Pharmacology of Recombinant Q-Aminobutyric Acid E Receptors

FEBS Letters 431 (1998) 400^404 FEBS 20566

Pharmacology of recombinant Q-aminobutyric acidA receptors rendered diazepam-insensitive by point-mutated K-subunits

Jack A. Benson, Karin Loëw, Ruth Keist, Hanns Mohler, Uwe Rudolph*

Institute of Pharmacology, University of Zuërich, Winterthurerstrasse 190, CH-8057 Zuërich, Switzerland

Received 8 June 1998

point mutation. In the present study, the BZ-responsiveness of Abstract Amino acids in the K- and Q-subunits contribute to the point-mutated receptors was tested electrophysiologically fol- benzodiazepine binding site of GABAA-receptors. We show that the mutation of a conserved histidine residue in the N-terminal lowing the co-expression of the mutated K1-, K2-, K3- and K5- extracellular segment (K1H101R, K2H101R, K3H126R,and subunits with the wild-type L2- (or L3-) and Q2-subunits in K5H105R) results not only in diazepam-insensitivity of the HEK 293 cells. respective KxL2,3Q2-receptors but also in an increased potentiation of the GABA-induced currents by the partial agonist bretazenil. Furthermore, Ro 15-4513, an inverse agonist at wild- 2. Materials and methods type receptors, acts as an agonist at all mutant receptors. This conserved molecular switch can be exploited to identify the 2.1. Construction of GABAA receptor subunit expression vectors The rat K1-subunit cDNA [6] (1.6-kb XhoI fragment in XhoI site of pharmacological significance of specific GABAA-receptor sub- pSK-K1) was subcloned as a HindIII/KpnI fragment into M13mp19 types in vivo. and point-mutated using the Amersham Sculptor kit and oligonucleo- z 1998 Federation of European Biochemical Societies. tide UR39: 5P-GAC TTT TTT CCA TTC CGG AAA AAT GTA TCT-3P. The mutated cDNA was resequenced, subcloned ¢rst into Key words: Neurotransmitter; Receptor; Q-Aminobutyric pKS (HindIII/KpnI), then with a complete BglII and partial BamHI acid; Benzodiazepine; Recombinant; Central nervous system digest into the BamHI site of the expression vector pBC12/CMV [8]. The K2-cDNA (1.4-kb fragment in M13PICH-rK2) underwent olignu- cleotide-directed mutagenesis with KL4: 5P-TGA CTT TTT CCC GTT CCG GAA GAA GGT GTC AGG AGT-3P, and was resequenced and subcloned as a BamHI/BglII fragment into the BamHI 1. Introduction site of pBC12/CMV. The K3-cDNA [9] (3.4-kb EcoRI fragment in M13mp18) was mutated using oligonucleotide UR40: 5P-AGA TAC Classical benzodiazepines (BZ) such as diazepam are in CTT CTT CCG GAA CGG TAA AAA ATC-3P. The mutated cDNA was sequenced and subcloned into pKS with SacI (now as a 1.7-kb wide clinical use as anxiolytics, hypnotics, myorelaxants and fragment). From there it was partially digested with SstI and com- anticonvulsants. These activities are based on the enhance- pletely with BamHI and subcloned into pSP72 (Promega), from where ment of GABAergic transmission at BZ-sensitive GABAA-re- it was recovered as a BamHI/BglII fragment and subcloned into the ceptors [1], which are composed of an K-subunit variant (K1, BamHI site of pBC12/CMV. The K5-cDNA [9] (EcoRI partial frag- K2, K3, or K5) in combination with a L-subunit (L1^3) and a ment in M13PIC-19H) was mutated using oligonucleotide UR41: 5P- GAC TTC TTC CCA TTC CGG AAG AAT GTG TCT-3P, se- Q-subunit (Q1^3) [2^4]. The receptor subtypes K1L2,3Q2, quenced and subcloned into pKS as a 2.1-kb HindIII/SstI fragment. K2L2,3Q2, K3L2,3Q2 and K5L2,3Q2 are considered to be the It was then subcloned into pSP72 with SstI/Asp718, from where it was major mediators of BZ actions in the brain [5]. To identify recovered as a BamHI/BglII fragment and subcloned into the BamHI the pharmacological signi¢cance of individual receptor sub- site of pBC12/CMV. Corresponding expression vectors containing the wild-type K1-, K2-, K3- and K5-subunit cDNAs were prepared in par- types, point mutations were sought by which individual recep- allel. The L2-, L3- and Q2-subunits were also used in pBC12/CMV. tor subtypes might be rendered diazepam-insensitive in vivo. In previous mutational analyses, a histidine to arginine sub- 2.2. Cell culture and transfection stitution in position 101 of the K1-subunit (K1H101R) strongly Human embryonic kidney cells 293 were grown in MEM medium reduced the diazepam response of the recombinant K1L2Q2- supplemented with 10% fetal bovine serum, 50 Wg/ml gentamicin and 20 mM L-glutamine (all from Life Technologies, Basel, Switzerland) receptor expressed in human embryonic kidney (HEK) 293 and transformed transiently with rat cDNA KLQ combinations (ratio cells in vitro [6,7]. Since a homologous histidine residue also 1:1:1) by calcium phosphate precipitation [10]. occurs in the K2-, K3- and K5-subunits (Fig. 1), we investi- gated whether the histidine to arginine mutation would rep- 2.3. Electrophysiology and data analysis The whole-cell con¢guration of the patch-clamp technique was used resent a common molecular switch to render the respective to record GABA-induced Cl3 currents. The GABA dose-response receptor subtypes diazepam-insensitive. A common molecular curves were obtained by applying 2-s pulses of GABA every 2 min switch could be exploited for the analysis of the contribution to the patch-clamped HEK-293 cells, using a multibarrelled micro- of individual receptor subtypes to the pharmacological spec- applicator pipette, as previously described [11]. The maximum current amplitudes from individual cells were ¢rst ¢tted separately using the trum of diazepam by generating knock-in mice carrying this Hill equation I/Imax = 1/(1+(EC50/[GABA]) ), where I = GABA-evoked current, Imax = the maximum of the ¢t, EC50 = the GABA concentra- *Corresponding author. Fax: (41) (1) 63 55708. tion evoking the half maximal response, and Hill = the Hill coe¤cient. E-mail: [email protected] The individual dose-response curves were then normalized to Imax, and the data replotted using the mean values for each concentration.

