CA2186788A1 - Method for the permanent expression of glutamate receptors - Google Patents

Abstract

The invention concerns a method for the preparation of eucaryotic cell lines for the permanently ectopic expression of glutamate receptors, the cell lines being prepared by the transformation of cells containing nucleic acids coding for glutamate receptors. The method is characterized in that, during the establishment of the cell lines, at least one of the following conditions is satisfied for the culture: a) the transformed cells are cultured in a culture medium containing a glutamate precursor; b) the transformed cells are cultured in the presence of a glutamate-receptor antagonist: c) the transformed cells ant cultured in a first phase under conditions in which glutamate-receptor expression is repressed and in a second phase under conditions in which the repression is discontinued. The invention also concerns the cell lines thus obtained and their use.

Description

6~
The perm~nent expres~ion of glutamate receptors Descript ion The invention relates to the permanent ectopic expression of glutamate L~le~ ,Ol# in eukaryotic cells, to the preparation of suitable L~_ ' ins-nt cell lines with the a~u. it-n--d properties, and to the use thereof.
Glutamate is the most important excitatory neurotransmitter in the central nervous system (q'rends ir~ Pharmacological Sciences 11, 1990, 126-132; Pharmacological Re~views 40, 1989, 143-210;
Trends in Pharmacological Sciences 13, 1992, 291-296), and is 15 involved in numerous pathophysiological processes such as epilepsy, schizophrenia and ischemia.
Glutamate Lec0p~vLIi are therefore potential sites of attack by drugs for treating these disorders.
A distinction is made in glutamate L~cé~uL~, between ionotropic (NMDA, AMPA, kainate~ and metabotropic receptors.
However, the invention relates only to the class of ionotropic 2 5 g lutamate L ~ .UL :S .
The primary structure of some subunits of AMPA, kainate and NMDA
re~eptors, and of metabotropic Le~:eu~ol:~, has been elucidated (Nature 342, 1989, 643; Science 249, 1990, 556; Neuron 8, 1992, 30 169 ) .
To date, four rat AMPA glutamate receptor subunits have been described in the literature, GluR-A, GluR-B, GluR-C and GluR-D, each of which occurs in two splicing variants, "flip~ and "flop~, 35 (Science 249, 1990, 1580).
In addition, RNA editing has been shown for mouse and rat GluR-B, which relates to the Q/R site in the second tr~ ne domain.
These two GluR-B variants dif fer con~iderably in their ~0 ele~:~Lu~lly~icological properties (Cell 67, 1991, ll-l9; Neuron 8, 1992, 189--198) . The human cDNAs for GluR-Aflip and GluR-Aflop have likewise been published (PNAS USA 88, 1991, 7557-7561; PNAS
USA 89, 1992, 1443-1447).
45 AMPA receptcr subunits are able to form both homo- and heteromeric channels.

2 1 ~67,Q,8 NMDA receptors may form heteromeric structures whLch consist of one NRl subunit (Nature 354, 3 (1991) ) and one of four NR2 subunits (2A, 2B, 2C, 2D) (Science 256, 1217 (1992~; Nature 358, 36 (1992); Nature 357, 70 (1992); FE}~S Lett. 313, 34 (1992); J.
5 siol. Chem. 268, 2836 (1993)).
NMDA channels shcw slower kinetics than AMPA channels but have a high Ca2+ permeability, have a volta~ de~ d~..L Mg2+ block and require glycine as coagonist . Functional kainate ~ ep~oI s may 10 consist of the ~ubunits GluR5, GluR6, GluR7 (Neuron 5, 583 (1990); Nature 351, 745 (1991); EMB0 J 11, 1651 (1992); Neuron 8, 257 (1992); FE~S Lett. 307, 139 (1992)) and RA1 and RA2 (Na-ture 351, 742 (1991); Neuron 8, 267 and 775 (1992)). These channels are characterized by very rapid kinetics and activation 15 of very rapidly desensitizing currents by AMPA and kainate.
}litherto only transient ectopic expression of glutamate r~ L,LoL~
and subunits in eukaryotic cells has been described ( Science 246, 1990, 556-560 ) .
E~owever, the suitability of such transiently expressing cells as test cells for identifying glutamate receptor antagonists i9 low be~ause they cannot be prepared entirely reproducibly and, as a cnnceqll~n~e, the results obtained therewith are comparable only 25 with great difficulty, if at all.
Permanent ectopic expression of glutamate receptors has not been possible to date. Glutamate i~ an essential amino acid and its presence as nutrient in the culture medium for the cell leads to 30 permanent stimulation of the glutamate L~ p~vLS, which may re~ult rapidly in cell death as a ~ , ..r~ of the resulting ion influx. This applies particularly to ion channels which permit a high ion flux or channels which are Ca2+ permeable (e.g.
NMDA channels or Ca2+ perme~ble AMPA channels).
It is an object of the present invention to provide a process for preparing eukaryotic cell lines with permanent ectopic expression of glutamate receptors.
40 We have found that this object is achieved by a process for preparing eukaryotic cell lines with permal~ent ectopic expre~sion of glutamate receptors by transforming cells with a nucleic acid which codes for glutamate re~ LoL~, wherein at least one of the following culture conditions is maintained during establishment 45 of the cell line:

0 4g7 005 / ~1 2l~7~

a) cultivation of the transformed cells in a culture medium which contains a glutamate precursor, b) cultivation of the transformed cells in the presence of a glutamate receptor antagonist, c) cultivation of the transformed cells in a first phase under conditions with which glutamate receptor expression is repressed, and in a second phase under conditions with which the repression is abolished.
It is generally pos~ible to use eukaryotic cells as cells suitable for the process according to the invention. For example, it is possible to use cells from lower eukaryotes such as yeasts 15 and fungi, or insect cells.
n cell8 are preferably employed, ~or example ~ER 293, ~t~R, COS 7 .
20 These cells are transformed with one or more nucleic acids which code f or a glutamate receptor .
The process according to the invention is suitable ~or permanent expression both of single glutamate receptor subunits and of 25 combinations of several subunits. The~e may be naturally occurring combinations of subunits or novel combinations of subunits which do not occur thus in nature. ~~ ; n~tion of subunits from different species is also po~sible. It is likewise possible to combine glutamate receptor subunits from different 30 s~lh~ os ( for example from AMPA, kainate and NMDA) .
Preferably employed are the single ~ubunits or combinations of single subunits of human glutamate receptors.
35 The nucleic acids used for the transformation are, as a rule, employed linked to an expression vector. The choice of a suitable expression vector depends, inter alia, on the cell to be transformed. Thus, it should be ensured that the regulation signals of the expression vector are also recognized by the cell.
A large number of vectors can be used ~or expression in the eukaryotic cells, with vectors having ~yt , lnvirus promoters showing particularly good expression.

The expression constructs can be introduced into the cells by various msthods, for example electroporation, calcium phosphate precipitation or with liposome mediation.

5 Eukaryotic expression systems have the advantage of expressing corresponding expression products efficiently and usually in native form and of carrying out post-translational modification.
At least one of the culture conditions described above under a), b) and c) must be complied with to establlsh the cell lines 10 according to the invention.
However, it is also possible for more than one of these culture conditions to be maintained simultaneously or else sequentially.
Th9 culture medium used for cell cultivation in the establishment of cell lines under culture conditions according to a) contains no glutamic acid or its salts ( glutam~tes ) . The culture medium contains one or more glutamate precursors which are converted 20 into glutamate in the cells.
Suitable glutamate precursors are compounds which are taksn up as such by the cell and from which glutamine is liberated inside the cell and is subsequently converted into glutamate by metabolic 25 reactions.
Examples of such compounds are glutamine-containing oligopeptides or oligopeptide derivatives such as the corresponding esters or amides. Dipeptides of an apolar amino acid and glutamine are 30 particularly suitable.
Media which contain glutamate precursors are also commercially available, e.g. the Glutamax medium from Gibco BRL, which contains the dipeptide L-alanyl-L-glutamine as glutamate 35 precursor.
Where premixed commercial culture media with glutamate precursors are not employed, the glutamate precursors are, as a rule, added to the culture medium in a final conoentration in the IIM range, 40 preferably of 1 - 100 IIM.
Another possible culture condition for the process according to the invention is the cultivation mentioned under b) of the transformed cells in the presence of a glutamate receptor 45 antagonist.

