the effect of subunit composition of rat brain gabaa receptors on channel function

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 Bar- nard, 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 nonco- operative 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 exhibit- ing 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 See- burg, 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 accommo- dated 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 Xeno- pus oocyte, only a weak sensitivity was found (Mal- herbe 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 [Khrest- chatisky 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

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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 as- signment of some of the functional properties to defined subunit isoforms. In addition, a minimal composition of a5B2y2 has been found to display the be- havior 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 pro- ceed 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). How- ever, 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 cooper- ative gating by GABA. This encouraged us to try other combinations with a smaller number of subunits. Ex- pression 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. Subse- quently, 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 prop- ertiesof 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 em- phasis 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 Properties of Different Subunit lsoform Combinations after Expression in the Xenopus Oocyte

CABA

Subunits injected I,,, (nA) K, (cLM) H”

Picrotoxin Pentobarbital Stimulation by

I (nA; 10 PM) I (%I,,,; 1 mM) Diazepam (o/o; 1 PM)

21 1.7 130

0.9 * 0.1 1.0 f 0.0 1.2 * 0.1 1.1 * 0.1

n

n (10/2) n (3/I) n (3/l)

408 (4716) n (6/I) n (3/I)

28 (712) 5 (712)

10 (912) n (6/I) n (13/2)

13 f 4

132 f 13

44 f 13

196 (9/l) n (6/I)

1.6 f 0.0

1.7 * 0.1 1.0 f 0.0 1.0 * 0.1 1.2 * 0.0 1.3 * 0.0 1.6 + 0.0 1.7 * 0.1

1.9 * 0.1 1.7 f 0.0 2.0 f 0.0

1.7 * 0.1 1.4 f 0.1 1.4 * 0.1 1.2 * 0.1 1.5 * 0.1 1.5 * 0.1

1.6 f 0.2

20 (6/2) n (6/2)

30 f 10

32 f 8

98 f 15 65 f 16

n (l/l)

Chick Braina - 2500

al n (10/2) a3 n (8/2)

;;15b n (6/2) n (8/2)

P2 n (10/2)

Y2 - 3 (6/2)

alplc >3000 (25/6) 13 *7 (411) -2+5 VI a3DlC >3000 (25/4) 17 * 11 (3/W -1 f IO (6) a5PlC >3000 (26/4) 1.1 * 0.7 (5/l) -2*4 (7) aID - 890 (7/2) 139 * 18 (311) 15 * 6 (3) aly2 n (13/2) a3y2 n (7/2) a5y2 n (6/2)

W/2 - 16 (9/l)

P2Y2 - 155 (7/2) 64 f 11 (311) 198 * 49 (5)

aluSe >2000 (7/l) 18 *3 (3/l) 4 * 16 (3) ala5y2 n (7/2) a3a5y2 - 470 (8/2) 147 * 39 (4/Z) 176 * 61 (4) alply2e >3000 (34/6) 74 + 13 (5/2) 15 * 7 (13) alpZy2 >3000 (12/4) 985 f 352 (3/I) 108 * 37 (IO) a3ply2 >3000 (3/l) 240 f 136 (3/l)’ 23 * 25 (3) a3P2y2 >2000 (4/l) 487 f 42 (3/l) 299 * 50 (3) a5ply2 >3000 (3/l) 17 *7 (3/l) 31 * 14 (3) a5P2y2 >3000 (23/3) 14 *3 (7/2) 136*41 (4)

ala3P2y2 >3000 (8/l) 145 * 75 (3/l) 355 * 163 (4) ala5P2y2 >3000 (7/l) 21 *I (311) 191 * 26 (3) a3a5!32y2 >3000 (7/l) 34 *5 (311) 16Oi26 (3)

ala3a5plp2s >3000 (3/l ) 9 (l/V -5 f 5 (3) ala3a5fily2s >3000 (4/l) 44 * 22 (3/l) 57 * 13 (3) ala3a5P2y2s >3000 (3/l) 20 (l/l)h 150 _t 25 (3) ala3plp2y2s >3000 (3/l) 88 (l/W 113 * 21 (3) ala5Blp2y2s >3000 (5/l) 6 +I cwh a+3 (3) a3a5blp2y2s >3000 (4/l) 17 *7 (3/l) 35 f 14 (3)

ala3a5plp2y2 >3000 (1012) 62 f 28 (5/2) 36 f 18 (4)

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).

