Allosteric modulation of [3H]flunitrazepam binding to recombinant GABAA receptors

European Journal of Pharmacology Molecular Pharmacology Secuon 291 (1995) 99-105

ELSEVIER

molecular pharmacology

Allosteric modulation of [3H]flunitrazepam binding to recombinant GABA A receptors Astrid Slany, Jiirgen Zezula, Karoline Fuchs, Werner Sieghart * Department of Bmchemtcal Psychmtry, Umverstty Chmc for Psychtatry, Wahrmger Gurtel 18-20, 1090 Vienna, Ausma Recetved 22 February 1995, rewsed 14 June 1995, accepted 20 June 1995

Abstract The allostenc modulatmn of [3H]flumtrazepam binding by y-ammobutync aod (GABA), pentobarbltal, (+)-etomldate, etazolate, alphaxalone, propofol and chlormethmzole was mvesugated m cerebellar membranes and membranes from human embryomc k~dney (HEK) 293 cells transfected with a I/3372 or a t y 2 subumts Results obtmned indicate that [3H]flumtrazepam binding to recombinant GABA A receptors consisting of a I/3372 subunlts could be modulated by these compounds m a way and w~th a potency smular to that observed m cerebellar membranes In addmon, ~t was demonstrated that not only receptors consisting of a~/3372, but also those consisting of a~ Y2 subumts exhthlted [3H]flunitrazepam binding which could be stimulated by GABA In contrast to a~/3~Y2 receptors, however, [3H]flumtrazepam binding to recombinant a l y 2 receptors was mhthlted by pentobarb~tal, (+)-etomldate, etazolate, alphaxalone, propofol and chlormethmzole Th~s seems to mthcate that binding s~tes for these compounds are present on cqy 2 receptors, but that their allostenc interaction w~th [3H]flumtrazepam bmdmg s~tes is different from that of a~/3372 receptors Keywords GABA A receptor, Flumtruzepam binding, Alphaxaione, Propofol, Chlonnethmzole, (+)-Etonudate, Etazolate

1. Introduction

GABA A receptors are hgand gated chloride channels that mediate the actions of many pharmacologically and cllmcally important drugs, such as benzodiazepmes, barbiturates, neurosterolds, anesthetics, and convulsants (Sleghart, 1992a) A variety of evidence indicates that these compounds do not interfere directly with the GABA binding site, but exert their actaon by binding to separate and distinct allosteric modulatory sites at GABAA receptors Interaction of a compound with one of these s~tes causes a change in the conformation of the receptors, resulting in an enhancement or reduction of GABA-Induced chloride ion flux and in a modulataon of the binding properties of the other allosteric binding s~tes at these receptors (Sieghart, 1992a) So far only three binding sites present on GABA A receptors can be directly investigated by binding studies the GABA-(Olsen, 1982), the benzodIazepine-(Braestrup and Squires, 1977), and the picrotoxmin/t-butylbicyclophosphorothIonate-(TBPS) binding site (Squires et

' Corresponding author Tel 43-1-40400-3575, Fax 43-1-40400-3629 0922-4106/95/$09 50 © 1995 Elsewer Science B V All rights reserved .~.~/)I t)q22-4 1 0 6 ( 9 5 ) 0 0 0 9 7 - 6

al, 1983) The binding of compounds to other s i t e s on GABA A receptors can be investigated only indirectly by their allosteric modulation of the binding properties of these three binding sites The allosteric interactions of ligands with GABA A receptors m brain membranes, however, are complex, and interpretation of results is difficult due to multiple interactions of different binding s~tes within the same receptors and due to the heterogeneity of GABA A receptors in the brain (Olsen, 1982, Leeb-Lundberg and Olsen, 1983, Pnnce and Simmonds, 1993) Thus, the exact site of interaction of most of the allosteric modulators of GABA A receptors so far is not known Recently, at least 17 different GABA g receptor subunits have been cloned and sequenced from vertebrate brmn (Butt and Kamatchl, 1991, Sleghart, 1992b) and It has been demonstrated that five subunIts seem to assemble to form intact GABA A receptors (Nayeem et al, 1994) Other studies have indicated that depending on the subunit combination used for transfectlon of cell hnes, recombinant GABA A receptors wRh different pharmacological and electrophysIologlcal properties do arise (Burt and Kamatchi, 1991, Sleghart, 1992b) So far, the binding properties of recombinant GABA A receptors have only incompletely been characterized For