These ~~ prevent the contlnuous stimulation of the permanently expressed glutamate receptors. ~his makes it possible to prevent the cell death which otherwise occurs on continuous stimulation. Examples of suitable glutamate receptor antagonists 5 are N3QX or CNQX. The final concentration of these uilds in the culture medium is, as a rule, in the IIM range.
Another possible culture condition is the temporary repression 10 mentioned under c) of glutamate receptor expression. This is done by using an inducible expression system which i8 switched on or off ~1~r~nfling on the culture conditions. The culture conditions chosen in a first phase of the establishment and cultivation of cell lines are ones in which the glutamate receptor expression is 15 repressed. Then, in a second phase, the repression i9 abolished, which results in receptor expression.
Preferred inducible expression systems are those controlled by the tetracycline operon.
The invention furthermore relates to the cell lines which have r~.rr n~.nl ectopic ~xpression of glutamate L~ /LuL~ and which can be prepared by the process described above.
25 Cells lines of this type are particularly suitable for identifying functional ligands of glutamate receptors.
Receptor-expressing cell lines are an important in,LL, t in screening for specific receptor ligands. It is possible for this 30 purpose to employ, for example, ~ne3s of the cell lines in receptor binding assays.
Information about the mode of action (agonism/antagonism) of receptor ligands is obtained by introducing reporter syctems into 35 cell lines according to the invention. Suitable reporter systems are those in which a promoter which is regulated by c _ ntlc of the slgnal tlall~du~ LLon pathway ~second - ; ) is functionally connected to a gene for a product which can easily be detected, such a~ luciferase. Reporter systems of this type 40 are disclo~ed, for example, in Science 252, 1424 (1991); Proo.
Natl. Acad. sci. USA 88, 5061 ~1991) or J. Rec. Res. 13, 79 (1993). A suitable promoter which is, for example, regulated by the intracellular ca2+ .:ull< ~.~LL~tion is that of the fos gene.
Likewise ~uitable is the metallothionein promoter which is 45 stimulated, inter alia, by Zn2+.

0050/4~7~1 6 ~8~78~
~he change brought about in the intracellular ion concentration by the binding of a ligand to a receptor can be measured via fluorescent dyes (for example FURA 2AM, sodium green, calcium green, aequorin (Analyt. Biochem. 209, 343 (1993~) or via 5 chemLcal detection reactions (such as precipitation of ion~ (Neu-ron 7, 509 ~1991))~.
It is likewise possible to measure the current flow through the cell membrane as a function of the liyand binding.
The expressed receptor proteins can be used, after appropriate purification, also as antigens for generating polyclonal or monoclonal antibodies which are used for diagnostic purposes or as aids to rational drug design. The pure polypeptide can also be 15 used, after crystallization and x-ray structure analysis or other physical methods such as NMR or scanning tunnelling microscopy, for elucidating the spatial structure of the receptor and of the ligand binding site. The cell lines according to the invention can also be implanted in recipient systems such as transgenic 20 animals or host organisms in order to influence signal transduction or to analyze the ligand concentration.
The invention is illustrated further by the following examples.
2S Example 1 Stable expression of glutamate receptor subunits in HEK 293 cells (A'rCC) .
30 Human embryonic kidney cells transformed with adenovirus Type 5 were cultivated in RPMI 1640 glutamax medium I (Gibco 2RL) with lOY6 dialyzed FCS (Gibco BRL) under 5~ CO2.
The appropriate glutamate receptor cDNA molecules (WO 93~23536;
35 Science 249, 556-560, 1990 J. Biol. Chem. 268, 3728-3733, 1993;
WO 91/06648 ) were cloned into the eukaryotic expressi on vector pcDNA3 tfrom Invitrogen). These expression constructs were introduced into HEK 293 cells singly or in combination by electroporation according to the following protocol: for the 40 electroporation, 107 cells in 0 . 8 ml of PBS were transfected with 20 llg of the expression construct using an electroporator ( BTX, electro cell manipulator 600, 3 mF, 130 V, 72 ohm).
The cells were incubated in culture medium for 24 - 36 h and 45 subsequently transferred into selection medium (culture medium with 600 - 800 ~Ig/ml G418 sulfate, Geneticin, or 50 ~Ig/ml hygromy-cin ) . Stable Geneticin- or hygromyci.n-resistant cell clones were 7~

