Neuroseienee Letters, 130 (1991) 169-172

169

© 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100502J NSL 08020

Inverse but not full benzodiazepine agonists modulate recombinant 6f1272 GABAAreceptors in transfected human embryonic kidney cells C. Kleingoor, M. Ewert, G. von Blankenfeld, P.H. Seeburg and H. Kettenmann Department of Neurobiology and ZMBH, University of Heidelberg, Heidelberg ( F.R.G.) (Received 24 January 1991; Accepted 6 June 199 I)

Key words: GABAA receptor; Benzodiazepine; Patch clamp; cDNA; Cell transfection We compared the modulation of GABA 0,-aminobutyric acid)-activated currents by benzodiazeplnes in recombinant GABAA receptors containing either one of two ~t subunits, ~q or %. Lfiddens et al. (Nature, 346 (1990) 648-651) have previously demonstrated that the % subunit is part of a cerebellar receptor subtype which selectively binds Ro 15-4513, an antagonist of alcohol-induced motor ataxia. Here we report that the imidazobenzodiazepine Ro 15-4513 (ethyl 8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo-(1,5-a) (l,4)benzodiazepine-3-carboxylate) reduced GABA-activated currents in recombinant 0~2 h and ~qfl2½ receptors, thus acting consistently as an inverse agonist. Moreover, another well characterized negative modulator, DMCM (methyl-4-ethyl-6,7-dimethoxy-fl-carboline-3-carboxylate), also reduces GABA activated-currents in both receptors. In contrast, flunitrazepam (FNZM), a benzodiazepine agonist, increases GABA-activated currents in etlflzy2 receptors, but not in ~t~fl2h receptors. This study lends further support to the hypothesis that the binding sites of full and partial inverse agonists are different.

The GABAA receptor complex is the main inhibitory neurotransmitter receptor in the central nervous system of mammals. The Cl- channel intrinsic to this receptor is gated by GABA and channel activity is modulated by different classes of hormones and clinically important drugs, i.e. steroids, barbiturates, and benzodiazepines [3, 10]. The latter are thought to bind to specific domains determined by the subunit composition of the receptor complex [9, 13, 14, 17]. Full benzodiazepine agonists such as flunitrazepam or diazepam enhance GABA-activated currents; inverse agonists such as DMCM have the opposite effect [4, 5]. The partial inverse agonist Ro154513 reverses, in part, central nervous depressant effects of ethanol [15]. Its action is thought to be mediated via the GABAA/benzodiazepine receptor complex since it can be blocked by the antagonist Ro15-1788 (flumazenil) [2]. In a study comparing recombinant GABAA receptors, Lfiddens et al. [9] demonstrated that receptor complexes containing the or6 subunit selectively bind Ro 15-4513 with high affinity, but not the full benzodiazepine agonists such as flunitrazepam; DMCM binding shows decreased affinity relative to 0q subunit containing receptors. In this study, we compared the physiological action of these drugs on cells expressing 0t6f12~2and 0tlfl2~2

Correspondence: H. Kettenmann, Department of Neurobiology, University of Heidelberg, Im Neuenheimer Feld 345, 6900 Heidelberg, F.R.G.

receptors. We demonstrate that both the partial inverse agonist Ro 15-4513 and the inverse agonist DMCM, but not the full agonist flunitrazepam, modulate GABA responses in receptors containing the 0~6subunit. Transfo/med human embryonic kidney cells 293 (ATCC CRL 1573) [7] were grown at 37°C on coverglass coated with fibronectin (25/~g/gl in phosphate-buffered saline; PBS) in minimum essential medium (MEM, Gibeo) supplemented with 10% fetal bovine serum containing 100 U of penicillin (Gibco) and 100/zg of streptomycin (Gibco) per ml in a 5% CO2/95% air incubator. Exponentially-growing cells were trypsinized and seeded at 2 x 104 per coverglass in l ml of growth medium. The transfection was performed by the calcium phosphate precipitation technique [6]. The cloned cDNAs of rat GABAA receptor subunits, inserted singly into the eukaryotic expression vector CIS2 [7, 11] or CDM8 [9, 17], were used for transfection. The cells were incubated in the presence (50 ng/500 ~l) of one or several supercoiled expression plasmids for 12-16 h at 37°C under 3% CO2/ 97% air. The medium was removed, and the cells were rinsed twice, refed and incubated for 24 h at 37°C under 5 % CO2/95 % air before beginning the electrophysiological studies. For recording of membrane currents, cells were transferred to the stage of an inverted microscope and maintained at about 25°C in a recording chamber which was continuously perfused permitting rapid application of drugs. 90% of the bath exchange was accomplished

