on single oocytes using cumulative application of drugs and dose-response
curves obtained using a different oocyte for each dose.
Acknowledgments
Olsen, R. W., and Tobin, A. J. (1990). Molecular
GABAA receptors. FASEB J. 4, 1469-1480.
biology
of
Osmanovic, S. S., and Shefner, S. A. (1990). y-Aminobutyric acid
responses in rat locus coeruleus neurones in vitro: a currentclamp and voltage-clamp study. J. Physiol. 427, 151-170.
Parker, I., Cundersen, C. B., and Miledi, R. (1986). Actions of pentobarbital on rat brain receptors expressed in Xenopus oocytes.
J. Neurosci. 6, 2290-2297.
Pritchett, D. B., and Seeburg, H. (1990). y_Aminobutyric acidA
receptor a5subunit creates novel type II benzodiazepine receptor pharmacology. 1. Neurochem. 54, 1802-1804.
Pritchett, D. B., Liiddens, H., and Seeburg, P H. (1989a). Type I
and type II CABAA/benzodiazepine
receptors produced in
transfected cells. Science 245, 1389-1392.
We are grateful to Professor H. Reuter, in whose laboratory this
work was carried out, for continuous support and for comments
on earlier versions of the manuscript. The support to E. S. of the
Swiss National Science Foundation (grant No. 3.078-0.87) is gratefully acknowledged.
The costs of publication of this article were defrayed in part
by the payment of page charges. This article must therefore be
in accordance with 18 USC Sechereby marked “advertisement”
tion 1734 solely to indicate this fact.
Prichett, D. B., Sontheimer, H., Shivers, B. D., Ymer, S., Kettenmann, H., Schofield, P R., and Seeburg, P H. (1989b). Importance of a novel CABAA subunit for benzodiazepine pharmacology. Nature 338, 582-585.
Received June 21, 1990; revised August 2, 1990.
Schofield, P R. (1989). GABAA receptor complexity. Trends Pharmacol. Sci. IO, 476-478.
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