GABA-A RECEPTOR SUBUNITS ( alpha -2, alpha -3, alpha -5, alpha -6, beta -2) AND TRANSFECTED CELLS EXPRESSING THEM

Abstract: The present invention relates to a stably co-transfected eukaryotic cell line capable of expressing a GABAA receptor, particularly a human GABAA receptor, which receptor comprises at least one alpha, one beta and one gamma subunit; to the cloning of novel cDNA sequences encoding the alpha 2, alpha 3, alpha 5, alpha 6 and beta 2 subunits of the human GABAA receptor; and to the use of the cell line in designing and developing GABAA receptor subtype-selective medicaments.

Description: GABA-A RECEPTOR SUBUNITS (-*-2 _,-3,<__-5p_.-6,@-2) AND TRANSFECTED CELLS EXPRESSIN THEM This invention concerns a cell line, and in particular relates to a stable cell line capable of expressing human or animal GABA^. receptors. The invention further concerns the cloning of novel cDNA sequences encoding particular subunits of the human GABA^ receptor. In addition, the invention relates to the use of the cell line in a screening technique for the design and development of subtype-specific medicaments. Gamma-amino butyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system. It mediates fast synaptic inhibition by opening the chloride channel intrinsic to the GABA^ receptor. This receptor comprises a multimeric protein of molecular size 230-270 kDa with specific binding sites for a variety of drugs including benzodiazepines, barbiturates 0 and /3-carbolines, in addition to sites for the agonist ligand GABA (for reviews see Stephenson, Biochem. J.. 1988, 249 / 21; Olsen and Tobin, Faseb J.. 1990, A. 1469; and Sieghart, Trends in Pharmacol. Sci.. 1989, 10, 407). Molecular biological studies demonstrate that 5 the receptor is composed of several distinct types of subunit, which are divided into four classes (α, β , j , and S) based on their sequence similarities. To date, six types of α (Schofield et al. , Nature (London) . 1987, 328. 221; Levitan et al. , Nature (London) . 1988, 335. 76; 0 Y er et al.. EMBO J.. 1989, 8., 1665; Pritchett & Seeberg, J. Neurochem.. 1990, 54., 802; Luddens et al.. Nature (London.. 1990, 346. 648; and Khrestchatisky et al.. Neuron. 1989, 3., 745), three types of β (Ymer et al.. EMBO J.. 1989, 8., 1665), two types of % (Ymer et al. , EMBO J.. 1990, 9., 3261; and Shivers et al.. Neuron. 1989, 3., 327) and one δ subunit (Shivers et al.. Neuron. 1989, 3., 327) have been identified. The differential distribution of many of the subunits has been characterised by in situ hybridisation (Sequier et al.. Proc. Natl. Acad. Sci. USA. 1988, 85., 7815; Malherbe et_al. , J. Neurosci.. 1990, 10, 2330; and Shivers et al. , Neuron. 1989, 3., 327) and this has permitted it to be speculated which subunits, by their co-localisation, could theoretically exist in the same receptor complex. Various combinations of subunits have been co- transfected into cells to identify synthetic combinations of subunits whose pharmacology parallels that of bona fide G B ^ receptors in vivo (Pritchett et al. , Science. 1989, 245. 1389; Malherbe et al.. J. Neurosci.. 1990, 10, 2330; Pritchett and Seeberg, J. Neurochem.. 1990, 54, 1802; and Luddens et al.. Nature (London) . 1990, 346. 648) . This approach has revealed that, in addition to an a and β subunit, either i or _^2 (Pritchett et al.. Nature (London) . 1989, 338. 582; Ymer et al.. EMBO J.. 1990, 9., 3261; and Malherbe et al.. J. Neurosci.. 1990, 10. 2330) or ^ 3 (Herb et aJL. - Proc. Natl. Acad. Sci. USA. 1992, 89., 1433; Knoflach et al. , FEBS Lett.. 1991, 293. 191; and Wilson-Shaw et al. , FEBS Lett.. 1991, 284. 211) is also generally required to confer benzodiazepine sensitivity, and that the benzodiazepine pharmacology of the expressed receptor is largely dependent on the identity of the and subunits present. Receptors containing a δ subunit (i.e. aβδ) do not appear to bind benzodiazepines (Shivers et al. , Neuron. 1989, 3., 327). Combinations of subunits have been identified which exhibit the pharmacological profile of a BZ-t_ type receptor (α13-1 2) and a BZ2 type receptor or o * 3 ,-' l _ / 2 . Pritchett et al.. Nature (London) . 1989, 338. 582) , as well as two GABA^ receptors with a novel pharmacology, 0.582 2 (Pritchett and Seeberg, J. Neurochem.. 1990, 54.. 1802) and ας (Luddens et al.. Nature (London) . 1990, 346- 648) . Although the pharmacology of these expressed receptors appears similar to that of those identified in brain tissue by radioligand binding, it has nonetheless not been shown that these receptor subunit combinations exist in vivo. The present invention is concerned with the production of permanently transfected cells containing the GABAA receptor, which will be useful for screening for drugs which act on this receptor. The GABA^ receptor has previously been expressed in Xenopus oocytes (Sigel et al.. Neuron. 