A CELL EXPRESSING A GLUTAMATE RECEPTOR

Abstract: Mammalian cell transfected with a DNA sequence encoding a protein with electrophysiological and pharmacological properties characteristic of a glutamate receptor, or a functional fragment of said protein, and capable of permanently expressing said DNA sequence.

Description: A CELL EXPRESSING A GLUTAMATE RECEPTOR FIELD OF INVENTION The present invention relates to mammalian cells capable of stably expressing glutamate receptors, as well as to a method of screening for substances interacting with glutamate receptors by means of said cell. BACKGROUND OF THE INVENTION Since the early 1960's, it has been known that the amino acids glutamate and aspartate exert excitatory effects on central nervous system neurons, and since then, efforts have been made to explore the mechanisms by which neurotransmission takes place. More recently, it was discovered that glutamate and other excitatory amino acids bind to receptors present on neurons. Efforts have also been made to study the interaction of the receptors with agonists and antagonists, primarily with a view to identifying substances which are able to block the excitation of neurons induced by glutamate or other ligands, and which may therefore have anticonvulsant activity, muscle relaxant activity, anticonflict effects or effects against brain anoxia and ischemia. A review of various excitatory amino acid receptors and their antagonists is found in T. Honore, Medicinal Research Reviews £(1), 1989, pp. 1-23. To provide useful tools in the screening for glutamate receptor agonists or antagonists, glutamate receptor subunits have recently been prepared by recombinant DNA techniques. The cloning of DNA coding for glutamate receptor subunits and expression thereof in Xenopus oocytes is described in WO 91/06648. Transient expression of four glutamate receptor subunits in human embryonic kidney cells is reported by T.A. Verdoorn et al., Neuron 4_, 1990, p. 919, and Keinanen et al., Science 249, 1990, pp. 556-560. However, no cells stably expressing glutamate receptors have previously been reported. SUMMARY OF THE INVENTION It has surprisingly been found possible to provide a mammalian cell which is capable of permanently expressing glutamate receptors. Accordingly, the present invention relates to a mammalian cell transfected with a DNA sequence encoding a protein with electrophysiological and pharmacological properties characteristic of a glutamate receptor, or a functional fragment of said protein, and capable of permanently expressing said DNA sequence. In the present context, the term "glutamate receptor" refers to a membrane component present on neurons and certain other cells and activated by glutamate. The term "electrophysiological and pharmacological properties characteristic of a glutamate receptor" is intended to indicate a protein which generates a signal or signals comparable to the signal or signals generated by native glutamate receptors when activated in the presence of glutamate, and which can be affected by substances known to affect native glutamate receptors. In the following, the protein is usually referred to as a glutamate receptor-like protein or a GluR-like protein. The term "functional fragment" is intended to indicate a fragment with properties corresponding to those of a glutamate receptor. As indicated above, the cells of the present invention are distinguished from those described in T.A. Verdoorn et al., supra. or Keinanen et al, supra. by being capable of stably expressing glutamate receptor-like proteins. This is an important advantage when such cells are to be used to screen for substances interacting with the receptors as stable cell lines are less expensive to produce and more convenient to use because no large-scale production RNA or DNA is required for transfection of cells. Cells capable of permanently expressing glutamate-receptor-like proteins have been found to express a membrane component which is able to bind glutamate with high affinity but which has a different pharmacology from any known glutamate receptor in that it is not activated by glutamate. The presence of this component, believed to be a glutamate transporter, i.e. a protein transporting glutamate from the exterior to the interior of the cell, causes less free glutamate, which is a common constituent in cell culture media, to be present in the vicinity of the cells. Consequently less excitation of the glutamate receptors expressed