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A Slan ~ et al / European Journal of Pharmacologp - Mole~ ular Pharmacolog~ Set tton 291 (I 995) 99-105

instance, httle Inlormat~on is available on the allostenc modulation of [3H]flumtrazepam binding and its subunlt requirement In order to investigate whether all the allosterlc modulatory sites identified on GABA g receptors in brain tissue are present on recombinant receptors, in the present study the possible modulation by pentobarbltal, alphaxalone, (+)-etomidate, etazolate, propofol and chlormethlazole of [3H]flumtrazepam binding to membranes from ot I f13T2 or OLIT2 transfected HEK cells was investigated

HEK 293 cells were transfected with cDNAs encoding for rat al-, fl~-or yz-subunlts subcloned individually into pCDM8 expression vectors, using the calcmm phosphate precipltatmn method (Chen and Okayama, 1988) Since in prehmmary experiments ~t was found that a 2 1 1 ratio for the a-, fl-, and y-subunlts yielded the highest binding level for [~H]flunltrazepam in the transfected cells, 3 × 10 6 cells were routinely transfected with 12 6 6 /xg cDNA When cells were transfected with a-and y-subunits only, s~mllar results were obtained whether a cDNA ratio of 1 1 or 2 1 was used The medmm was changed 20 h after transfectlon

2. Materials and methods

2 1 Matertals [3H]flunltrazepam (specffic activity 87 C l / m m o l ) was purchased from Amersham, Buckinghamshire, UK Other compounds were obtmned from the following sources dlazepam (Hoffmann La Roche, Basle, Switzerland), alphaxalone (Glaxo Group Research, Middlesex, England), propofol (ICI-Pharma, Milan, Italy), chlormethmzole (Astra Arcus, Sodertalje, Sweden), etazolate (E R Squibb, Princeton, NJ, USA), (+)-etomldate (Janssen Pharmaceuticals, Belgium)

2 2 Clonmg of al-, fl3-and y2-subumts of GABA a receptors A rat brain cDNA library was constructed in AZAP (Stratagene, La Jolla, CA, USA) from poly A ÷ mRNA ~solated from the brains of 8-10-day-old rats as detailed in the protocol from Stratagene al-, /33-and T2-subunlts of GABA A receptors were cloned from this cDNA hbrary and their sequence proved to be ldenUcal with the respective sequence pubhshed previously (Khrestchatlsky et al, 1989, Ymer et al, 1989, Shivers et al, 1989) Cloned cDNA's encoding for the al-, fl3-or 72-subunits of GABA A receptors plus varying amounts of 5' (4-400 bp) and 3' (0-150 bp) untranslated regions were subcloned into the pCDM8 expression vector (Invltrogen, San Diego, CA, USA) by using standard recombinant DNA procedures Each plasmld was purified after growth from a single bacterial colony 2 3 Culturing and transfecttng of human embryomc kidney 293 cells

Human embryonic kidney 293 cells (American Type of Culture Collection, Rockville, MD, CRL 1573) were maintamed in Dulbecco's modified Eagle medium (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum (JRH Blosciences, Lenexa, KS, USA), 2 mM glutamine, 50 p.M fl-mercaptoethanol, 100 unlts/ml penlcllhn G, and 100 /xg/ml streptomycin in 75-cm 2 Petri dishes by using standard cell culture techniques