170

within 10 s. The standard salt solution contained in mM: NaCl 130.0, glucose 5, KC1 5.4, CaClz 1.8, MgClz 1.O, buffered at pH 7.2 with 5 mM HEPES. GABA (1 ,uM), Flunitrazepam (1 PM), Ro 15-45 13 (1 PM) and DMCM (1 ,uM) were added to the standard salt solution. All voltage-clamp recordings were made with an EPC7 patch-clamp amplifier (List Electronics, Darmstadt, F.R.G.) using the tight-seal, whole-cell recording configuration [8]. Voltage and current traces were digitized and stored on an AT-compatible computer system. Control of data acquisition and the data analysis were achieved with the aid of a program developed in our laboratory. Recording pipettes were fabricated from borosilicate capillaries (Hilgenberg, Malsfeld, F.R.G.) with resistances ranging from 2 to 5 MQ. Pipettes were filled with a solution containing in mM: CsCl 130, MgC12 1, CaCl* 0.5, EGTA 5, HEPES 10. Ca*+ activity was calculated to be approximately 11 nM. In cultured cells transiently expressing cl&y2 or a&y* receptors, GABA (1 ,uM) elicited pronounced inward currents (as recorded with the patch-clamp technique). To characterize the effect of benzodiazepines on GABAevoked currents we selected cells which showed stable

GABA

GABA

GABA

GABA

current amplitudes after successive GABA applications. The GABA pulses were applied for 30 s separated by 5 min intervals to avoid desensitization of the response. Flunitrazepam (1 ,uM) was preincubated for 2 min to saturate the binding sites. A further control response was recorded to ensure that the effect of flunitrazepam was fully reversible. In a&y2 receptors, GABA-induced currents were increased by 64% (SD. 45, n=2 out of 4) in the presence of flunitrazepam relative to controls, as previously described (Fig. 1) [12]. In contrast, GABA responses from a&y1 receptors were either not affected by the presence of flunitrazepam (10e6 M; n= 13) or were decreased by 33% (Fig. 1; Table I) (SD. 16, n=6). We also varied the flunitrazepam concentration between low7 and 10e5 and obtained similar results (n = 7). Only in one case did we observe an increase of the GABAinduced current by lhrnitrazepam. The effect of Rol5-4513 and DMCM on GABAinduced currents was tested with a similar experimental protocol as described for flunitrazepam. In cells expressing a&y2 receptors, GABA-induced currents were decreased by 22% (S.D. 6.9, n=7 out of 11) and 46.2% (S.D. 18.2, n=2 out of 3) in the presence of Ro15-4513

GABA

GABA FNZM

FNZM

30 set

GABA

GABA

GABA

FNZM 250pA I

30 set

Fig. 1. The effect of flunitrazepam

on GABA currents

fected with the a,/~& (left traces) and a&y2 subunits with the patch-clamp applied

as indicated

technique.

Successive

by bar. While GABA

applications responses

on a,,!$y, and a&y2 receptors. (right traces).

The membrane

were separated

GABA potential

by 5 min intervals

were increased by flunitrazepam in a&y2 receptors.

(1 PM) was applied was clamped to minimize

as indicated

at -70

desensitization.

on alay2 receptors,

by bars on cells trans-

mV and currents Flunitrazepam

it was uneffective

were recorded (1 PM) was

or led to a decrease

171 TABLE I PHARMACOLOGICAL GABAA RECEPTORS

PROPERTIES

OF

RECOMBINANT

Binding affinities (K0 in n M of selected benzodiazepines to membranes of human embryonic kidney cells expressing ~taB272and ~tl,8~72receptors are displayed in the left column (from ref. 9). On the right, the modulation of GABA-activated currents by the benzodiazepines is compared to controls (application of G A B A alone). Negative values denote a decrease, positive values an increase in percentage of the G A B A activated current. The first values indicate the mean from several experiments, the second the standard deviation. Binding studies [nM]

Electrophysiology [% change of current]

Compounds

alf1272

a6~2~2

~1fl272

Ct6fl2)J2

Flunitrazepam

2 + 0.3

> 10,000

+ 6 4 __+45

Ro15-4513 DMCM

15 _ 4 5 __+2

5.4 + 0.4 210 __+ 50

-22 _ 7 - 4 6 __+ 18

0 - 3 3 + 16 - 2 9 _ 14 - 2 9 + 16

and DMCM, respectively (Fig. 2). A similar result was observed for ct6f1272receptors (Fig. 2; Table I); GABA responses were decreased by 28.5% (S.D. 14.4, n= 11) and 28.5% (S.D. 16, n=7) in the presence of Ro15-4513 and DMCM, respectively.

