WO1995026401A1

WO1995026401A1 - Method for the permanent expression of glutamate receptors

Info

Publication number: WO1995026401A1
Application number: PCT/EP1995/001029
Authority: WO
Inventors: Sylvia Sterrer; Andreas Ultsch; Thomas Höger; Hans-Georg Lemaire; Alfred Bach
Original assignee: Basf Aktiengesellschaft
Priority date: 1994-03-29
Filing date: 1995-03-20
Publication date: 1995-10-05
Also published as: MX9604382A; EP0753064A1; CA2186788A1; DE4410882A1; JPH09510870A

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 are 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

Process for the permanent expression of glutamate receptors

Description 5

The invention relates to the permanent ectopic expression of glutamate receptors in eukaryotic cells: the production of suitable recombinant cell lines with the abovementioned. Properties and their use. 0

Glutamate, the most important excitatory neurotransmitter in the central nervous system (Trends in Pharmacological Sciences 11, 1990, 126-132; Pharmacological Reviews 40, 1989, 143-210; Trends in Pharmacological Sciences 13, 1992, 291-296) is in numerous 5 pathophysiological processes such as Epilepsy, schizophrenia, ischemia involved.

Receptors for glutamate are therefore potential targets for pharmaceuticals to treat these diseases. 0

With glutamate receptors, a distinction is made between ionotropic (N DA, AMPA, kainate) and metabotropic receptors.

However, the invention relates only to the class of ionotropic glutamate receptors.

So far, some subunits of AMPA, kainate, NMDA and metabotropic receptors in their primary structure have been elucidated (Nature 342, 1989, 643; Science 249, 1990, 556; Neuron 8, 30 1992, 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 in two splicing variants, "flip" and "flop", 35 occur (Science 249, 1990, 1580).

For GluR-B from mouse and rat, "RNA editing" has additionally been shown, which relates to the so-called Q / R site of the second transmembrane membrane. These two GluR-B variants differ 40 considerably in their electrophysiological properties (Cell 67, 1991, 11-19; Neuron 8, 1992, 189-198). The human cDNA's for GluR-Aflip and GluR-Aflop have also been published (PNAS USA 88, 1991, 7557-7561; PNAS USA 89, 1992, 1443-1447).

45 AMPA receptor subunits can form both homo- and heteromeric channels. NMDA receptors can form heteromeric structures which consist of an NR1 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); FEBS Lett. 313, 34 (1992); J. Biol. Chem. 268, 2836 (1993)).

NMDA channels have slower kinetics than AMPA channels, but have a high Ca ²⁺ permeability, have a voltage-dependent Mg ²⁺ block and require glycine as a coagonist. Functional Kaina receptors can be derived from the subunits GluR5, GluR6, GluR7 (Neuron 5, 583 (1990); Nature 351, 745 (1991); EMBO J. 11, 1651 (1992); Neuron 8, 257 (1992); FEBS Lett . 307, 139 (1992)) and KAI and KA2 (Nature 351, 742 (1991); Neuron 8, 267 and 775 (1992)). These channels are characterized by very rapid kinetics and the activation of very rapidly desensitizing currents by AMPA and Kainat.

So far, only the transient ectopic expression of glutamate receptors or subunits in eukaryotic cells has been described (Science 246, 1990, 556-560).

Such transiently expressing cells are, however, not very suitable as test cells for the identification of glutamate receptor antagonists, since they cannot be produced in a completely reproducible manner and, as a result, the results obtained therefrom cannot be compared or can only be compared with great difficulty.

So far, permanent ectopic expression of glutamate receptors has not been achieved. The essential amino acid glutamate present as a nutrient in the culture medium of the cell leads to permanent stimulation of the glutamate receptors, as a result of which the resulting ion influx can lead to rapid cell death. This applies in particular to ion channels that allow a high ion flow or channels that are Ca ²⁺ permeable (eg NMDA channels or Ca ²⁺ permeable AMPA channels).

The object was therefore to provide a method for producing cell lines expressing eukaryotic permanent ectopic glutamate receptors.

The invention relates to a process for the production of eukaryotic cell lines expressing permanently ectopic glutamate receptors by transforming cells with a nucleic acid coding for glutamate receptors, characterized in that at least one of the following culture conditions is maintained when establishing the cell lines: a) culturing the transformed cells in a culture medium which contains a glutamate precursor,

b) culturing the transformed cells in the presence of a glutamate receptor antagonist,

c) Culturing the transformed cells in a first phase under conditions in which the glutamate receptor expression is repressed and in a second phase under conditions in which the repression is released.

Eukaryotic cells can generally be used as cells which are suitable for the method according to the invention. For example, cells from lower eukaryotes such as yeasts and fungi, or insect cells can be used.