1990, 5 , 703-711) and in transiently transfected mammalian cells (Pritchett et al. , Science. 1989, 245. 1389-1392). However, both of those systems involve transient expression and are unsuitable for screening purposes. We have now achieved the stable expression of the receptor. Accordingly, the present invention provides a stably co-transfected eukaryotic cell line capable of expressing a G BA^ receptor, which receptor comprises at least one alpha, one beta and one gamma subunit. This has been achieved by co-transfeeting cells with three expression vectors, each harbouring cDNAs encoding for an α, β or GABA^ receptor subunit. In a further aspect, therefore, the present invention provides a process for the preparation of a eukaryotic cell line capable of expressing a GABA^ receptor, which comprises stably co-transfeeting a eukaryotic host cell with at least three expression vectors, one such vector harbouring the cDNA sequence encoding for an alpha, another such vector harbouring the cDNA sequence encoding for a beta, and a third such vector harbouring the cDNA sequence encoding for a gamma G B ^ receptor subunit. The stable cell-line which is established expresses an aβ)f GABA^ receptor. Each receptor thereby expressed, comprising a unique combination of a, β and )f subunits, will be referred to hereinafter as a GABA^ receptor "subunit combination". Pharmacological and electrophysiological data confirm that the recombinant aβJA receptor expressed by the cells of the present invention has the properties expected of a native GABA^ receptor. Expression of the GABA^ receptor may be accomplished by a variety of different promoter- expression systems in a variety of different host cells. The eukaryotic host cells suitably include yeast, insect and mammalian cells. Preferably the eukaryotic cells which can provide the host for the expression of the receptor are mammalian cells. Suitable host cells include rodent fibroblast lines, for example mouse Ltk " , Chinese hamster ovary (CHO) and baby hamster kidney (BHK) ; HeLa; and HEK293 cells. It is necessary to incorporate at least one α, one β and one / subunit into the cell line in order to produce the required receptor. Within this limitation, the choice of receptor subunit combination is made according to the type of activity or selectivity which is being screened for. For example, benzodiazepines (designated BZ) represent one class of drugs which act upon the GABA^. receptor. The presence of an c-i subunit is specific for a class of benzodiazepines having the pharmacology designated BZ ; whereas c-2 to c.5 define different pharmacological profiles , broadly designated as BZ2. The type of β subunit is not critical in defining the class of benzodiazepine, although a β subunit is required. The % subunit is also important in defining BZ selectivity. It is likely that differentiation between subunit selectivity is conferred by the identity of the particular }f subunit present. In order to employ this invention most effectively for screening purposes, it is preferable to build up a library of cell lines, each with a different combination of subunits. Typically a library of 5 or 6 cell line types is convenient for this purpose. Preferred subunit combinations include: ' a lβ2 2 >' <-2/*l<h. - c. 2 /3-J " 2 " > -.501 2 ? and a β β lc y 2 >' especially c-iSi^L- The DNAs for the receptor subunits can be obtained from known sources, and are generally obtained as specific nucleotide sequences harboured by a standard cloning vector such as those described, for example, by Maniatis et al. in Molecular Cloninσ. A Laboratory Manual. Cold Spring Harbor Press, New York, 2nd edition, 1989. Preferably the cDNA sequences are derived from the human gene. However, for screening purposes, cDNAs from other species are also suitable, such as bovine or rat DNA. Known sources of GABA^ receptor subunit cDNAs are as follows: Schofield et al.. Nature. 1987, 328. 221-227. Schofield et al.. FEBS Lett.. 1989, 244. 361-364. c_2 rat Khrestchatisky et al. , J. Neurochem. , 1991, 56, 1717. - 6 - -2 bovine ) Levitan et_al. , Nature, 1988 , 3_35 , c_3 bovine ) 76-79. 0:4 rat Wisden et al. - FEBS Lett.. 1991, 289., 227. cr.4 bovine Ymer et al.. FEBS Lett.. 1989, 258. 119-122. a 5 rat Pritchett and Seeburg, J . N eurochem. . 1990 , 5 , 1802-1804. e g rat Luddens et al.. Nature. 1990, 346. ot bovine 648-651. 