GABA

GABA

We tested the effect of 20 mM alcohol on GABAinduced currents in cells transfected with the ~lf12Y2 and ~6fl272 subunits. We did not observe a significant effect of alcohol on GABA-induced currents either on ~lfl2Y2 (n=4) or ct6f1272(n = 19) receptors. Here we demonstrate that the inverse benzodiazepine agonist DMCM and the partial inverse agonist Ro154513 act similarly on GABA-induced currents in ~1fl272 and ~t6f1272receptors. This is consistent with the observation that Ro15-4513 and DMCM show binding affinity in the nM range to at6fl2Y2receptors (Table I) [9]. The difference in affinity between DMCM and Ro15-4513 observed in binding studies could not be resolved in our physiological studies, since significant effects on GABAinduced currents were only consistently observed with drug doses far above the Kd value obtained in the binding study. Such a discrepancy between dose response curves obtained from binding studies and physiological studies have been observed for a variety of drugs including benzodiazepines. Our study supports the hypothesis that the inverse and the partial inverse agonists bind to different domains than full agonists. Our data suggest that the inverse and the partial inverse, but not the full benzodiazepine binding domain is functionally present in ~6fl272 receptors.

GABA GABA

Ro 15-4513

GABA Ro 15-4513

"1

GABA

] 250pA 30 see

f

oopA I GABA

GABA

30 sec

GABA

DMCM 1 nA

GABA 30 sec

GABA

GABA

DMCM

Fig. 2. The effect of Ro15-4513 and D M C M on cq,8272 and ~t~f1272receptors. In a similar experimental protocol as described in the legend to Fig. 1, the effect of Ro15-4513 (1 gM) and D M C M (1/tM) was tested on cells transfected with the ~1//272 (left traces) and ct~2?: subunits (right traces). GABA-induced currents were decreased in the presence of Ro 15-4513 (upper traces) and D M C M (lower traces).

172

This view is supported by the finding that the co-expression of the ~2 subunit is required for the modulation of GABA responses by full agonists whereas inverse benzodiazepines modulate GABA currents in both, y~ and Y2 containing receptors [1]. The observation that in 50% of the measurements flunitrazepam induced an inverse effect in ~rf12Y2 opposite to 0qf1272 receptors might be explained by a cross-reactivity of flunitrazepam on the Ro15-4513 site. Ro 15-4513 is described as an alcohol antagonist effective in electrophysiological, behavioral and neurochemical paradigms [2]. In a study on two strains of mice, long-sleep and short-sleep, alcohol increased GABAactivated currents in the former population and decreased it in the latter [16]. GABA responses had been studied by injecting mRNA into oocytes isolated from the corresponding mouse strain. In our study on recombinant receptors with defined subunits we could not observe a significant effect of alcohol on the GABAinduced currents. This would indicate that other GABAA receptor complexes with subunit compositions different from the ones used in our study mediate the alcohol effects on GABAA receptors. Moreover, the antagonistic effect of Ro 15-4513 on alcohol-induced responses is mediated by receptive sites different from the ones investigated in the present study. This research was supported by Bundesministerium fiir Forschung und Technologie, Deutsche Forschungsgemeinschaft (Heisenberg-Stipendium to H.K., SFB 317) and Boehringer-Ingelheim Fonds (M.E.). The authors thank Dr. H. Liiddens for discussion, T. Berger, S. Griinewald and P. Sick for excellent technical assistance. 1 Blankenfeld, G. von, Sontheimer, H., Ewert, M., Seeburg, P.H. and Kettenmann, H., Differential benzodiazepine pharmacology of recombinant GABAA receptors, Neurosci. Lett., 115 (1990) 269273. 2 Bonetti, E.P., Burkard, W.P., Gabl, M., Hunkeler, W., Lorez, H., Martin, J.R., Moehler, H., Osterrieder, W., Pieri, L., Polc, P., Richards, J.G., Schaffner, R., Scherschlicht, R., Schoch, P. and Haefely, R., Ro15-4513: partial inverse agonism at the BZR and interaction with ethanol, Pharmacol. Biochem. Behav., 31 (1989) 733-749.

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