Abbreviations: GABAA, Q-aminobutyric acid type A; BZ, benzodia- For each experiment, at least three GABA control responses were zepine; Ro 15-4513, ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H- evoked and only cells showing stable GABA responses were selected imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate; DMCM, methyl- for the drug testing. Prior to microapplication of a GABA-drug mix- 6,7-dimethoxy-4-ethyl-L-carboline-3-carboxylate ture, the same concentration of the drug alone was applied by bath

FEBS 20566 24-7-98 J.A. Benson et al./FEBS Letters 431 (1998) 400^404 401 perfusion for at least 2 min. Stock solutions of the test compounds were prepared in 100% DMSO and diluted 1000-fold before use. Dur- ing the experiment, all bath solutions contained 0.1% DMSO, which by itself was without detectable e¡ect on the GABA responses. The benzodiazepine ligands bretazenil, Ro 15-4513, and diazepam were kindly provided by F. Ho¡mann-La Roche Ltd. (Basel).

3. Results

3.1. GABA-sensitivity of the point-mutated recombinant GABAA-receptors A histidine to arginine codon substitution was introduced into homologous positions of rat K1-, K2-, K3- and K5-subunit cDNAs (positions 101, 101, 126 and 105, respectively) (Fig. 1) by site-directed mutagenesis. The wild-type and point-mutated K-subunits were co-expressed in HEK 293 cells with the Q2- subunit and either the L2- or the L3-subunit, depending on the associations most commonly detected in rat brain [12^14], in order to generate the following subunit combinations: K1L2Q2, K1H101RL2Q2, K2L3Q2, K2H101RL3Q2, K3L3Q2, K3H126RL3Q2, K5L2Q2 and K5H105RL2Q2. The EC50 values for GABA for the receptors incorporating wild-type K-subunits were 23 þ 2 WM for K1L2Q2, 74 þ 12 WM for K2L3Q2, 165 þ 68 WM for K3L3Q2 and 14 þ 1 WM for K5L2Q2 (Fig. 2). These values largely correspond to the po- tencies of GABA reported previously: the published EC50 values are 4.5^20 WM for K1L2Q2 [15^17], 25 WM for K2L3Q2 [18], and 4.2^16 WM for K5L2Q2 [9,11,15,17]. Only the EC50 value determined for the K3L3Q2 (165 þ 68 WM) di¡ered appreciably from the published values, 28 WM and 33 WM [17,18]. The GABA EC50 values for receptors incorporating the point-mutated K-subunits were all modestly higher than H101R the EC50 values for the wild-type receptors: K1 L2Q2 yielding an EC50 value of 31 þ 1 WM (compared to 23 þ 2 WM for K1L2Q2); K2H101RL3Q2 yielding 154 þ 4 WM (compared to 74 þ 12 WM for K2L3Q2); K3H126RL3Q2 yielding 253 þ 51 WM (compared to 165 þ 68 WM for K3L3Q2) and K5H105RL2Q2 yielding 27 þ 1 WM (compared to 14 þ 1 WM for K5L2Q2) (Fig. 2).

3.2. Modulation of point-mutated GABAA-receptors by ligands of the benzodiazepine site To assess the modulation of the GABA-evoked currents by benzodiazepine site ligands, GABA concentrations of 3 WM (K1-, K3- and K5-receptors) and 30 WM(K2-receptor) were chosen, corresponding to maximally EC30 values. The modulation of the GABA response by the full agonist diazepam is Fig. 2. GABA dose-response curves for wild-type and point-mutated summarized in Fig. 3; representative individual traces for the GABAA-receptor subtypes expressed in HEK 293 cells transiently transfected with the cDNAs coding for the wild-type (wt) or mu- K2L3Q3 subtype are demonstrated in Fig. 4. The benzodiaze- tated K-subunits in combination with the L2- or L3- and Q2-subunits pine full agonist diazepam (1 WM) potentiated GABA-induced (K1L2Q2, K1H101RL2Q2; K2L3Q2, K2H101RL3Q2; K3L3Q2, K3H126RL3Q2; K5L3Q2, and K5H105RL3Q2). The data points and error bars represent the means and standard errors for the di¡erent GABA concentrations (n = 3^5).

currents of receptors incorporating wild-type K1-, K2-, K3- and K5-subunits by 29 þ 11%, 72 þ 5%, 108 þ 29% and 118 þ 19%, respectively (Figs. 3 and 4). In contrast, the GABA-evoked responses mediated by receptors containing the point-mutated K1-, K2-, K3- and K5-subunits were diaze- Fig. 1. Alignement of partial N-terminal extracellular sequences of pam-insensitive (Figs. 3 and 4) at doses that produced max- the rat GABAA-receptor K-subunits (K1^K6) (modi¢ed from [6]). imum enhancement at wild-type receptors. These results dem- The conserved histidine residue in the K1-, K2-, K3- and K5-subunits (positions 101, 101, 126, and 105, respectively) are highlighted. The onstrate that K2-, K3- and K5-GABAA-receptors can be K4- and K6-subunits contain an arginine residue at the correspond- rendered diazepam-insensitive by a histidine to arginine point ing positions (99 and 100, respectively). mutation. Thus, the structure-activity relationship for the in-