on the surface of the cells, eventually resulting in cell death, will occur (continuous stimulation of glutamate receptors has been reported to be the major cause of cell death in ischaemia, cf. D.W. Choi, Neuron 1 , 1988, pp. 623- 634). In another aspect, the present invention relates to a method of screening for a substance capable of interacting with a glutamate receptor, the method comprising incubating a cell according to the invention with a sample suspected of containing a substance capable of interacting with a glutamate receptor or with an agonist of a glutamate receptor, and detecting any interaction of said substance with a protein with electrophysiological and pharmacological properties characteristic of a glutamate receptor, or a functional fragment of said protein, the protein or fragment being present on said cell, or detecting any interaction of said substance with said agonist. In the present context, the term "interacting with" is intended to include substances which are able to bind to a glutamate receptor or a possible associated protein, thereby either stimulating the receptor or blocking agonist effects on the receptor, or which otherwise allosterically affect receptor function. DETAILED DISCLOSURE OF THE INVENTION The DNA sequence encoding the glutamate receptor-like protein introduced into the cell of the invention may be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by S.L. Beaucage and M.H. Caruthers, Tetrahedron Letters 22. 1981, pp. 1859-1869, or the method described by Matthes et al., EMBO Journal 3. 1984, pp. 801-805. According to the phosphoamidite method, oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in suitable vectors. The DNA sequence may also be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the GluR-like protein by hybridization using synthetic oligonucleotide probes in accordance with standard techniques (cf. Sambrook et al.. Molecular Cloning: A Laboratory Manual. 2nd Ed. , Cold Spring Harbor, 1989) . In particular, the DNA sequence may be obtained by procedures described in WO 91/06648. Finally, the DNA sequence may be of mixed synthetic and genomic, mixed synthetic and cDNA or mixed genomic and cDNA origin prepared by annealing fragments of synthetic, genomic or cDNA origin (as appropriate) , the fragments corresponding to various parts of the entire DNA sequence, in accordance with standard techniques. The DNA sequence may also be prepared by polymerase chain reaction using specific primers, for instance as described in US 4,683,202, or Saiki et al., Science 239. 1988, pp. 487-491. The resulting DNA sequence may be introduced into the cell by transfection of the cell with a recombinant expression vector comprising the sequence. The expression vector may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated. In the vector, the DNA sequence encoding the GluR-like protein should be operably connected to a suitable promoter sequence. The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the DNA insert in mammalian cells are the SV 40 promoter (Subramani et al., Mol. Cell Biol. jL, 1981, pp. 854-864) , the MT-1 (metallothionein gene) promoter (Palmiter et al.. Science 222. 1983, pp. 809-814) or the adenovirus 2 major late promoter. The DNA sequence should also be operably connected to a suitable terminator, such as the human growth hormone terminator (Palmiter et al., op. cit.) . The vector may further comprise elements such as polyadenylation signals (e.g. from SV 40 or the adenovirus 5 Elb region) , transcriptional enhancer sequences (e.g. the SV 40 enhancer) and translational enhancer sequences (e.g. the ones encoding adenovirus VA RNAs) . The recombinant expression vector may also comprise a DNA sequence enabling the vector to replicate in the host cell in question. An examples of such a sequence is the SV 40 origin of replication. The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the gene coding for dihydrofolate reductase (DHFR) or one which confers resistance to a drug, e.g. neomycin, hygromycin or methotrexate. The procedures used to ligate the DNA sequence encoding the GluR-like protein with the DNA sequences coding for the promoter and the terminator, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf. , for instance, Sambrook et al., op.cit.) . A preferred cell for the for transfection with the expression vector containing the DNA sequence described above is one which, although it does not in nature express glutamate receptors, comprises endogenous transport systems capable of transporting glutamate or related amino acids to the interior of the cell. Examples of cells which are suitable for the present purpose are kidney cells, in particular those expressing a glutamate binding site other than the glutamate receptor, as described above, such as a baby hamster kidney (BHK) cell line (ATCC CRL 1632, ATCC CCL 10), the related BHK 570 cell line (ATCC CRL 6282 and ATCC CRL 10314 (described in WO 91/11514)) being particularly preferred. Methods of transfecting mammalian cells and expressing DNA sequences introduced in the cells are described in e.g. Kaufman and Sharp, J. Mol. Biol. 159. 