2 4 Radtohgand bmdmg studies m cerebellar membranes or m membranes from HEK cells Adult rats were sacrifized by decapitation and the cerebella were rapidly dissected and homogenized in 0 32 M sucrose, 10 mM phosphate buffer, pH 7 4 The suspension was centrifuged at 1000 × g for 10 min at 4°C and the supernatant was then centrifuged at 100000 × g for 60 mm The pellet was resuspended in 50 mM Trls-cltrate buffer, pH 7 4 and the membranes were recentrlfuged at 100000 × g for 60 mln Membranes were resuspended in 50 mM TrlS-cltrate buffer, pH 7 4 and stored frozen at - 2 0 ° C Before use, the frozen suspension was thawed, membranes were centrifuged and resuspended in the same volume of 50 mM Trls-citrate buffer, pH 7 5 Non-transfected HEK 293 cells or cells 96 h after transfectlon with plasmlds encoding for GABA A receptor subunlts were washed twice and then harvested by scraplng into phosphate buffered sahne After centnfugation at 12 000 X g for 10 min the cell pellets were homogenized in 50 mM Trls-cltrate buffer, pH 7 4 by using an Ultraturax, followed by three centrlfugatlon (200 000 x g for 20 mln) resuspenslon cycles, and were then used for hgand binding or were stored at - 2 0 ° C For binding studies, both freshly prepared or trozen membranes from non-transfected cells or from cells transfected with plasmlds encoding for GABA A receptor subunits were used with similar results Frozen membranes were thawed, centrifuged and resuspended in 50 mM Tris-cltrate buffer, pH 7 4, at a protein concentration of about 1 m g / m l as measured by the BCA-protein assay kit of Pierce Chemical with bovine serum albumin as standard Membranes (0 5 ml) were then incubated in a total of 1 ml of a solution containing 50 mM Trls-citrate buffer, pH 7 4, 150 mM NaC1 and various concentrations of [3H]flunitrazepam in the absence or presence of 10 /xM dlazepam or various concentrations ef GABA A receptor hgands for 90 mln at 4°C (Sieghart and Schuster, 1984) Membranes were then filtered through Whatman G F / B filters and the filters were rinsed twice with 5 ml of ice-cold 50 mM Tris-citrate buffer Filters were transferred to scintillation vials and subjected to scintillation counting after addition of 3 5 ml Hydrofluor (National Diagnostics,

A Slany et al/European Journal of Pharmacology - Molecular Pharmacology Sectwn 291 (1995) 99-105

NJ, USA) scintillation fluid Non-specific binding determined in the presence of 1 0 / z M diazepam was subtracted from total [3H]flunitrazepam binding to obtain specific binding In control experiments the same results were obtamed whether l0 p.M diazepam or 1 0 / x M clonazepam was used to define non-specific binding

3. Results

Recently, we were able to demonstrate the existence of spec]fic [~H]flunltrazepam binding sites on membranes from non-transfected human embryonic kidney (HEK) 293 cells These bindmg sites, however, were not assocmted with endogenous G A B A A receptors (Fuchs et al, 1995) possibly extstmg in these cells (Kirkness and Fraser, 1993) Since most of these sites did not belong to 'pertpheral' benzodmzepine binding sites either, they rmght have been assocmted with metabohc enzymes or transporters possibly present in embryonic kadney cell membranes (Fuchs et al, 1995) When HEK cells were transfected with various combinations of o[1-, and 3"2-subunits of G A B A A receptors, a m o n g all combinations including homoohgomeric sets, only those containing 0/i 3'2 or o~1 /333'2 subunlts of G A B A h receptors resulted in an increase in [~H]flumtrazepam binding over that endogenously present in these cells Thus, at 2 nM [3H]flunitrazepam, an endogenous [3H]flunltrazepam binding of 20 + 1 f m o l / m g protein (mean + S D , n = 1 l) was observed Transfectton of cells with o/13'2 or 0/!/333'2 subunits increased [~H]flunltrazepam binding to 4 6 4 - 9 f m o l / m g protein (mean + S D , n = 12) or 246 4- 19 f m o l / m g protein, (mean _+ S D , n = 19) respectively Membranes from non-transfected HEK 293 cells or from cells transfected with 0/13'2 or 0/1/333'2 subunits were then incubated with various concentrations of [~H]flumtrazepam m the absence or presence of l0 /xM