FEBS 20566 24-7-98 402 J.A. Benson et al./FEBS Letters 431 (1998) 400^404

enhancement of the GABA-evoked currents by Ro 15-4513 applied to the point-mutated receptors (K1L2Q2, potentiation: 171 þ 45%; K2L3Q2, potentiation: 124 þ 6%; K3L3Q2, potentiation: 168 þ 17%; K5L2Q2, potentiation: 124 þ 16%) was equal to (K5) or greater than (K1, K2, K3) the potentiation induced by the same dose of diazepam applied to the wild-type receptors (Figs. 3 and 4). The point mutation thus changed the mode of interaction of Ro 15-4513 from inverse agonism to agonism. These results suggest that the mode of interaction of Ro 15-4513 with the benzodiazepine binding site di¡ers from that of either diazepam and bretazenil. A switch in the apparent e¤cacy of a benzodiazepine site ligand has previously been observed for methyl-6,7-dimethoxy-4-ethyl-L-carboline-3-carboxylate (DMCM), based, however, on an unusual dose-response behavior on various wild- type receptors (K1L1Q2, K1L2Q2, and K5L2Q2); DMCM acted Fig. 3. Potentiation of the GABA-evoked currents by BZ site li- as an inverse agonist at low doses and as an agonist at high gands for wild-type and point-mutated GABAA-receptor subtypes expressed in HEK 293 cells transiently transfected with the cDNAs doses [9]. To exclude the possibility that the agonism observed coding for the wild-type (wt) or mutated K-subunits in combination for Ro 15-4513 on the point-mutated receptors is due to a with the L2- or L3- and Q2-subunits (for details see legend to Fig. dose-response phenomenon, an entire dose-response curve 2). The modulation of GABA-evoked currents by the BZ site li- was recorded for the e¡ect of Ro 15-4513 on one of the gands diazepam (1 Wm), bretazenil (1 Wm) and Ro 15-4513 (1 Wm) is H101R expressed relative to the control currents at the given GABA con- mutant receptors, K2 L3Q2 (Fig. 5). For each dose tested centration (mean þ standard error, n = 3^7). over the range of 1 nM to 1 WM, Ro 15-4513 displayed an agonistic e¡ect on the mutated receptors. Thus, the agonistic activity of Ro 15-4513 on receptors containing the mutated K- subunits cannot be attributed to a dose-response phenomen- teraction of diazepam with the BZ site appears to be con- on. The histidine to arginine mutation in the K1- K2-, K3- and served among the receptor subtypes tested. K5-subunits rather switches the e¤cacy of Ro 15-4513 from To assess the in£uence of the histidine to arginine point inverse agonism to agonism. mutation on the responsiveness to other benzodiazepine site ligands, the e¡ect of the partial agonist bretazenil was ana- 4. Discussion lyzed. Application of bretazenil (1 WM) to the receptors containing the wild-type K1-, K2-, K3- and K5-subunits character- The BZ binding site is considered to be located at the inter- istically potentiated the GABA-evoked response, but to a face of the K- and the Q2-subunit of the GABAA receptor lesser degree than the full agonist diazepam applied at the (reviewed in [23]). Here we demonstrate that the histidine same concentration (K1L2Q2, potentiation: 12 þ 1%; K2L3Q2, residue in