02 bovine Ymer et al.. EMBO J.. 1989, 8., 1665-1670 , β 2 rat 03 bovine 03 rat fa human Ymer et al.. EMBO J.. 1990, 9_, 3261-3267 /l bovine 2 human Pritchett et al.. Nature, 1989, 338, 582-585. % bovine Whiting et al. , Proc. Natl. Acad. Sci. USA. 1990, 52, 9966-9970. 3 rat Herb et al. , Proc. Natl. Acad. Sci. USA. 1992, 89., 1433; and Knoflach et al. , FEBS Lett.. 1991, 293. 191. 3 mouse Wilson-Shaw et al. , FEBS Lett.. 1991, 284. 211. δ rat Shivers et al. , Neuron. 1989, 3. / 327. Certain cDNA sequences encoding various subunits of the human GABAA receptor have hitherto been unavailable. These include in particular the sequences encoding the 0 * 2r α 3 r 5 > α 6 and 02 subunits, which nucleotide sequences are accordingly novel. We have now ascertained the cDNA sequences of the 0-2, 0*3, 0.5, α- and 02 subunits of the human GABAA receptor. These nucleotide sequences, together with the deduced amino acid sequences corresponding thereto, are depicted in Figures 2 to 6 of the accompanying drawings. The present invention accordingly provides in several additional aspects DNA molecules encoding the a , 03, 05, αtg and 02 subunits of the human GABAA receptor comprising all or a portion of the sequences depicted in Figures 2, 3, 4, 5 and 6 respectively, or substantially similar sequences. The sequencing of the novel cDNA molecules in accordance with the invention can conveniently be carried out by the standard procedure described in accompanying Example 3; or may be accomplished by alternative molecular cloning techniques which are well known in the art, such as those described by Maniatis et al. in Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Press, New York, 2nd edition, 1989. In another aspect, the invention provides a recombinant expression vector coaprising the nucleotide sequence of a GABAA receptor subunit together with additional sequences capable of directing the synthesis of the said GABAA receptor subunit in cultures of stably co-transfected eukaryotic cells. The term "expression vectors" as used herein refers to DNA sequences that are required for the transcription of cloned copies of recombinant DNA sequences or genes and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic genes in a variety of hosts such as bacteria, blue-green algae, yeast cells, insect cells, plant cells and animal cells. Specifically designed vectors allow the shuttling of DNA between bacteria-yeast, bacteria- plant or bacteria-animal cells. An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selective markers, a limited number of useful restriction enzyme sites, a high copy number, and strong promoters. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and to initiate RNA synthesis. A strong promoter is one which causes mRNAs to be initiated at high frequency. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses. The term "cloning vector" as used herein refers to a DNA molecule, usually a small plasmid or bacteriophage DNA capable of self-replication in a host organism, and used to introduce a fragment of foreign DNA into a host cell. The foreign DNA combined with the vector DNA constitutes a recombinant DNA molecule which is derived from recombinant technology. Cloning vectors may include plasmids, bacteriophages, viruses and cosmids. The recombinant expression vector in accordance with the invention may be prepared by inserting the nucleotide sequence of the chosen G B subunit into a suitable precursor expression vector (hereinafter referred to as the "precursor vector") using conventional recombinant DNA methodology known from the art. The precursor vector may be obtained commercially, or constructed by standard techniques from known expression vectors. The precursor vector suitably contains a selection marker, typically an antibiotic resistance gene, such as the neomycin or a picillin resistance gene. The precursor vector preferably contains a neomycin resistance gene, adjacent the SV40 early splicing and polyadenylation region; an ampicillin resistance gene; and an origin of replication, e.g. pBR322 ori. The vector also preferably contains an inducible promoter, such as MMTV-LTR (inducible with dexamethasone) or metallothionin (inducible with zinc) , so that transcription can be controlled in the cell line of this invention. This reduces or avoids any problem of toxicity in the cells because of the chloride channel intrinsic to the GABAA receptor. One suitable precursor vector is pMAMneo, available from Clontech Laboratories Inc. (Lee et al.. Nature. 1981, 294. 228; and Sardet et al. , Cell. 1989, 56. 