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diazepam In order to measure [3H]flunitrazepam spectfically bound to G A B A A receptors formed by the transfection of cells and to compensate for endogenous [3H]flunitrazepam binding in HEK cells, specific radioactivity ( f m o l / m g protein) bound to non-transfected cells was subtracted from that of transfected cells Scatchard analysis of the data obtained indicated that [3H]flunltrazepam exlubited a K D of 3 5 3 _ 0 75 nM and a Bmax of 705 + 86 f m o l / m g protein (mean 4- S D , n = 6) for receptors containing a I/333, 2 subunits Similarly, receptors consisting of 0/172 subunlts exhibited a K o of 10 9 + 0 7 nM and a Bmax of 240 _ 121 f m o l / m g protein (mean + S D , n = 4) In cerebellar membranes, however, G A B A A receptors exhtbited a K o of 2 8 + 0 1 nM and a Bmax of 1332 + 64 f m o l / m g protem (mean + S D , n = 4) In order to compare the allosterlc modulation of [3H]flunttrazepam binding to recombinant receptors consistmg of 0/~/3372 subuntts with that of receptors present in brain membranes, cerebellar membranes or membranes from HEK 293 cells transfected with a I/3t3,2 subuntts were incubated with 2 nM [3H]flunitrazepam m the absence or presence of various concentrations of allostenc G A B A A receptor hgands As shown in Fig 1, G A B A was able to stimulate specific [3H]flumtrazepam blndmg to HEK 293 cells transfected with 0/1/333,2 subunlts with a maximum effect of about 150% and an ECs0 of 0 5 _ 0 1 /xM (mean -t- S D , n = 4) Similarly, [3H]flumtrazepam binding to cerebellar membranes was stimulated by G A B A with a maximum effect of about 100% and an ECs0 of 043-t-01 /xM(mean+SD, n=4) Alphaxalone, presumably mteracting with the steroid binding site of G A B A A receptors (Sleghart, 1992a), caused a concentration-dependent increase in [3H]flunttrazepam binding with a maximum effect of about 40-50% and an ECs0 of 0 8 - t - 0 2 5 /zM (mean + S D , n = 4) or 077-t0 12 /xM (mean _ S D , n = 7) In a I/333'2 or cerebellar receptors, respectively Similarly, pentobarbltal, by bmdmg

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Fig 1 Effects of GABA, alphaxalone, (+)-etonudate, and pentobarbltal on [3H]flumtrazepambinding to membranes from rat cerebellum or from HEK ceils transfected with a I/333,2 subumts of GABAA receptors Membranes from cerebellum or from HEK 293 cells transfected with oq/333,2 subumts of GABAA receptors were incubated with 2 nM [3H]flumtrazepamm the absence or presence of 10 /~M dlazepam or various concentrations of drugs as indicated Radloactwlty bound to membranes was then determined as descnbed m Matenals and methods Data shown represent mean values + S D from 3-7 different expenments performed m duphcate

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Fig 2 Effects of etazolate, propofol and chlormethtazole on [3H]flumtrazepam binding to membranes from rat cerebellum or from HEK cells transfected wnh t~I/3ay 2 subumts of GABAA receptors Membranes from cerebellum or from HEK 293 cells transfected with oq/33y 2 subumts of GABA A receptors were incubated with 2 nM [SH]flumtrazepam m the absence or presence of 10 /xM dmzepam or various concentrations of drugs as indicated Radioactivity bound to membranes was then determined as descnbed in Matenals and methods Data shown represent mean values + S D from 3 - 5 different experiments performed m duphcate