1982, pp. 601-621; Southern and Berg, J. Mol. Appl. Genet. 1 , 1982, pp. 327-341; Loyter et al., Proc. Natl. Acad. Sci. USA 79. 1982, pp. 422-426; Wigler et al.. Cell 14. 1978, p. 725; Corsaro and Pearson, Somatic Cell Genetics 1_, 1981, p. 603, Graham and van der Eb, Virology 5_2, 1973, p. 456; and Neumann et al. , EMBO J. 1, 1982, pp. 841-845. The DNA sequence encoding the GluR-like protein may, for instance, encode a glutamate receptor of the N-methyl-D- aspartate (NMDA) or the non-N-methyl-D-aspartate (non-NMDA) type. Both NMDA and non-NMDA receptors are defined in T. Honore, supra. More preferably, the DNA sequence may encode an ionotropic glutamate receptor, by which is meant a receptor forming ion channels in the membrane of cells expressing the receptor so as to make the membrane permeable to ions in response to agonist exposure, whereby an electrical signal is generated and the cells are depolarized; see i.a. M. Hollmann et al.. Science 252, 1991, pp. 851-853, and T.A. Verdoorn et al, Science 252, 1991, pp. 1715-1718. Examples of ionotropic glutamate receptors are Glul-4 flip and flop (cf. Sommer et al., Science 249, 1990, pp. 1580-1585), otherwise known as α- amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors, Glu 5-7 (WO 91/06648) , or kainate 1 or 2 or γ-2 receptors, or a functional fragment of any of these receptors, or a combination of two or more of said receptors or functional fragments thereof. In a particularly preferred embodiment of the present invention, the cell is a BHK 570 cell comprising a DNA sequence encoding an ionotropic glutamate receptor or a functional fragment thereof, or a combination of two or more ionotropic glutamate receptors or functional fragments thereof. For use in the screening method of the invention, the cells may be separated from the medium in which they are cultured to express the GluR-like receptor by any conventional method such as filtration or centrifugation and subsequently incubated with the sample. Suitable samples may be obtained from any material of biological origin, preferably in fluid form, e.g. a microbial fermentation broth, a plant or part(s) thereof, a tissue extract or solutions or suspensions of synthetic compunds of potential interest. In one embodiment, the invention relates to a method of screening for an antagonist of ligand binding to the receptor, wherein the cell of the invention is incubated with a sample suspected of containing such an antagonist in the presence of a labelled ligand for the receptor. Alternatively, in another embodiment, the invention relates to a method of screening for a substance interacting with glutamate receptor function via a binding site different from the glutamate receptor, i.e. a substance which is an allosteric modulator. In this embodiment, the cell of the invention is incubated with a sample suspected of containing such a substance in the presence of an agonist followed by determination of any effect of said substance by detection methods such as those indicated below. The label substance with which the ligand is labelled is preferably selected from the group consisting of enzymes, coloured or fluorescent substances, or radioactive isotopes. Examples of enzymes useful as label substances are peroxidases (such as horseradish peroxidase) , phosphatases (such as acid or alkaline phosphatase) , β-galactosidase, urease, glucose oxidase, carbonic anhydrase, acetylcholinesterase, glu- coamylase, lysozyme, malate dehydrogenase, glucose-6-phosphate dehydrogenase, β-glucosidase, proteases, pyruvate de- carboxylase, esterases, luciferase, etc. Enzymes are not in themselves detectable but must be combined with a substrate to catalyse a reaction the end product of which is detectable. Examples of substrates which may be em¬ ployed in the method according to the invention include