271) . Alternatively the precursor vector pMSGneo can be constructed from the vectors pMSG and pSV2neo as described in Example 1 herein. The recombinant expression vector of the present invention is then produced by cloning the GABAA receptor subunit cDNA into the above precursor vector. The required receptor subunit cDNA is subcloned from the vector in which it is harboured, and ligated into a restriction enzyme site, e.g. the Hindlll site, in the polylinker of the precursor vector, for example pMAMneo or pMSGneo, by standard cloning methodology known from the art, and in particular by techniques analogous to those described in Example 1, step (b) herein. Before this subcloning, it is often advantageous, in order to improve expression, to modify the end of a subunit cDNA with additional 5 « untranslated sequences, for example by modifying the 5' end of the / L subunit DNA by addition of 5' untranslated region sequences from the oχ subunit DNA. One suitable expression vector of the present invention is illustrated in Fig. 1 of the accompanying drawings, in which R represents the nucleotide sequence of a given alpha, beta or gamma subunit of the GABAA receptor, and the remainder of the expression vector depicted therein is derived from the precursor vector pMSGneo and constructed as described in accompanying Example 1, steps (a) and (b) . For each cell line of the present invention, three such vectors will be necessary, one containing an α subunit, one containing a 0 subunit, and the third containing a <Y subunit. Cells are then co-transfected with the desired combination of three expression vectors. There are several commonly used techniques for transfeetion of eukaryotic cells in vitro. Calcium phosphate precipitation of DNA is most commonly used (Bachetti et al., Proc. Natl. Acad. Sci. USA. 1977, 74., 1590-1594; Maitland et al.. Cell. 1977, 14., 133-141), and represents a favoured technique in the context of the present invention. A small percentage of the host cells takes up the recombinant DNA. In a small percentage of those, the DNA will integrate into the host cell chromosome. Because the neomycin resistance gene will have been incorporated into these host cells, they can be selected by isolating the individual clones which will grow in the presence of neomycin. Each such clone is then tested to identify those which will produce the receptor. This is achieved by inducing the production, for example with dexamethasone, and then detecting the presence of receptor by means of radioligand binding.. In a further aspect, the present invention provides protein preparations of GABAA receptor subunit combinations, especially human GABAA receptor subunit combinations, derived from cultures of stably transfected eukaryotic cells. The invention also provides preparations of membranes containing subunit combinations of the GABAA receptor, especially human GABAA receptor subunit combinations, derived from cultures of stably transfected eukaryotic cells. In particular, the protein preparation and membrane preparations according to the invention will suitably contain the αsiøi subunit combinations of the human G B A receptor, and will preferably contain a human GABAA receptor consisting of the _ι01 2L subunit combinations. In an especially preferred embodiment, the invention provides cell membranes containing a human GABAA receptor consisting of the α * ι0i/j2L subunit combinations isolated from stably transfected mouse Ltk~ fibroblast cells. The cell line, and the membrane preparations therefrom, according to the present invention have utility in screening and design of drugs which act upon the GABAA receptor, for example benzodiazepines, barbiturates, 0-carbolines and neurosteroids. The present invention accordingly provides the use of the cell line described above, and membrane preparations derived therefrom, in screening for and designing medicaments which act upon the GABAA receptor. Of particular interest in this context are molecules capable of interacting selectively with GABAA receptors made up of varying subunit combinations. As will be readily apparent, the cell line in accordance with the present invention, and the membrane preparations derived therefrom, provide ideal systems for the study of structure, pharmacology and function of the various GABAA receptor subtypes. The following non-limiting Examples illustrate the present invention. EXAMPLE 1 PREPARATION OF α-j8_- 2L TRANSFECTED CELLS a) Construction of eukaryotic expression vector pMSGneo The approx. 2500 base pair Hindlll-EcoRI fragment of the vector pMSG (purchased from Pharmacia Biosystems Limited, Milton Keynes, United Kingdom) , containing the gpt structural gene and SV40 polyadenylation signals was replaced by the approx. 