to the barbiturate bmdlng site (Sieghart, 1992a), stimulated [SH]flunltrazepam binding with a maximum effect of about 70% and an ECs0 of 65 5 5:12 /xM (mean + S D , n = 3) or 1 2 5 _ 1 7 /xM (mean 5 : S D , n = 5 ) in recombinant cq f1372 receptors or In cerebellum, respectively (Fig l) (+)-Etomidate, however, which also seems to act via the barbiturate binding site (Thyagarajan et al, 1983), dose-dependently increased [3H]flunitrazepam binding with a maximum effect of about 50% (ECs0 of 4 5 _+ 0 2 /xM (mean_+SD, n = 3 ) o r T 0 + 2 1 / x M ( m e a n S : S D , n = 3) f o r a I /~3"/2 o r cerebellar receptors, respectively), and reduced [3H]flunitrazepam binding at higher concentrations (Fig l) A similar effect was observed when the modulation of [~H]flunttrazepam bindmg by etazolate (again interactmg wtth the barbiturate site, Leeb-Lundberg et al, 1981) or propofol (possibly interacting with a novel anesthetic binding site at GABA A receptors, Sleghart, 1992a) was investigated (Fig 2) These c o m p o u n d s stimulated [~H]flunitrazepam binding at low concentrattons (ECs0 of 1 0 _ + 0 2 /xM ( m e a n s : S D , n = 4 ) or 1 2 5 : 0 4 /xM (mean 5: S D, n = 5) for etazolate, ECso of 7 2 5:1 0 /xM (mean-I-SD, n = 3 ) or 1 6 3 5 : 2 6 /zM ( m e a n + S D , n = 3) for propofoi, in recombinant a~/33y 2 or cerebellar receptors, respectively At higher concentrattons these compounds Inhibited [3H]flunltrazepam binding (Fig 2) Chlormethiazole, however, possibly interacting wtth the same site as propofol (Moody and Skolnlck, 1989, Sieghart, 1992a), only shghtly stimulated [3H]flumtrazepam binding at a concentration of about 100 /xM in both tissues and strongly mhibited this binding with an IC50 of 1 8 5:0 1 m M ( m e a n S : S D , n = 3 ) or 1 4 4 - 0 1 m M ( m e a n + S D , n = 3) in recombinant a~/33y 2 or cerebellar receptors, respectively (Fig 2) In other experiments the modulation of [SH]flunltrazepam binding to membranes from HEK cells transfected with a 13,2 subumts was investigated (Fig 3) Since spe-

cific [3H]flunltrazepam bmdlng was low and unspecific binding was relatively high in these membranes, no exact dose response curves could be measured and thus, the

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Fig 3 Effect of various compounds on [~H]flumtrazepam binding to membranes from HEK cells transfected with a t/3a-y2 or ott'y2 subunlts of GABA A receptors Membranes from HEK 293 cells transfected with oq fls"/2 or oqy 2 subumts of GABA A receptors were incubated wnh 2 nM [3H]flumtrazepam m the absence or presence of l 0 / x M dlazepam or 100p.M GABA. 100/xM alphaxalone, 333 /xM pentobarbltal, l0 /xM (+)-etonudate, 10p.M etazolate, 33 /xM propofol, or 1 mM chlormethlazole Radioactivity bound to membranes was then determined as descnbed m Materials and methods Data shown represent mean values _+ S D from 3 - 6 different experiments performed m duphcate Values measured m O~l'Y2 transfected cells were slgmficantly different from control values in unpaired Student's t-test with Bonferrom-Holm adjustment (Holm, 1979) for pentobarbital ( p = 0 ~ l
A Slany et al/European Journal of Pharmacology - Molecular Pharmacology Sectwn 291 (1995) 99-105

effects of single drug concentrations were investigated GABA was able to shghtly stimulate [3H]flumtrazepam binding to these receptors (maximal effect of about 20%) In contrast, alphaxalone, pentobarbltal, (+)-etomldate, etazolate, and propofol, inhibited binding of [3H]flunltrazepam to membranes from HEK cells transfected with a 13'2 subunits (Fig 3) Under the same conditions (parallel experiments), these compounds stimulated [3H]flumtrazepam binding to membranes from HEK cells transfected with ott/333'2 subunlts (Fig 3), and &d not influence [3H]flunitrazepam binding to membranes from non-transfected H E K cells ( e x p e r i m e n t s not s h o w n ) [aH]Flunitrazepam binding to HEK cells transfected with 0¢1 /3"~3'2 as well as that of cells transfected with a13'2 subunits was inh~bited by chlormethtazole at similar concentratmns (Fig 3)