hydrogen peroxide/tetramethylbenzidine or chloronaphthole or o-phenylenediamine or 3-(p-hydroxyphenyl) propionic acid or luminol, indoxyl phosphate, p-nitrophenylphosphate, nitro- phenyl galactose, 4-methyl umbelliferyl-D-galactopyranoside, or luciferin. Alternatively, the label substance may comprise coloured or fluorescent substances, including gold particles, coloured or fluorescent latex particles, dye particles, fluorescein, phycoerythrin or phycocyanin. In a particularly favoured embodiment, the ligand is labelled with a radioactive isotope. Radioactive isotopes which may be used for the present purpose may be selected from 1-125, I- 131, In-Ill, H-3, P-32, C-14 or S-35. The radioactivity emitted by these isotopes may be measured in a beta- or gamma- counter or by a scintillation camera in a manner known per se. To screen for antagonists of ligand binding to glutamate receptors, the labelled ligand may suitably be a glutamate receptor agonist or antagonist binding to the receptor. Examples of possible ligands are glutamate, α-amino-3-hydroxy- 5-methyl-4-isoxazole propionic acid (AMPA) , 6-cyano-7- nitroquinoxaline-2,3-dion (CNQX) or kainate (kainic acid). Alternatively, the method of the invention may be used to screen for a negative or positive modulator of glutamate receptor function, wherein said cell is incubated with a sample suspected of containing such a modulator in the presence of an agonist of the receptor, and wherein interaction of said substance with the glutamate receptor-like protein expressed by the cell is detected by measuring the ion flux across the cell membrane. In particular, the calcium or sodium may be measured, since glutamate receptors are selective for these two cations (cf. Hume et al., Science 253. 1991, pp. 1028-1031). To measure the ion flux across the cell membrane, various techniques may be used. A convenient technique comprises monitoring changes in the membrane potential of the cell by means of whole cell clamping, e.g. as described in Voltage and Patch Clamping with Microelectrodes. T.G. Smith et al. (Eds.), American Physiological Society, Maryland, USA, 1985. Another suitable technique comprises isolating membranes of cells according to the invention, and monitoring changes in the membrane potential on such isolated membranes incorporated in an artificial bilayer, e.g. as described in Ion Channel Reconstitution, CM. Miller (Ed.), Plenum Press, New York, 1986. A further suitable technique might be to monitor changes in intracellular concentrations of sodium, potassium or calcium ions by means of fluorescent probes, e.g. SBFI (S- 1264) or Fura (F-1225) , respectively (available from Molecular Probes) , followed by detection in a fluorospectrophotometer. BRIEF DESCRIPTION OF THE DRAWINGS The present invention is further illustrated in the following examples with reference to the appended drawings, wherein Fig. 1 shows the presence of ionic conductances activated by glutamic acid or analogues thereof in BHK 570 cells expressing glutamate receptor cDNAs; and Fig. 2 shows a glutamate dose-response curve in a BHK 570 cell line expressing a glutamate receptor. EXAMPLE 1 Screening for cells capable of permanently expressing glutamate receptors BHK 570 cells (ATCC CRL 10314, described in WO 91/11514) were grown to 80-90% confluence in 24-well microtiter plates (Nunc #146485) in Dulbecco's Modified Eagle's Medium (DMEM; Gibco 041-1965) containing 1 mM pyruvate, 1% penicilllin/streptomycin (Gibco BRL #043-05070) , 2 mM glutamine and 10% foetal calf serum. The cells were then washed twice in DMEM at 37°C. One ml of DMEM was then added to each well followed by 25 μl of 3 H-glutamate (available from NEN, Boston, MA, USA, NET-490) to a final concentration of 10 nM, after which the plates were left at 37°C for 10 minutes. The cells were then washed twice in ice-cold DMEM and lysed by adding 200 μl/well of 0.1 M NaOH. An aliquot of the lysed cells was counted in a beta counter for diffusion of 3 H- glutamate into the cells, and another aliquot was used for protein determination. Non-carrier mediated diffusion of glutamate into the cells was defined as uptake of 3 H-glutamate by the cells when incubating at 0°C. Cells found to have an endogenous transport system for glutamate and related amino acids (e.g. aspartate) were found to be capable of expressing glutamate receptors. EXAMPLE 2 Characterization of cells capable of stably expressing glutamate receptors 3 H-glutamate binding was carried as