Cells from mammals are preferably used, for example HEK 293, BHK, COS 7.

These cells are transformed with one or more nucleic acids which code for a glutamate receptor.

The method according to the invention is suitable both for the permanent expression of individual glutamate receptor subunits and for combinations of several subunits. These can be naturally occurring combinations of subunits as well as new, not naturally occurring combinations of subunits. It is also possible to recombine subunits of different species. The combination of glutamate receptor subunits from different subclasses (for example from AMPA, Kainat, NMDA) is also possible.

The individual subunits or combinations of individual subunits of the human glutamate receptors are preferably used.

The nucleic acids used for the transformation are generally used linked to an expression vector. The choice of a suitable expression vector depends, among other things, on the cell to be transformed. This should ensure that the regulatory signals of the expression vector are also recognized by the cell. A large number of vectors can be used for expression in eukaryotic cells, wherein vectors with cytomegalovirus promoters show particularly good expression.

The expression constructs can be introduced into the cells by means of various methods, for example electroporation, calcium phosphate precipitation or liposome-mediated.

Eukaryotic expression systems have the advantage of expressing the corresponding expression products effectively and mostly in native form and also of modifying them post-translationally. To establish the cell lines according to the invention, at least one of the culture conditions described under a) b) and c) must be fulfilled.

However, several of these culture conditions can also be observed at the same time or in different stages.

When establishing cell lines under culture conditions according to a), a culture medium is used for cell growth which does not contain glutamic acid or its salts (glutamates). The culture medium contains one or more glutamate precursors which are converted into glutamate in the cells.

Suitable glutamate precursors are compounds which are taken up as such by the cell and from which intracellularly releases glutamine, which is subsequently converted into glutamate by metabolic reactions.

Such compounds are, for example, glutamine-containing oligopeptides or oligopeptide derivatives such as the corresponding esters or amides. Dipeptides composed of an apolar amino acid and glutamine are particularly suitable.

Such media containing glutamate precursors are also commercially available, e.g. the Gluta ax medium from Gibco BRL, which contains the dipeptide L-alanyl-L-glutamine as the glutamate precursor.

If no pre-mixed commercial culture media with glutamate precursors are used, the glutamate precursors are generally added to the culture medium in a final concentration in the μM range, preferably from 1 to 100 μM. Another possible culture condition for the method according to the invention is the culturing of the transformed cells mentioned under b) in the presence of a glutamate receptor antagonist.

These compounds prevent permanent stimulation of the permanently expressed glutamate receptors. This makes it possible to prevent cell death that otherwise occurs in the case of continuous stimulation. Suitable glutamate receptor antagonists are, for example, NBQX or CNQX. The final concentrations of these compounds in the culture medium are usually in the μM range.

Another possible culture condition is the temporary repression of glutamate receptor expression mentioned under c). An inducible expression system is used for this, which is switched on or off depending on the culture conditions. In a first phase of the establishment and cultivation of cell lines, culture conditions are selected in which the glutamate receptor expression is repressed. In a second phase, the repression is then released, which causes a receptor expression.

Preferred inducible expression systems are those which are controlled by means of the tetracycline operon.

The invention further relates to the cell lines which permanently express ectopic glutamate receptors and which can be prepared using the method described above.

Soche cell lines are particularly suitable for the identification of functional ligands of glutamate receptors.

Receptor-expressing cell lines are an important tool in the screening for specific receptor ligands. For this purpose, membranes of the cell lines can, for example, be used in receptor binding tests.

Information about the mode of action (agonism / antagonism) of receptor ligands is obtained by introducing reporter systems into cell lines according to the invention. Suitable reporter systems are those in which a promoter which is regulated by compounds of the signal transduction pathway (second messenger) is functionally linked to a gene for an easily detectable product such as luciferase. Such reporter systems are, for example, from Science 252, 1424 (1991); Proc. Natl. Acad. Be. USA 88, 5061 (1991) or J. Rec. Res. 13, 79 (1993). A suitable promoter, which is regulated, for example, by the intracellular Ca ²⁺ concentration, is that of the fos gene. The me- Tallothionein promoter, which can be stimulated by Zn ²⁺ , is also suitable.

The change in the intracellular ion concentration caused by the binding of a ligand to a receptor can be determined via fluorescent dyes (for example FURA 2AM, sodium green, calcium green, aequorin (Analyt. Biochem. 209, 343 (1993)) or via chemical detection reactions (such as precipitation by ions (Neuron 7, 509 (1991)).

The current flow through the cell membrane depending on the ligand binding can also be measured.