4. Discussion

In the present study prewous lnvestlgatmns on the allosterlc modulation of [3H]flumtrazepam bmdmg to brain membranes were confirmed Thus, it was demonstrated that GABA (Supavdm and Karobath, 1981, Leeb-Lundberg and Olsen, 1983), alphaxalone (Prince and Slmmonds, 1993), and pentobarbltal (Leeb-Lundberg et al, 1981, Thyagarajan et al, 1983) stimulated [3H]flunltrazepam binding to cerebellar membranes w~th a potency similar to that described prewously. Furthermore, not only the potency but also the shape of the dose response curve of (+)-etomldate (Thyagarajan et al, 1983), etazolate (Supawlai and Karobath, 1981, Leeb-Lundberg and Olsen, 1983), propofol (Prince and Slmmonds, 1992) or chlormethiazole (Moody and Skolnlck, 1989) was close to that observed previously Interestingly, these compounds were also able to modulate [3H]flumtrazepam binding to recombinant GABA A receptors consisting of al/333'2 subunits, and their potency and mode of mteractmn was closely similar to that for cerebellar receptors These results indicate that bmdmg s~tes for alphaxalone, pentobarbltal, (+)-etom~date, etazolate, propofol, and chlormethmzole not only are present on these receptors, but also interact with [3H]flumtrazepam binding sites m a way slmdar to naUve receptors Recombinant receptors consisting of a-, /3-and y2-subunits thus again proved to be an excellent model system for GABA A receptors m the bram (Sieghart, 1992a,b) In agreement with a prewous report (Wong et al, 1992), not only receptors consisting of oq/333'2, but also those consisting of o/13"2 subumts exhlbtted a high affimty [~H]flunitrazepam binding The presence of benzodlazepine binding sites on a r t 2 receptors is supported by the observation that GABA-lnduced chloride ion flux in HEK cells transfected with aT subumts could be modulated by benzodmzepmes (Pula et al, 1989, Knoflach et al, 1992, Wong et al, 1992, Im et al, 1993) In contrast,

103

receptors consisting of a I/33 or /33T2 subunlts m the present study were not able to bind [3H]flunltrazepam, under conditions where the actual expression of these receptors was demonstrated by the appearance of binding sites for [35S]TBPS (Fuchs et ai, 1995) This is m partial agreement with electrophyslologlcal studies on recombinant receptors Whereas no reproducible modulation of GABA-mduced chloride ion flux could be observed in cells transfected with a 3/31 (Knoflach et al, 1992) or al/3z subunits (Im et al, 1993), an atypical modulation by benzodiazepmes was observed in recombinant /31Y2 receptors (Knoflach et al, 1992) In recombinant receptors consisting of /32Tz subumts, however, depending on the benzo&azepme hgand investigated, either no potentiatmn, or a potentiation with a magnitude similar to that observed for cqy z receptors was obtained (Im et ai, 1993) Differences m the experimental con&tlons used m binding studies and in electrophysmloglcal stu&es, a low affinity of the /33Y2 receptor for [3H]flumtrazepam, or a difference in the subumt stolchiometry of recombinant receptors might have been the reason for this &screpancy Recently, the presence of endogenous mRNA for /3~subunits of GABA A receptors was demonstrated in nontransfected HEK cells (Kirkness and Fraser, 1993) Several hnes of evidence argue against the possibihty that [3H]flumtrazepam binding observed in HEK cells transfected with ott3,2 subumts was actually due to receptors formed by a coassembly of these subunlts with endogenous /33-subunlts Thus, receptors found in HEK cells transfected with cqT 2 subumts, m contrast to those consisting of oq/33T2 subunlts, were not able to bind [35S]TBPS (Fuchs et al, 1995) In addmon, although binding sites for pentobarbital, alphaxalone, (+)-etom~date, etazolate or propofol could be demonstrated on at Yz transfected HEK cells, allosteric coupling of these s~tes with the binding site for [3H]flunitrazepam was different from that observed in a~/33Y2 transfected cells These results and the affinity of [3H]flunitrazepam for a~Y2 transfected HEK cells, which was different from that of oq/33Y2 transfected cells, support the actual formatmn of recombinant GABA A receptors consisting of oq Y2 subunits Interestingly, [3H]flunitrazepam binding to c~13'2 transfected HEK cells was inhibited by pentobarbital, alphaxalone, (+)-etomldate, etazolate or propofol under con&tmns where these compounds caused a stimulation of [3H]flumtrazepam binding to oq/333"2 transfected cells These results are consistent with a recent report (Hams et a l , 1995) demonstrating a shght inhibition of [3H]flunltrazepam binding by alphaxalone and pentobarbital m HEK cells permanently transfected with oq 3"2 subunlts The mteractxon with [3H]flunitrazepam binding indicates a conformatlonal change m oqT 2 receptors reduced by these compounds Since benzodiazepine binding sites are neither involved m nor essential for opening of GABA A receptor associated chloride channels, a reduction