the BZ site, originally identi¢ed in the K1-subunit potentiation: 28 þ 6%; K3L3Q2, potentiation: 72 þ 14%; and as a photoa¤nity target [24,25], is a functional characteristic K5L2Q2, potentiation: 37 þ 4%) (Figs. 3 and 4). However, ap- common to all major BZ-sensitive GABAA-receptors. Muta- plication of bretazenil (1 WM) to the receptors containing the tion of this residue to arginine in either the K1-, K2-, K3- or point-mutated K-subunits resulted in a potentiation of the the K5-subunit leads to a complete loss of potentiation of GABA-evoked response that was 2- to 9-fold higher than GABA-induced currents by diazepam when co-expressed that observed for receptors incorporating wild-type K-subunits with L2- or L3- and Q2-subunits, in accordance with the dia- (K1H101RL2Q2, potentiation: 107 þ 16%; K2H101RL3Q2, potentia- zepam-insensitivity of the K4L2Q2 and K6L2Q2 wild-type recep- tion: 93 þ 15%; K3H126RL3Q2, potentiation: 148 þ 29%, and tors which contain an arginine residue in the corresponding K5H105R, potentiation: 239 þ 8%) (Figs. 3 and 4). Bretazenil position [19,26^28]. Since diazepam does not bind to either thus showed an increased potentiating e¡ect when tested on the K4- and K6-receptors [26,27,29] or the K1H101RL2Q2-recep- the diazepam-insensitive receptors compared to wild-type re- tors [6], the lack of diazepam-responsiveness of the point-mu- ceptors. Its potency was about equal to (K2H101RL3Q2, tated K2-, K3- and K5-receptors can most likely be attributed K3H126RL3Q2) or signi¢cantly higher (K1H101RL2Q2, to a lack of a¤nity for diazepam. K5H105RL2Q2) than that of diazepam (1 WM) at the correspond- In contrast to diazepam, the potentiation of GABA-evoked ing wild-type receptors. Thus, the point mutation which ren- responses of the point-mutated receptors by bretazenil is uni- ders the K1-, K2-, K3- and K5-receptors diazepam-insensitive formly increased compared to the respective wild-type recep- does not abolish the e¡ectiveness of bretazenil at any of the tors. This is unlikely to be due to an increase in the a¤nity of receptors. bretazenil, since bretazenil displays a nanomolar a¤nity to the The partial inverse agonist Ro 15-4513 is known to reduce diazepam-insensitive K6L3Q2-receptor (Ki = 12.7 nM) which is the amplitude of the GABA-evoked responses at K1L2Q2, lower than its a¤nity to the diazepam-sensitive K1L3Q2-recep- K1L1Q2, K2L1Q2, K5L3Q2 and K5L3Q3 receptors [19^22]. Here, tor (Ki = 0.35 nM) [27]. Thus, the histidine to arginine point we show that the inverse agonism of Ro 15-4513 extends to mutation does not appear to increase the a¤nity but rather to the K2L3Q2-, K3L3Q2-, and K5L2Q2-receptors (Figs. 3 and 4). a¡ect the signal transduction process in the interaction be- However, on receptors containing the point-mutated K1-, K2-, tween bretazenil and the mutant receptors. These di¡erences K3- and K5-subunits, Ro 15-4513 (1 WM) was found to in the interaction of diazepam and bretazenil appear to be strongly potentiate the GABA-evoked currents. The percent common to all receptor subtypes tested and point to distinct