2800 base pair Hindlll-EcoRI fragment of pSV2neo (Southern, P. . and Berg, P.J., Molecular and Applied Genetics. 1, 327-341, 1982) containing the neomycin resistance gene Neo r and SV40 polyadenylation signals. The EcoRI and Hindlll sites were then removed by restriction digesting, blunt ending with klenow polymerase, and religating. EcoRI and Hindlll cloning sites were then inserted at the Xhol and Smal sites of the polylinker by conventional techniques using EcoRI and Hindlll linkers. b) Cloning of subunit cDNAs into pMSGneo Bovine α and χ GABAA receptor cDNAs were obtained from the Molecular Neurobiology Unit, MRC Centre, Hills Road, Cainbridge (Scholfield, P. et al. Natur . 328. 221-227, 1987). Bovine 2 cDNA was cloned by the method of Whiting, P. et al. (Proc. Natl. Acad. Sci. USA. 87. 9966-9970, 1990). Bovine c-i was subcloned from pbGRαsense by digestion with EcoRI, blunt ending the DNA with klenow polymerase, addition of Hindlll linkers by ligation, digestion with Hindlll and ligation into the Hindlll site of pMSGneo. Bovine 0χ was subcloned from pbGR sense by restriction digestion with EcoRI (partial digestion) , klenow polymerase blunt ending, ligation of Hindlll linkers, restriction digestion with Hindlll and ligation into Hindlll site of pMSGneo. Before subcloning into pMSGneo, the bovine X cDNA was modified from the published sequence as follows. The 5 1 untranslated region of the bovine α cDNA (bases 60-200 of the published sequence) was added to the 5' end of the published % 2 sequence by amplifying the αi untranslated region using polymerase chain reaction, and then subcloning the product into the 5" BamHI (site in the polylinker of the Bluescript Sk~ cloning vector; Bluescript vector purchased from Stratagene, San Diego, U.S.A.) Hindlll sites of the 2 cDNA. The modified fo cDNA was then subcloned into pMSGneo by digestion with Xbal (site in the polylinker of the cloning vector) , blunt ending with klenow polymerase, ligation of Xhol linkers, digestion with Xhol (site in the polylinker of the cloning vector) , and ligation into Xhol site of pMSGneo. c) Co-transfection of mouse Ltk *" cells Ltk " cells were obtained from the Salk Institute for Biological Studies, San Diego, California. Cells were grown at 37 β C, 5-8% CO2, in Modified Eagles Medium containing penicillin, streptomycin and 10% fetal calf serum. The expression vector harbouring the GABAA receptor subunit DNAs for co-transfection was prepared by a standard protocol (Chen, C. and Okayama, H. , BioTechnigues. 6 . , 632-638, 1988). For co-transfection, Ltk " cells were plated in dishes (approx. 2xl0 5 cells/dish) and grown overnight. The transfeetion was performed by calcium phosphate precipitation using a kit (purchased from 5 Prime -> 3 Prime Products, Westchester, Pennsylvania) . Co-transfection was performed according to manufacturers' instructions, using 5μg of each subunit DNA construct per 10cm dish of cells. After 2 days in culture the cells were divided 1:8 into culture medium containing lmg/ml neomycin [Geneticin (obtainable from Gibco BRL, Paisley, Scotland, U.K.)]. After a further week the concentration was increased to l.5mg/ml, and then 2mg/ml 1 week after that. Resistant clones of cells were isolated and subcloned using cloning cylinders. Subclones were analysed using radioligand binding: subclones were grown in 10cm culture dishes, and when confluent changed into culture medium containing lμM dexamethasone (obtainable from Sigma Chemical Company, Poole, Dorset, United Kingdom) . 3-5 days later the cells were harvested, membranes prepared and used for radioligand binding (see Example 2, step (a) below) using the benzodiazepine antagonist 3 H Rol5-1788 (obtained from New England Nuclear, Du Pont (U.K.) Ltd, Stevenage, United Kingdom) . The clone expressing the highest amount of 3 H Rol5-1788 binding was subcloned from a single cell by limiting dilution. The resultant clonal population of cells described below is referred to as population A. EXAMPLE 2 CHARACTERIZATION OF TRANSFECTED CELLS a) Radioligand binding The nature of the recombinant αiøi^L GABAA receptors prepared as described in Example 1 was addressed by characterization of the benzodiazepine (BZ) binding pharmacology, using the BZ antagonist 3 H Rol5- 1788. For radioligand binding assays, cells which had been induced by culture in dexamethasone containing medium for 3-5 days were scraped off into 50mM Tris, pH7.5, lOOmM NaCl in the form of Tris buffered saline (TBS) and pelleted (20,000rpm, Sorvall