by the method described in Example 6 below for 3 H-AMPA binding. BHK 570 cells were found to express large quantities (30-50 pmole/mg membrane protein) of a putative membrane component which binds glutamate with high affinity (30-50 nM at 37°C). This binding site has a pharmacology differing from that of glutamate receptors (e.g. IC 50 (quisqualate) > 10 μM) . The presence of this putative membrane component is believed to be important for the ability of a cell stably to express glutamate receptors. BHK 570 is therefore the currently preferred cell for the present purpose. EXAMPLE 3 Cloning and expression of an AMPA selective glutamate receptor Total RNA was prepared from rat hippocampus or cerebellum by the acidic phenol method described by P. Chomczynski and N. Sacchi, Anal. Biochem. 162. 1987, pp. 156-159. First strand cDNA was prepared from 10 μg total rat brain RNA using oligo- dT primer and Superscript™ RNase H-reverse transcriptase as recommended by the manufacturer (BRL, Life Technologies) . Polymerase chain reactions (PCR) were carried out as described in K.B. Mullis et al., Meth. Enzvmol. 155. R. Wu (Ed.), 1987, pp. 335-350, using 5 μg of cDNA (a fourth of the first strand cDNA synthesized above) as template, sequence specific oligonucleotide primers (based on the sequence disclosed in K. Keinanen et al., Science 249, 1990, pp. 556-560), and AmpliTaq™ DNA polymerase (Perkin Elmer-Cetus) . The sequence of the oligonucleotide primers was the following: Glul: TTAGGATCCACCATGCCGTACATCTTTGCCTTT CCACTCCACTCGAGTATGGTCTCGGGAGTC GACCATACTCGAGTGGACTGGAAGAGGCCA GCCTGCCTCGAGTGTCCCATAAGCAATTTC TATGGGACACTCGAGGCAGGCTCCACTAAG AATTCTAGATTACAATCCTGTGGCTCCCAAGGG Glu2: ATATATGGATCCACCATGCAAAAGATTATGCATATTTC AAGGTTACGGAAAGCTTCAGTCATCAC GTGATGACTGAAGCTTTCCGTAACCTT ACCTCTTCTCTCACTAGTGTGATAGTTAATGGAGCA TATCACACTAGTGAGAGAAGAGGTGATTGACTTCTC TTATTAGGTACCTCAAATTTTAACACTCTCGATGCCAT Glu3: TTAGGATCCACCATGGGGCAAAGCGTGCTCCGG CATGACTCTCTCGAGTAAAATGTCAGT ACTGACATTTTACTCGAGAGAGTCATG TCAGGAGGGCTTTGTGGGTCTCGAGGTTC GAACCTCGAGACCCACAAAGCCCTCCTGA AATTCTAGATTAGATCTTAACACTTTCTGTTCC Glu4: TTAGGATCCACCATGAGGATTATTTGCAGGCAG AGTCAGCCCTTGAATTCGAACCTGCTTCAG CTGAAGCAGGTTCGAATTCAAGGGCTGACT GTCACTGGGCCCTTCCTTCCCATCCTCAGG CCTCAGGATGGGAAGGAAGGGCCCAGTGAC AATTCTAGATTATGGTAGGTCCGATGCAATGAC PCR was carried out for 30-40 cycles of amplification for 1 minute at 94°C, 1 minute at 50°C and 3 minutes at 72°C followed by 10 minutes at 72°C. Each amplified PCR product which was typically 0.8-1.1 kb in length was subcloned in the cloning vector pSKII+ (available from Stratagene) . cDNAs encoding the AMPA-selective receptors (Glul-Glu4) were assembled from three PCR fragments subcloned in pSKII+ in the mammalian expression vector Zem219b which carries an MT promoter, SV40 origin of replication and MTX marker. The vector is described in detail in Danish Patent Application No. 3023/88. The nucleotide sequence of the entire coding region was determined by the dideoxy chain termination method of F. Sanger et al., Proc. Natl. Acad. Sci. USA 74. 1977, pp. 5463- 5467, using the double stranded plasmids as templates, a Sequenase kit (available from United States Biochemical) and sequence specific primers derived from the sequence shown by Keinanen et al, supra. EXAMPLE 4 Transfection of cells BHK 570 cells were grown to 30-50% confluence in a tissue culture dish (Nunc #168381) in DMEM containing 1% penicillin/streptomycin (Gibco BRL #043-05070) . Prior to transfection, the cells were washed twice with calcium-free PBS after which 20 ml of DMEM was added to the cells. Transfection was carried out as described previously (product description: Lipofectin, Gibco BRL cat. No. 8292SA) . Briefly, 2 μg of the cDNA obtained in Example 3 was diluted in 300 μl of sterile water. 30 μg of Lipofectin (Gibco BRL) was diluted in 300 μl of sterile water. The cDNA and Lipofectin solutions were mixed and left at room temperature for 15 minutes. The Lipofectin/cDNA mixture was added dropwise to the cells while gently swirling the plates. The cells were then incubated for 16-24 hours, after which the medium was replaced with standard medium containing 1 nM methotrexate. Resistant colonies appearing after 1-2 weeks were isolated and propagated for further characterization. EXAMPLE 5 Transfection of cells BHK 570 cells were grown at 37°C in 5% C0 2 in Costar ® 75 cm 2 vented culture flasks in a DMEM medium as described in Example 1. Prior to transfection,. the cells were transferred to Nunc 10 tissue culture dishes and grown to 60-80% confluence. 