29 f 9

16 f a 34 f 14 12 f 3

n 6r2

subunit combinations alp2y2 and a5P2y2, respectively. Fitting of the entire dose-response curves gave Hill

coefficients almost identical to those determined

from the above type of experiments.

It is interesting to note that all subunit combina-

tions that resulted in GABA responses showed desensitization of the current elicited by GABA. The rate

and extent of desensitization depended on the GABA

concentration, but not on the subunit combination expressed.

Effect of Actinomycin D Recently it has been found that the Xenopus oocyte endogenously transcribes an mRNA coding for an a subunit of the nicotinic acetylcholine receptor channel (Buller and White, 1990). This subunit is able to

Neuron 706

-7 -6 -5 -4 -3 -2 log GABA (M)

Figurel. Determination of the Hill Coefficients for CABA- Dependent 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 Proce- dures. 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 in- terpretation 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 in- hibitor 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 some- what inhibited, whereas the affinity of the CABA gat-

Table 2. Effect of Actinomycin D

Relative Expression (% of Control)

Subunit Combination loAsA fP,CrOtDXl”

81 17 * 4 (1012) alB1 55 + 12 (611) 103 f 10 (311) a5Bl a7 k 45 (4/i) 73 ir 34 (311) P2Y2 95 * 24(4/l) - a5Bly2 146 f 40 (4/l) - aSB2y2 172 * 44 (10/2) -

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 of oocytes and number of different batches of oocytes).

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 competi- tion of endogenous mRNA for the translational ma- chinery 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. Differ- ent concentrations of CABA were applied for the times indicated by the bar. The con-

12.5 25 50 centrations are given (micromolar) above 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,

log GABA (MI and >I0 uM, respectively. (b) Individual dose-response curves were fitted using

the equation given in Experimental Procedures. Curves obtained from different oocytes were standardized to the fitted Imax and refit- ted using the same equation. Each point represents the mean and SD of at least three oocytes. Closed circles, u5B2~2; open circles, al B2y2.


a5492 y2

J 200 nA

40s :

diazepam

r diozepam

II v: a5191 y2

-I 500 nA

40s

L Figure 3. Diazepam Stimulation of the CABA Current Expressed in Oocytes Injected with the a5P2y2 or the a5ply2 Subunit Com- bination

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

G e9

I -8 -7 -6 -5

Diazepam (Ml

Figure4. Concentration Dependence of the Response to Di- azepam

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-6,7- dimethoxy4ethyl-&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 diagnos- tic 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 (Fig- ure 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 -// -9 -8

DMCili (Ml -6 -5

Figure 5. Concentration Dependence of the Response to DMCM

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 oo-

cytes 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

i

1, ’ ‘f 0 \ A-

-7 -6 -5 -4 Pi crotox i n (M)

Figure 6. Concentration Dependence of the Inhibition by Picro- toxin

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, re- sult 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 de- serve 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 con- cerning cooperativity of GABA gating, K, for GABA, and diazepam and pentobarbital effects on GABA- gated currents available in the literature. And in those cases in which such parameters have been measured, the subunit composition is not known. For conve- nience, 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


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 surpris- ing 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 ap- propriate subunits to build a functional channel. -Different receptor channel subunits may interact al- losterically and, as a consequence, acquire properties that cannot be assigned to individual subunits. -It cannot be excluded that the Xenopus oocyte car- ries 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 in- duction 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 GABA- dependent 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 [31 subunit, 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 perturba- tion 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 (Pritch- ett 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 effi- cient 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 corre- sponds 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 local- ization studies for the individual subunit isoforms at the single-cell level and, if possible, immunoprecipita- tion 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 use- ful 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 fu- ture 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 ny- lon 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) = Imax c”/(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. Be- tween 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 mea- surement 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 al- lowed 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 Biochemi- cals (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


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

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 grate- fully acknowledged.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Sec- tion 1734 solely to indicate this fact.

Received June 21, 1990; revised August 2, 1990.

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the effect of subunit composition of rat brain gabaa receptors on channel function

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