RC5C centrifuge). The cell pellet was resuspended in 50mM Tris, pH7.5, homogenised using an Ultra-Turrax homogeniser and then pelleted as above. This was repeated once more, and the cells then resuspended in TBS (0.4ml per original 10cm dish of cells) . Radioligand binding was performed in 0.1ml final volume TBS, containing 5-15 fmols of 3 H Rol5- 1788 binding sites. After 1 hour incubation on ice the membranes were harvested onto filters using a Brandel cell harvester, washed with cold TBS, and bound radioactivity determined by scintillation counting. The recombinant αiøi^L receptors bound 3 H Rol5-1788 with high affinity (K-, 0.4nM), at levels of up to 200fmols/10cm dish of cells. No binding was seen to either untransfected Ltk " cells, or population A cells which had not been induced by addition of dexamethasone to the culture medium, confirming that the 3 H Rol5-1788 was binding to recombinant GABAA receptors. The 3 H Rol5-1788 binding was inhibited by flunitrazepam, CL218872, FG8205, 0CCM, zolpide and Rol5-4513, confirming the BZ pharmacology of the recombinant receptor. Since it is established that only GABAA receptors containing an α, a 0 and a / subunit exhibit BZ binding (Pritchett, D. et al. , Nature. 338., 582-585, 1989) these data confirm the nature of the recombinant α 10 1 -' 2 GABAA receptors expressed by population A cells. b) E1ectrophvsio1ogy The nature of the GABAA receptor expressed by population A cells has been extensively characterised by electrophysiological techniques, using whole cell patch clamp. Only cells induced by culture in the presence of dexamethasone showed responses to GABA. Concentration response curves to GABA gave a log EC5 0 of 5.2, and a Hill coefficient of 1.9. The response to GABA was potentiated by BZs flunitrazepam and CL218872, by the barbiturate pentobarbitone, and by the steroid alphaxalone. The response to GABA was antagonised by both bicuculline and picrotoxin. All these electrophysiological data confirm that the recombinant GABAA receptor expressed by population A cells has all of the properties expected of a bona fide GABAA receptor. EXAMPLE 3 ISOLATION AND SEQUENCING OF CDNAS ENCODING HUMAN GABA A RECEPTOR α 2 , α 3 , α 5 , αg & 2 SUBUNITS a) cDNA libraries cDNAs were cloned from human foetal brain (α 2 , 0 3 ) , hippocampal (a^ , β 2 ) and cerebellum (αg) lambda bacteriophage cDNA libraries. All cDNA libraries were constructed in the lambdaZAP vector, and were purchased from Stratagene (San Diego, California) . For screening, the cDNA libraries were plated according to the manufacturer's instructions, at 40,000 pfu per 137 mm plate. Filter lifts were taken using Hybond N filters (Amersham) according to the manufacturer's instructions. b) Isolation of cDNA encoding human ct subunit A bovine α2 cDNA (obtained from E. Barnard, Molecular Neurobiology, University of Cambridge, Hills Road, Cambridge; Levitan et al. , Nature. 1988, 335. 76) was labelled to high specific activity (>1.10 9 cpm/μg) with 3 P by random priming and used as a probe. Library filters (8 replica filters) were prehybridised for 3-6 hours at 42"C in 5x SSPE (lx SSPE is 0.18M NaCl, 0.01M Na 3 P0 4 [pH7.4], lmM EDTA), 5x Denhardt's solution, 100 μg/ml salmon sperm DNA, 0.1% sodium dodecyl sulphate (SDS) , 30% formamide. Hybridisation was performed in the same buffer for 18 hours at 42"C, including 0.5-1.10 6 σpm 32 P-labelled probe per ml of hybridisation buffer. Filters were washed at 55 " C in 5x SSPE (2x 15 minutes) and lx SSPE (2x 15 minutes) and exposed to Kodak XAR film for 1-3 days. Positive clones were plaque purified using standard techniques, and the Bluescript plasmid (Stratagene) "rescued" according to manufacturer's instructions. cDNA clones were sequenced on both strands by standard techniques using Sequenase II enzyme (United States Biochemicals) . The nucleotide sequence of the cDNA encoding the human GABAA receptor α2 subunit, together with the deduced amino acid sequence corresponding thereto, is shown in Fig. 2 of the accompanying drawings. c) Isolation of cDNA encoding human α subunit A bovine α 3 cDNA (obtained from E. Barnard, Molecular Neurobiology, University of Cambridge, Hills Road, Cambridge; Levitan et al. , Nature. 1988, 335. 