10 μg of the plasmid DNA obtained above was diluted to 250 μl in HBS (20 mM HEPES, pH 7.4, 150 mM NaCl) and mixed with 50 μl DOTMA liposomes (Boehringer Mannheim) diluted to 250 μl with HBS. The mixture and 8 ml medium were incubated with the cells overnight. The cells were diluted 50 fold and transferred to Nunc 15 cm tissue dishes in the same medium as described above to which 5 μM methotrexate had been added. Methotrexate resistant colonies appearing in 1-2 weeks were isolated with cloning rings and propagated for further characterization. EXAMPLE 6 ^-AMPA binding to transfected BHK 570 cells BHK 570 cells obtained as described in Example 4 or 5 above were grown to 90-100% confluence in standard 15 cm tissue culture dishes (Nunc #168381) . The dishes were scraped and the cells were spun at 5000 x g for 10 minutes. The cell pellet was homogenized by means of an Ultra Turrax and centrifuged at 25000 x g for 10 minutes. This step was repeated twice. The final pellet was resuspended in 10 ml buffer and incubated at 37°C for 30 minutes and once more centrifuged at 25000 x g for 10 minutes. The pellet was resuspended in 10 ml buffer and frozen for 30 minutes on dry ice. After thawing, the suspension was centrifuged at 25000 x g for 10 minutes. The pellet was rehomogenised in buffer additionally containing 100 mM KSCN and used immediately in a binding assay. 500 μl of the homogenate was added to 25 μl 3 H-AMPA (final concentration 5 nM) and incubated for 30 minutes on ice. For further details, see T. Honore et al., J. Neuroche . 38, 1982, pp. 173-178, and Nielsen et al., Eur. J. Pharmacol. 157 , 1988, pp. 197-203. EXAMPLE 7 Pharmacological profile of stably transfected BHK 570 cell lines The pharmacological profile of BHK cell lines stably expressing glutamate receptors was investigated by the 3 H-AMPA binding assay described in Example 6. The profile of the Glul and Glu2 flip subunit stably expressed in BHK cells is shown in the following table. Cell line expressing Drug Glul flip Glu2 flip IC50 (nM) 13 3700 290 230 200 58 .5 6 >30000 The Kd and Bmax values in these two cell lines were 12 nM and 0.6 pmol/mg membrane protein (Glul flip) and 20 nM and 6 pmol/ng membrane protein (Glu2 flip) . These results clearly indicate that the stably expressed glutamate receptor subunits exhibit the expected AMPA receptor pharmacology as described previously for transiently expressed receptors (cf. for instance Keinanen et al, supra) . EXAMPLE 8 Functional glutamate receptors expressed in BHK 570 cells transfected with GluR cDNA BHK 570 cells, transfected with GluR subunits as described above, were grown on 35 mm plastic dishes (Falcon 3001) in DMEM (supplemented with 10% heat inactivated calf serum, antibiotics, non-essential amino acids, sodium pyruvate, and 5 μM methotrexate) . For recording, cells were continuously perfused with mammalian Ringer (135 mM NaCl, 5.3 mM KC1, 1 mM MgCl 2 , 1.8 mM CaCl 2 , 10 mM HEPES; pH 7.2) at 1 ml/min. Whole cell patch recordings were made using a computer controlled amplifier, using pipettes pulled from borosilicate capillaries to have a tip resistance of 4 to 7 MΩ when filled with internal solution of the following composition: 125 mM CsCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 10 mM EGTA, 10 mM HEPES; pH 7.3. Kainate was applied from an array of small pipes (outer diameter 610 μm) switched by a stepper motor. Recordings were made at room temperature (20 to 23°C). Prior to recording cells were pretreated with concanavalin A (0.2 mg/ml for 10 min.). Glutamate was applied from an array of small pipes (outer diameter 610 μm) . Electrophysiological analysis of BHK 570 cells transfected with GluR cDNAs revealed the presence of ionic conductances activated by the neurotransmitter glutamic acid or its analogues. The conductances indicate the de novo synthesis and functional expression of glutamate receptor-ion channel complexes on the surface of the BHK cells. Fig. 1 shows sample traces obtained in one cell expressing GluR flip receptors. The magnitude of the response increases with increasing concentration of applied glutamate, indicating the activation of more receptors. Fig. 2 shows a log-dose response analysis of responses (such as those shown in the first figure) recorded in up to 12 cells. The responses have been normalized by dividing by the magnitude of the response to 30 μM glutamate. The error bars represent the standard error of the mean.

