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WO1993024629A1 - Cellule exprimant un recepteur de glutamate - Google Patents

Cellule exprimant un recepteur de glutamate Download PDF

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Publication number
WO1993024629A1
WO1993024629A1 PCT/DK1993/000169 DK9300169W WO9324629A1 WO 1993024629 A1 WO1993024629 A1 WO 1993024629A1 DK 9300169 W DK9300169 W DK 9300169W WO 9324629 A1 WO9324629 A1 WO 9324629A1
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Prior art keywords
cell
glutamate
receptor
glutamate receptor
dna sequence
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PCT/DK1993/000169
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English (en)
Inventor
Peter Høngaard ANDERSEN
Jesper Skou Rasmussen
Carsten E. Stidsen
Lars Søegård NIELSEN
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Novo Nordisk A/S
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Priority to JP6500091A priority Critical patent/JPH07506970A/ja
Priority to EP93912656A priority patent/EP0672135A1/fr
Publication of WO1993024629A1 publication Critical patent/WO1993024629A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9406—Neurotransmitters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems

Definitions

  • 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.
  • 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
  • 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.
  • glutamate receptor refers to a membrane component present on neurons and certain other cells and activated by glutamate.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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) .
  • the DNA sequence may be obtained by procedures described in WO 91/06648.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • DHFR dihydrofolate reductase
  • 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.
  • 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.
  • ionotropic glutamate receptors are Glul-4 flip and flop (cf. Sommer et al., Science 249, 1990, pp. 1580-1585), otherwise known as ⁇ - amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors, Glu 5-7 (WO 91/06648) , or kainate 1 or 2 or ⁇ -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.
  • AMPA ⁇ - amino-3-hydroxy-5-methyl-4-isoxazole propionic acid
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • enzymes useful as label substances are peroxidases (such as horseradish peroxidase) , phosphatases (such as acid or alkaline phosphatase) , ⁇ -galactosidase, urease, glucose oxidase, carbonic anhydrase, acetylcholinesterase, glu- coamylase, lysozyme, malate dehydrogenase, glucose-6-phosphate dehydrogenase, ⁇ -glucosidase, proteases, pyruvate de- carboxylase, esterases, luciferase, etc.
  • peroxidases such as horseradish peroxidase
  • phosphatases such as acid or alkaline phosphatase
  • ⁇ -galactosidase urease, glucose oxidase, carbonic anhydrase, acetylcholinesterase, glu- coamylase, lysozyme, malate dehydrogen
  • Enzymes are not in themselves detectable but must be combined with a substrate to catalyse a reaction the end product of which is detectable.
  • 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.
  • the label substance may comprise coloured or fluorescent substances, including gold particles, coloured or fluorescent latex particles, dye particles, fluorescein, phycoerythrin or phycocyanin.
  • 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.
  • the labelled ligand may suitably be a glutamate receptor agonist or antagonist binding to the receptor.
  • glutamate ⁇ -amino-3-hydroxy- 5-methyl-4-isoxazole propionic acid (AMPA) , 6-cyano-7- nitroquinoxaline-2,3-dion (CNQX) or kainate (kainic acid).
  • AMPA ⁇ -amino-3-hydroxy- 5-methyl-4-isoxazole propionic acid
  • CNQX 6-cyano-7- nitroquinoxaline-2,3-dion
  • kainate kainic acid
  • 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.
  • 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).
  • 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.
  • fluorescent probes e.g. SBFI (S- 1264) or Fura (F-1225) , respectively (available from Molecular Probes) , followed by detection in a fluorospectrophotometer.
  • Fig. 1 shows the presence of ionic conductances activated by glutamic acid or analogues thereof in BHK 570 cells expressing glutamate receptor cDNAs
  • Fig. 2 shows a glutamate dose-response curve in a BHK 570 cell line expressing a glutamate receptor.
  • BHK 570 cells (ATCC CRL 10314, described in WO 91/11514) were grown to 80-90% confluence in 24-well microtiter plates (Nunc
  • DMEM Dulbecco's Modified Eagle's Medium
  • the cells were then washed twice in ice-cold DMEM and lysed by adding 200 ⁇ l/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.
  • 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 ⁇ M) . 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.
  • 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 ⁇ g total rat brain RNA using oligo- dT primer and SuperscriptTM RNase H-reverse transcriptase as recommended by the manufacturer (BRL, Life Technologies) .
  • PCR Polymerase chain reactions
  • Glul TTAGGATCCACCATGCCGTACATCTTTGCCTTT CCACTCCACTCGAGTATGGTCTCGGGAGTC
  • Glu2 ATATATGGATCCACCATGCAAAAGATTATGCATATTTC
  • Glu3 TTAGGATCCACCATGGGGCAAAGCGTGCTCCGG CATGACTCTCTCGAGTAAAATGTCAGT ACTGACATTTTACTCGAGAGTCATG
  • 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.
  • 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.
  • 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 ⁇ g of the cDNA obtained in Example 3 was diluted in 300 ⁇ l of sterile water. 30 ⁇ g of Lipofectin (Gibco BRL) was diluted in 300 ⁇ l 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.
  • 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 ⁇ g of the plasmid DNA obtained above was diluted to 250 ⁇ l in HBS (20 mM HEPES, pH 7.4, 150 mM NaCl) and mixed with 50 ⁇ l DOTMA liposomes (Boehringer Mannheim) diluted to 250 ⁇ l 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 ⁇ M methotrexate had been added. Methotrexate resistant colonies appearing in 1-2 weeks were isolated with cloning rings and propagated for further characterization.
  • 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 ⁇ l of the homogenate was added to 25 ⁇ l 3 H-AMPA (final concentration 5 nM) and incubated for 30 minutes on ice.
  • 500 ⁇ l of the homogenate was added to 25 ⁇ l 3 H-AMPA (final concentration 5 nM) and incubated for 30 minutes on ice.
  • 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.
  • 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) .
  • 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 ⁇ M methotrexate) .
  • DMEM supplied with 10% heat inactivated calf serum, antibiotics, non-essential amino acids, sodium pyruvate, and 5 ⁇ M methotrexate
  • 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.
  • 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 ⁇ M glutamate.
  • the error bars represent the standard error of the mean.