FEBS 20566 24-7-98 J.A. Benson et al./FEBS Letters 431 (1998) 400^404 403

Fig. 4. Single traces of the e¡ects of diazepam (DIAZ), bretazenil (BRET) and Ro 15-4513 on the GABA-evoked currents are exempli¢ed for H101R wild-type K2L3Q2 and point-mutated K2 L3Q2 GABAA-receptors. In K2L3Q2-receptors the agonist diazepam (1 Wm) and to a lesser degree the partial agonist bretazenil (1 Wm) potentiated the current, while the inverse agonist Ro 15-4513 (1 Wm) reduced the current. In K2H101RL3Q2 receptors, diazepam was without e¡ect and the current was strongly potentiated by bretazenil and Ro 15-4513. domains for interaction of diazepam and bretazenil at the BZ In summary, the mutation of a single histidine to arginine binding site. in the K1- K2-, K3- and K5-subunits induces a conformational The inverse agonist Ro 15-4513 interacts in yet another change with distinct pharmacological consequences common manner with the benzodiazepine binding site of GABAA-re- to all GABAA-receptors tested. The results underline the no- ceptors. In the point-mutated receptors, its e¤cacy is switched tion that the GABAA-receptor subtypes contain common and from inverse agonism to agonism. This is in line with the highly conserved structural determinants in£uencing the a¤n- agonistic action of Ro 15-4513 on the diazepam-insensitive ity and e¤cacy of BZ site ligands. K4- or K6-subunit-containing receptors K4L1Q2, K4L2Q2, Our ¢ndings open the possibility to de¢ne the pharmaco- K6L1Q2 and K6L2Q2 [19,26^28] and the retention of its high logical signi¢cance of GABAA-receptor subtypes in vivo. By a¤nity to the diazepam-insensitive K6-receptors (KD=10 nM) individually mutating the conserved histidine residue in the [6]. Ro 15-4513 has previously been shown to act as a partial K1-, K2-, K3- and K5-subunits to arginine by gene targeting, agonist at K1L1Q2-receptors containing a threonine to serine individual GABAA-receptor subtypes will be rendered insen- substitution in position 142 of the Q2-subunit [21]. Therefore, sitive to diazepam. The resulting mouse lines are expected to critical residues in both the K-subunits and the Q2-subunit display characteristic de¢cits in the pharmacological spectrum determine the e¤cacy of Ro 15-4513. of diazepam. The present results support the validity of this in