Claims: CLAIMS 1. A mammalian cell transfected with a DNA sequence encoding a protein with electrophysiological and pharmacological properties characteristic of a glutamate receptor, or a functional fragment of said protein, and capable of permanently expressing said DNA sequence. 2. A cell according to claim 1 which is a kidney cell. 3. A cell according to claim 1 comprising endogenous transport systems capable of transporting glutamate or related amino acids. 4. A cell according to claim 3 which is a baby hamster kidney (BHK) cell. 5. A cell according to claim 4 which is a BHK 570 cell. 6. A cell according to any of claims 1-5, wherein the DNA sequence encodes a glutamate receptor of the non-N-methyl-D- aspartate (non-NMDA) type. 7. A cell according to claim 6, wherein the DNA sequence encodes an ionotropic glutamate receptor, such as a Glul-4 flip and flop, Glu 5-7, -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) or a kainate 1 or 2 or γ-2 receptor, or a functional fragment thereof, or a combination of two or more of said receptors or functional fragments thereof. 8. A cell according to claim 7 which is a BHK 570 cell comprising a DNA sequence encoding an ionotropic glutamate receptor or a functional fragment thereof, or a combination of two or more ionotropic glutamate receptors or functional fragments thereof. 9. A cell according to any of claims 1-5, wherein the DNA sequence encodes a glutamate receptor of the N-methyl-D- aspartate (NMDA) type. 10. A method of screening for a substance capable of interacting with a glutamate receptor, the method comprising incubating a cell according to any of claims 1-9 with a sample suspected of containing a substance capable of interacting with a glutamate receptor or with a ligand of a glutamate receptor, and detecting any interaction of said substance with a protein with electrophysiological and pharmacological properties characteristic of a glutamate receptor, or a functional fragment of said protein, the protein or fragment being present on said cell, or detecting any interaction of said substance with said ligand. 11. A method according to claim 10 of screening for an antagonist of ligand binding to the receptor, wherein said cell is incubated with a sample suspected of containing such an antagonist in the presence of a labelled ligand for the receptor. 12. A method according to claim 11, wherein the ligand is labelled with a radioisotope. 13. A method according to claim 11, wherein the ligand is glutamate, AMPA, CNQX or kainate. 14. A method according to claim 10 of screening for a negative or positive modulator of glutamate receptor function, wherein said cell is incubated with a sample suspected of containing such a modulator in the presence of an agonist of the receptor, and wherein interaction of said substance with said receptor is detected by measuring the ion flux across the cell membrane or by measuring the changes in intracellular ion concentrations. 15. A method according to claim 14, wherein the calcium flow is measured. 16. A method according to claim 14, wherein the sodium flow is measured. 17. A method according to claim 14, wherein changes in the membrane potential of the cell are monitored by means of whole cell clamping. 18. A method according to claim 14, wherein changes in the membrane potential of the cell are monitored on membranes of cells according to any of claims 1-8 incorporated in an artificial bilayer. 19. A method according to claim 14, wherein changes in the intracellular concentrations of Na + , K + or Ca 2+ are measured fluorospectrophotometrically, using fluorescent probes. 20. A method according to any of claims 14-19, wherein said sample is suspected of containing an antagonist of glutamate receptor function.

Inventors: ANDERSEN PETER HONGAARD, NIELSEN LARS SOEGAARD, RASMUSSEN JESPER SKOU, STIDSEN CARSTEN E, ANDERSEN, PETER, HOENGAARD, NIELSEN, LARS, SOEEGAARD, RASMUSSEN, JESPER, SKOU, STIDSEN, CARSTEN, E.
Applicants: ANDERSEN PETER HONGAARD, NIELSEN LARS SOEGAARD, NOVONORDISK AS, RASMUSSEN JESPER SKOU, STIDSEN CARSTEN E, NOVO NORDISK A/S
Application year: 1993
Main classification: C12N15
IPC codes: C07K14, C12N5, C12N15, C12Q1, G01N33