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m benzodlazepme bmdlng not necessarily corresponds with a reduction in GABA-lnduced chloride ton flux In agreement with this conclusion it has been demonstrated that pentobarNtal (Burt and Kamatchl, 1991) or anesthetics (Harrison et al, 1993) were able to ehcit an enhancement of GABA-mduced chloride ton flux m GABA g receptors consisting of c~3" subuntts A possible modulation by alphaxalone, (+)-etomldate, etazolate, propofol or chlormethtazole of these receptors so far has not been investigated Receptors conststlng o f OelT2 subunlts, however, seemed to assemble less effictently than receptors consistmg o! cq fl3Y~ subunlts This can be concluded from the difference m the number of htgh afflntty [3H]flunttrazepam binding sites reduced in cells transfected with these subunit combmatmns, It tt IS assumed that Of13'2 or a I /33T2 receptors contain equal numbers of [3H]flunltrazepam bmdmg sites This conclusion ts consistent with previous results trom electrophystologlcal studies, which indicated that the expression efficiency of recombinant GABA A receptors consisting of a l y 2 subuntts was dependent on the cell system used and was smaller than that of receptors consistmg ot cq/3~3'2 subunits (Verdoorn et al, 1990, Knoflach et al, 1992, Angelottl et al, 1993) The Bma x values tor [3H]flunltrazepam binding to a I f1372 transfected HEK cells, however, were only about three times larger than those observed for a 1"/2 transfected cells It is thus possible that significant amounts of receptors consistmg ot only ~x~-and Y2-subunlts are formed m cells translected with oq /33T2 subuntts The possible existence in the brain ot GABA A receptors conslstmg of oq-and 3'2subumts thus cannot be excluded (Fritschy et al, 1992) Interestingly, as observed m cerebellar membranes, [~H]fhmttrazepam binding to recombinant receptors conslstmg ot cq/333'2 subuntts not only could be stimulated at low, but could also be inhibited at high concentrations of (+)-etomldate, etazolate, and p r o p o f o l Since [~H]flunltrazepam bmdlng to receptors conslstmg of cq 3'2 subumts ts mhlblted by these compounds, and this inhibition might even be stronger at higher concentrations, one possible explanation for these results could be the presence ol ~ 3 ' 2 receptors m a~ /333'2 translected cells Alternatively, this btphastc effect could be explained by the extstence of different conformatlonal states of binding sites on the same GABA A receptor Smce the mhlbltlOn of [~H]dlazepam binding by etazolate could be attenuated by the inverse benzodlazepine agonist /3-carbohne-3carboxylate ethyl ester, it has been hypothesized that the blphastc effect of etazolate on [3H]dlazepam binding to bram membranes might have been due to the stablhzatmn ot an antagonist preferring conformatmn of a two-state ohgomertc receptor (Leeb-Lundberg and Olsen, 1983) And finally, the btphasic effect of these compounds on [~H]flunttrazepam bmdmg might have been due to the posslbthty that etazolate, (+)etomidate, propofol, and chlormethiazole are able to interact with the GABA A

receptor at two different homologous or heterologous binding sites, whmh are activated at different concentrations of these drugs and which could reduce opposite conlormatlonal changes m the GABA A receptor Further experiments will have to be performed to clartly this question, and to identify the sites of interaction of the various allostertc modulators with the GABA A receptor

Acknowledgements Work performed in the present study was supported by the 'Fonds zur Forderung der Wtssenschafthchen Forschung m Ausma'

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