After appropriate cleaning, the expressed receptor proteins can also serve as antigens for generating polyclonal or monoclonal antibodies which are used for diagnostic purposes or as aids for rational drug design. After crystallization and X-ray structure analysis or other physical processes such as NMR or scanning tunneling microscopy, the pure polypeptide can also be used to elucidate the spatial structure of the receptor and the ligand binding site. The cell lines according to the invention can also be implanted in recipient systems such as transgenic animals or host organisms in order to influence the signal transduction or to analyze the ligand concentration.

The invention is illustrated further by the following examples.

example 1

Stable expression of the glutamate receptor subunits in HEK 293 cells (ATCC), human embryonic kidney cells transformed with adenovirus type 5, were in RPMI 1640 Gluta ax Medium I (Gibco BRL) with 10% dialyzed FCS (Gibco BRL) under 5% CO cultured.

The corresponding glutamate receptor cDNA molecules (WO 93/23536; Science 249, 556-560, 1990; J. Biol. Chem. 268, 3728-3733, 1993; WO 91/06648) were converted into the eukaryotic expression vector pcDNA3 (Fa. Invitrogen) cloned. These expression constructs were introduced individually or in combination in HEK 293 cells according to the following protocol by electroporation: For the electroporation, 10 ⁷ cells in 0.8 ml PBS with 20 μg of the expression construct with an electroporator (BTX, electro cell manipulator 600, 3 mF, 130 V, 72 Ohm) transfected. The cells were incubated for 24-36 h in culture medium and then transferred to selection medium (culture medium with 600-800 μg / ml G418 sulfate, geneticin or 50 μg / ml hygromycin). Stable genetic or hygromycin-resistant cell clones were isolated after 10-12 days via single cell deposition, expanded and analyzed by means of a membrane binding assay.

Example 2

Receptor binding assays on membranes of the cell lines stably expressing 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 centrifuged (1200 rpm, 4 ° C.). The pellet was resuspended in 1 ml of ice-cold Tris (5 mM, pH 7.4), incubated for 15 min on ice and then centrifuged at 11500 rpm, 4 ° C. The pellet was resuspended in 1.5 ml binding buffer / 10 cm dish (30 mM Tris pH 7.2 at 10 ° C.), 2.5 mM CaCl, 100 mM KSCN), homogenized, centrifuged (11500 rpm, 30 min , 4 ° C). The supernatant was homogenized again and centrifuged. The membrane pellet is resuspended in 150 μl / 10 cm dish of binding buffer and used for the binding assay.

Binding batch: 50 μl membranes in binding buffer with 149 μl binding buffer and 1 μl ³ H-AMPA (600 nM) were incubated for 1 hour on ice, filtered through GFC filters, seen with binding buffer and counted. For the displacement reaction, 50 μl membranes were incubated in binding buffer with 147 μl binding buffer, 2 μl glutamate (100 mM) and 1 μl ³ H-AMPA (600 nM) and the procedure was the same as for the binding approach.

Example 3

Reporter systems with the aid of which the influencing of the signal transduction path (second messenger) caused by the binding of a ligand can be detected.

The cells were cultivated and transfected as described in Example 1.

After the transfection, the cell clones were tested for inducible expression. For this purpose, the cells were cultivated in 96 well plates (= opaque microtiter plates). 2-48 h after stimulation of the cells, the test was terminated by lysis of the cells. Lysis, production of cell extracts and determination of the Luciferase activity was reported and using the luciferase assay kit (Promega, # E1500). Measurement of the luciferase activity was carried out in conventional luminometers.

Example 4

Identification of functional ligands for glutamate receptors, the change in intracellular ion concentration caused by binding of a ligand being measured by means of fluorescent dyes.

The recombinant cell lines were cultivated as described in Example 1.

FURA 2 - labeling of cells: cells were detached with trypsin or EDTA and washed with labeling buffer (120 mM NaCl, 5 mM KCl, 1.5 mM MgCl ₂ , ImM CaCl, 25 mM HEPES, 10 mM Glucose). The cells (2 × 10 ⁶ per ml) in labeling buffer were labeled with 2 μM FURA-2-pentaacetoxymethyl ester for 15 min at 37 ° C. and then washed with labeling buffer. The marked cells were kept on ice and used for the measurements within 3 h.

Claims

claims

1. A process for the production of eukaryotic cell lines expressing permanently ectopic glutamate receptors by transforming cells with nucleic acids coding for glutamate receptors, characterized in that at least one of the following culture conditions is observed when establishing the cell lines:

a) culturing the transformed cells in a culture medium which contains a glutamate precursor,

2. The method according to claim 1, characterized in that a glutamine-containing dipeptide is used as the glutamate precursor.

3. Cell lines which permanently express ectopic glutamate receptors, obtainable by a method according to one of claims 1 to 2.

4. Use of the cell lines according to claim 3 for the identification of functional ligands for glutamate receptors.