isolated after 10-12 days by single cell deposition and were ex-panded and analyzed by the membrane binding assay.
Example 2 5 Receptor binding assays on membranes of the cell lines which stably express glutamate receptor.
The recombinant cell lines were cultivated as described in Example 1.
Membrane preparation: cells were scraped off in 2-3 ml of 5 mM
Tris pH 7.4/10 cm dish and spun down (1200 rpm, 4C). ~he pellet was rpc~ ppn~iprl in 1 ml of ice-cold Tris ~ 5 mM, pH 7 . 4 ), incubated on ice for 15 min and ~hs~Pntly spun down at 15 11500 rpm, 4C. The pellet was L~la~nded in 1.5 ml of binding buffer/lO cm dish (30 mM Tris pH 7.2 at 10C, 2.5 mM CaCl2, 100 mM
KSCN), ~ and centrifuged (11500 rpm, 30 min, 4C). The supernatant was again ': , i ~Pd and centrifuged. The membrane pellet is ~ ended in 150 ~ 10 cm di~h of binding buffer and 20 used for the binding assay.
E~inding mixture: 50 ~11 of membranes in binding buffer were incu-bated with 149 1ll of binding buffer and 1 1ll of 3H-AMPA ( 600 nM) on ice for 1 hour, filtered through GFC filters, washed with 25 binding buffer and counted. For the ~ lr ~ reaction, 50 ~
of membranes in binding buffer were incubated with 147 1ll of bind-ing buffer, 2 1ll of glutamate (100 mM) and 1 ~l of 3H-AMPA (600 nM) and treated as for the binding mixture.
30 Example 3 Reporter systems which can be used to detect the effect of binding of ~1 ligand on the l~ignal tran~duction pathway ( second ~sn !'')-35 The cells were cultivated and transfected as described in Example1.
After the transfection, the cell clones were assayed for inducible expression. For this purpoce, the cells were cultivated 40 in 96-well plates (- opaque microtitre plates). 2-48 h after stimulation of the cells, the assay was stopped by lysis of the cells. Lysis, preparation of the cell extracts and determination of the luciferase activity took plac~ using the luciferase assay (Promega, ~o. E1500) in accordance with its instructions. The 45 luciferase activity was measured in conventional 1 i L~:L~.

0050/g4741 8 21 ~,67~
Example 4 Identif;ra~ion of functional ligands for glutamate receptors, the intention being to measure, using fluorescent dyes, the change in the intrA~ 71~r ion concentration brought about by binding of a 5 ligand.
~he L~ in~nt cell lines were cultivated as described in Example 1 .
10 FURA 2 - labeling of cells: cells were detached with trypsin or EDTA and washed with labeling buffer ~120mM NaCl, 5mM KCl, 1.5 n7M
MgC12, lmM CaC12, 25mM }~EPES, lOmM glucose). The cell~ ~2x106 per ml) in labeling buffer were labeled with 2 IJM FURA-2-pentaacetoxy methyl ester at 37C for 15 min and ~ e.~ n~ly washed with la-15 beling buffer. The labeled ~amples were kept on ice and used forthe measurements within 3h.

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Claims (4)

we claim

1. A process for preparing eukaryotic cell lines with permanent ectopic expression of glutamate receptors by transforming cells with a nucleic acid which codes for glutamate receptors, wherein at least one of the following culture conditions is maintained during establishment of the cell line:
a) cultivation of the transformed cells in a culture medium which contains a glutamate precursor, b) cultivation of the transformed cells in a first phase under conditions with which glutamate receptor expression is repressed, and in a second phase under conditions with which the repression is abolished.

2. Process as claimed in claim 1, wherein a glutamine-containing dipeptide is used as glutamate precursor.

3. Cell line with permanent ectopic expression of glutamate receptors, obtainable by a process as claimed in either of claims 1 and 2.

4. The use of a cell line as claimed in claim 3 for identifying functional ligands for glutamate receptors.