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Abstract

L'invention concerne une cellule de mammifère modifiée par transfection avec une séquence d'ADN codant une protéine ayant les protéines électrophysiologiques et pharmacologiques caractéristiques d'un récepteur de glutamate, ou un fragment fontionnel de ladite protéine, et étant capable d'exprimer de manière permanente ladite séquence d'ADN.
PCT/DK1993/000169 1992-05-25 1993-05-19 Cellule exprimant un recepteur de glutamate WO1993024629A1 (fr)

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JP6500091A JPH07506970A (ja) 1992-05-25 1993-05-19 グルタミン酸レセプターを発現する細胞
EP93912656A EP0672135A1 (fr) 1992-05-25 1993-05-19 Cellule exprimant un recepteur de glutamate

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DK68592A DK68592D0 (da) 1992-05-25 1992-05-25 Celle
DK685/92 1992-05-25

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Cited By (4)

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WO1995026401A1 (fr) * 1994-03-29 1995-10-05 Basf Aktiengesellschaft Procede pour l'expression permanente de recepteurs du glutamate
WO1998055870A1 (fr) * 1997-06-04 1998-12-10 A+ Science Invest Ab Detection de molecules biologiquement actives a l'aide de capteurs biologiques pre-actives a base de cellule dans des systemes de separation a base de liquide
US5849895A (en) * 1993-04-20 1998-12-15 Sibia Neurosciences, Inc. Human N-methyl-D-aspartate receptor subunits, nucleic acids encoding same and uses therefor
US6228610B1 (en) 1993-09-20 2001-05-08 Novartis Corporation Human metabotropic glutamate receptor subtypes (hmR4, hmR6, hmR7) and related DNA compounds

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US5093525A (en) * 1986-07-10 1992-03-03 State Of Oregon, Acting By And Through The Oregon State Board Of Higher Education, Acting For And On Behalf Of The Oregon Health Sciences University N,N'-disubstituted guanidines and their use as excitatory amino acid antagonists

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US5093525A (en) * 1986-07-10 1992-03-03 State Of Oregon, Acting By And Through The Oregon State Board Of Higher Education, Acting For And On Behalf Of The Oregon Health Sciences University N,N'-disubstituted guanidines and their use as excitatory amino acid antagonists
WO1991006648A1 (fr) * 1989-10-27 1991-05-16 The Salk Institute For Biological Studies Compositions receptrices de glutamate, et procedes

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Science, Volume 249, August 1990, BERND SOMMER et al., "Flip and Flop: A Cell-Specific Functional Switch in Glutamate-Operated Channels of the CNS", page 1580 - page 1585. *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6376660B1 (en) 1993-04-20 2002-04-23 Merck & Co., Inc. Human N-methyl-D-aspartate receptor subunits, nucleic acids encoding same and uses therefor
US6825322B2 (en) 1993-04-20 2004-11-30 Merck & Co., Inc. Human N-methyl-D-aspartate receptor subunits, nucleic acids encoding same and uses therefor
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DK68592D0 (da) 1992-05-25
EP0672135A1 (fr) 1995-09-20

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