76) was labelled to high specific activity with 32 P by random priming and used as a probe. Library filters were prehybridised for 3-6 hours at 55 " C in 5x SSPE, 5x Denhardt's solution, 0.1% SDS, 100 μg/ml salmon sperm DNA, and hybridised for 18 hours, 55"C in the same buffer, containing 0.5-lx 10 6 cpm/ml of 32 P-labelled bovine α 3 cDNA as probe. Filters were washed and exposed to X-ray film as described above; cDNA clones were rescued and sequenced as described above. The longest a^ cDNA clone was missing in approximately 100 bp of the 5' end of the coding region. This was obtained by PCR using as primers an oligonucleotide "anchor" primer derived from the T7 primer sequence of Bluescript vector (5ΑGCGCGCGTAATACGACTCACTATAGGGCGAA3') and an oligonucleotide derived from sequence near the 5' end of the truncated 0 3 cDNA, containing an internal Hpal site (5'CAGCATGAATTGTTAACCTCATTGTA3') . Oligonucleotides were synthesised on an Applied Biosystems 380B synthesiser. PCR was performed as described above, and a 300bp PCR product obtained which was double digested with Hpal and Kpnl and subcloned into the similarly cut truncated 0 3 cDNA to yield a full length human 0 3 cDNA. The cDNA was sequenced on both strands as described above. The nucleotide sequence of the cDNA encoding the human GABAA receptor 0 3 subunit, together with the deduced amino acid sequence corresponding thereto, is shown in Fig. 3 of the accompanying drawings. d) Isolation of cDNA encoding human 05 subunit A rat 05 cDNA obtained by polymerase chain reaction (PCR) was used as a probe to screen the cDNA library. For PCR, sequences of the oligonucleotide primers were taken from the published aς sequences (Khrestchatisky et al. , Neuron. 1989, 2, 745) and incorporated a Hind III site for subcloning purposes: 5' ATTATTCAAGCTTGCCATGGACAATGGAATGCTC3' (bpll4-148) ; 5'GGTTTCCAGCTTACTTTGGAGAGGTAGC3 ' (bpl507-1535) . PCR and subcloning of the PCR product into Bluescript SK-vector (Stratagene) for analysis was performed as described elsewhere (Whiting et al. , Proc. Natl. Acad. Sci. USA. 1990, 8_7, 9966) except that rat brain cDNA was used as template. The rat 05 cDNA was labelled with 32 P and used to screen the human hippocampal cDNA library, and positive ctζ clones rescued and sequenced as described for α2 above. The nucleotide sequence of the cDNA encoding the human GABAA receptor 05 subunit, together with the deduced amino acid sequence corresponding thereto, is shown in Fig. 4 of the accompanying drawings. e) Isolation of cDNA encoding human α- subunit A rat αg cDNA obtained by PCR was used as a probe to screen the cDNA library. PCR was performed as described above for 05, using oligonucleotide primers derived from the published rat αg sequence (Luddens et al.. Nature. 1990, 346. 648) incorporating an EcoRI site for subcloning purposes: 5•GAGGAAGAATTCAGGAGGGTGACC 3 ' (bp48-72) ; 5•GAAAATAACGAATTCCAGTGTCCAGCTTT3 • (bpl376- 1404) . The rat αg cDNA clone isolated by PCR was labelled with * ". ** " ~* *P and used to screen a human cerebellum cDNA library, as described above for 2 . Positive α clones were purified, rescued and sequenced as described above. None of the cDNAs contained a complete coding region. To obtain a full length cDNA 3 clones were joined together using convenient restriction sites. The nucleotide sequence of the cDNA encoding the human GABAA receptor αg subunit, together with the deduced amino acid sequence corresponding thereto, is shown in Fig. 5 of the accompanying drawings. f) Isolation of cDNA encoding human 02 subunit Human 02 cDNA was isolated using as a probe a short human 02 cDNA obtained by PCR. PCR was performed as described above (except that the human cerebellum cDNA library was used as template) , using oligonucleotide primers derived from the published rat 02 sequence (Ymer et al. , EMBO J.. 1989, 8., 1665), incorporating EcoRI sites for subcloning purposes: 5' CAAAAGAATTCAGCTGAGAAAGCTGCTAATGC3' (bpl088-1119) ; 5' TCAGGCGAATTCTCTTTTGTGCCACATGTCGTTC3 , (bpl331-1364) . The human 2 clone obtained by PCR was radiolabelled wi •th ** _ * -?"P and used to screen a human hippocampal cDNA library, as described above for α2« The largest cDNA clone obtained lacked the 5' 500bp of the coding region of the 2 subunit. This was obtained by PCR using as primers an oligonucleotide "anchor" primer derived from the T7 primer sequence of the Bluescript vector (5' AGCGCGCGTAATACGACTCACTATAGGGCGAA3 " ) , and an oligonucleotide derived from sequence near the 5* end of the truncated 02 cDNA, containing a Kpnl site (5 1 CATCCAGTGGGTACCTCCTTAGGT3 « ) . PCR was performed as described above, and a 700bp PCR product obtained which was digested with kpnl and subcloned into the truncated cDNA clone (also Kpnl digested) to yield a full length human 02 cDNA. The nucleotide sequence of the cDNA encoding the human GABAA receptor 02 subunit, together with the deduced amino acid sequence corresponding thereto, is shown in Fig. 6 of the accompanying drawings.