FEBS 20566 24-7-98 404 J.A. Benson et al./FEBS Letters 431 (1998) 400^404

[3] Hadingham, K.L., Wingrove, P.B., Wa¡ord, K.A., Bain, C., Kemp, J.A., Palmer, K.J., Wilson, A.W., Wilcox, A.S., Sikela, J.M., Ragan, C.I. and Whiting, P.J. (1993) Mol. Pharmacol. 44, 1211^1218. [4] Puia, G., Ducic, I., Vicini, S. and Costa, E. (1992) Proc. Natl. Acad. Sci. USA 89, 3620^3624. [5] Fritschy, J.-M. and Mohler, H. (1995) J. Comp. Neurol. 359, 154^194. [6] Wieland, H.A., Luëddens, H. and Seeburg, P.H. (1992) J. Biol. Chem. 287, 1426^1429. [7] Kleingoor, C., Wieland, H.A., Korpi, E.R., Seeburg, P.H. and Kettenmann, H. (1993) NeuroReport 4, 187^190. [8] Bertocci, B., Miggiano, V., Da Prada, M., Dembic, Z., Lahm, H.W. and Malherbe, P. (1991) Proc. Natl. Acad. Sci. USA 88, 1416^1420. [9] Sigel, E., Baur, R., Trube, G., Mohler, H. and Malherbe, P. (1990) Neuron 5, 703^711. [10] Chen, C. and Okayama, H. (1987) Mol. Cell. Biol. 7, 2745^2752. [11] Kno£ach, F., Drescher, U., Scheurer, L., Malherbe, P. and Moh- ler, H. (1993) J. Pharmacol. Exp. Ther. 266, 385^391. Fig. 5. Dose-response curve for the e¡ect of Ro 15-4513 on the re- [12] Fritschy, J.-M., Benke, D., Mertens, S., Oertel, W.H., Baëchi, T. combinant K2H101RL3Q2 receptor-mediated current evoked by 30 WM and Mohler, H. (1992) Proc. Natl. Acad. Sci. USA 89, 6726^ GABA. The mean amplitudes of the potentiated currents relative to 6730. the control currents were normalized to a control value set at 1.0, [13] Benke, D., Fritschy, J.-M., Trzeciak, A., Bannwarth, W. and and plotted as a function of the drug concentration. The data Mohler, H. (1994) J. Biol. Chem. 269, 27100^27107. points and error bars represent the means and standard errors for [14] McKernan, R.M. and Whiting, P.J. (1996) Trends Neurosci. 19, di¡erent drug concentrations (n = 3^4). 139^143. [15] Ducic, I., Caruncho, H.J., Zhu, W.J., Vicini, S. and Costa, E. (1995) J. Pharmacol. Exp. Ther. 272, 438^445. [16] Nagata, K., Hamilton, B.J., Carter, D.B. and Narahashi, T. vivo approach by verifying three major preconditions. (i) The (1994) Brain Res. 645, 19^26. point mutation a¡ects the diazepam response at the corre- [17] Ebert, B., Wa¡ord, K.A., Whiting, P.J., Krogsgaard-Larsen, P. sponding four GABAA-receptor subtypes in the same manner. and Kemp, J. (1994) Mol. Pharmacol. 46, 957^963. (ii) The mutation does not appreciably a¡ect the potency of [18] White, G., Gurley, D., Hartnett, C., Stirling, V. and Gregory, J. GABA in activating the four receptor subtypes, suggesting (1995) Receptors Channels 3, 1^5. [19] Whittemore, E.R., Yang, W., Drewe, J.A. and Woodward, R.M. that the physiological responsiveness of the receptors to (1996) Mol. Pharmacol. 50, 1364^1375. GABA is not altered. (iii) The mutation does not interfere [20] Wa¡ord, K.A., Bain, C.J., Whiting, P.J. and Kemp, J.A. (1993) with subunit synthesis and assembly, which is in keeping Mol. Pharmacol. 44, 437^442. with the expression of diazepam-insensitive K4- and K6-recep- [21] Mihic, S.J., Whiting, P.J., Klein, R.L., Wa¡ord, K.A. and Har- ris, R.A. (1994) J. Biol. Chem. 269, 32768^32773. tors in the brain. The point-mutated mouse lines will provide [22] Hadingham, K.L., Wa¡ord, K.A., Thompson, S.A., Palmer, K.J. important insights into the neuronal circuits mediating selec- and Whiting, P.J. (1995) Eur. J. Pharmacol. 291, 301^309. tive actions of diazepam-induced behavior. [23] Sigel, E. and Buhr, A. (1997) Trends Pharmacol. Sci. 18, 425^ 429. Acknowledgements: We thank Drs. Hartmut Luëddens (Mainz, Ger- [24] Mohler, H., Battersby, M.K. and Richards, J.G. (1980) Proc. many) and Pari Malherbe (Basel, Switzerland) for the gift of Natl. Acad. Sci. USA 77, 1666^1670. [25] Duncalfe, L.L., Carpenter, M.R., Smilie, L.B., Martin, I.L. and GABAA-receptor K-subunit cDNAs. This work was supported by Grants 3100-049754.96/1 and 31-47050.96 from the Swiss National Dunn, S.M.J. (1996) J. Biol. Chem. 271, 9209^9214. Science Foundation. [26] Kno£ach, F., Benke, D., Wang, Y., Scheurer, L., Luëddens, H., Hamilton, B.J., Carter, D.B., Mohler, H. and Benson, J.A. (1996) Mol. Pharmacol. 50, 1253^1261. References [27] Hadingham, K.L., Garrett, E.M., Wa¡ord, K.A., Bain, C., Heavens, R.P., Srinathsinghji, D.J.S. and Whiting, P.J. (1996) [1] Mohler, H., Fritschy, J.-M., Benke, D., Benson, J., Rudolph, U. Mol. Pharmacol. 49, 253^259. and Luëscher, B. (1996) in: GABA: Receptors, Transporters and [28] Wa¡ord, K.A., Thompson, S.A., Thomas, D., Sikela, J., Wilcox, Metabolism (Tanaka, C. and Bowery, N.G., Eds.), pp. 157^171, A.S. and Whiting, P.J. (1996) Mol. Pharmacol. 50, 670^678. Birkhaëuser Verlag, Basel. [29] Benke, D., Michel, C. and Mohler, H. (1997) J. Neurochem. 69, [2] Puia, G., Vicini, S., Seeburg, P.H. and Costa, E. (1991) Mol. 806^814. Pharmacol. 39, 691^696.

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