Claims: CLAIMS : 1. A stably co-transfected eukaryotic cell line capable of expressing a GABA receptor, which receptor comprises at least one alpha, at least one beta and at least one gamma subunit. 2. A cell line as claimed in claim 1 wherein the receptor expressed is a human GABAA receptor. 3. A process for the preparation of a eukaryotic cell line capable of expressing a GABAA receptor, which comprises stably co-transfecting a eukaryotic host cell with at least three expression vectors, one such vector harbouring the cDNA sequence encoding for an alpha, another such vector harbouring the cDNA sequence for a beta, and a third such vector harbouring the cDNA sequence encoding for a gamma GABAA receptor subunit. 4. A process as claimed in claim 3 wherein the eukaryotic host cell employed is a rodent fibroblast cell. 5. A DNA molecule encoding the α2 subunit of the human GABA receptor comprising all or a portion of the sequence depicted in Figure 2 herein, or a substantially similar sequence. 6. A DNA molecule encoding the 0 3 subunit of the human GABAA receptor comprising all of a portion of the sequence depicted in Figure 3 herein, or a substantially similar sequence. 7. A DNA molecule encoding the ctζ subunit of the human GABAA receptor comprising all or a portion of the sequence depicted in Figure 4 herein, or a substantially similar sequence. 8. A DNA molecule encoding the αg subunit of the human GABAA receptor comprising all or a portion of the sequence depicted in Figure 5 herein, or a substantially similar sequence. 9. A DNA molecule encoding the 02 subunit of the human GABAA receptor comprising all or a portion of the sequence depicted in Figure 6 herein, or a substantially similar sequence. 10. A recombinant expression vector comprising the nucleotide sequence of a GABAA receptor subunit together with additional sequences capable of directing the synthesis of the said GABAA receptor subunit in cultures of stably co-transfected eukaryotic cells. 11. A protein preparation of GABAA receptor subunit combinations derived from a culture of stably co- transfected eukaryotic cells. 12. A membrane preparation containing subunit combinations of the GABAA receptor derived from a culture of stably co-transfected eukaryotic cells. 13. A preparation as claimed in claim 11 or claim 12 wherein the subunit combinations derived are human GABAA receptor subunit combinations. 14. A preparation as claimed in claim 13 wherein the subunit combination derived is the a-jjSi/-- subunit combination of the human GABA receptor. 15. A preparation as claimed in claim 14 containing a human GABAA receptor consisting of the αi i^L subunit combination isolated from stably transfected mouse Ltk " fibroblast cells. 16. The use of the cell line as claimed in claim 1 or claim 2, and membrane preparations derived therefrom, in screening for and designing medicaments which act upon the GABAA receptor.

Inventors: HADINGHAM KAREN LOUISE, LE BOURDELLES BEATRICE, WHITING PAUL JOHN, WINGROVE PETER BAXTER, HADINGHAM, KAREN, LOUISE, LE BOURDELLES, BEATRICE, WHITING, PAUL, JOHN, WINGROVE, PETER, BAXTER
Applicants: MERCK SHARP & DOHME, MERCK SHARP & DOHME LIMITED
Application year: 1992
Main classification: C07K13
IPC codes: A61K38, C07K14, C12N5, C12N15, C12P21, C12R1, G01N33