WO1999033973A2

WO1999033973A2 - Cloned octapamine-receptor-producing cells

Info

Publication number: WO1999033973A2
Application number: PCT/EP1998/008411
Authority: WO
Inventors: Arnd Baumann; Stephan Frings
Original assignee: Forschungszentrum Jülich GmbH
Priority date: 1997-12-23
Filing date: 1998-12-22
Publication date: 1999-07-08
Also published as: DE19757695A1; DE19758545A1; WO1999033973A3

Abstract

The invention relates to a method for producing octopamine receptor-producing cells. To this end, at least one gene with coding for an octopamine receptor is transformed into a host cell. The cloning of neurotransmitter receptor genes and their stable expression makes it possible to investigate individual neurotransmitter receptor subtypes with precisely defined structures, for the first time. The cells transformed by the neurotransmitter receptor gene can be used in a simple method for investigating the characteristics of neurotransmitter receptors. According to said method, a ligand to be investigated is allowed to act on these cells and the effect is determined by analysing intracellular messenger substances.

Description

Cloned cells producing octopamine receptor

The invention relates to a method for producing octopamine receptor-producing cells according to claims 1 to 10, to octopamine receptor genes according to claims 11 to 14, octopamine receptors according to claims 15 and 16, gene constructs according to claims 17 and 18, transformed cells Claims 19 to 23 and a method for examining the properties of neurotransmitter receptors according to Claims 24 to 27.

Nerve cells or neurons form the functional units of the brain. The main task of a neuron is to transfer information to other neurons or to muscle and gland cells. To accomplish this task, each neuron not only has a diverse repertoire of highly specialized, membrane-bound protein molecules (proteins), but also an extraordinary cellular structure. The following cell areas are morphologically differentiated: dendrite, cell body, axon and synaptic end button. The information is recorded in the dendritic area of the neuron and is passed on as an electrical signal. If a certain threshold value of electrical excitation is exceeded, an action potential is triggered just below the cell body - in the area of the axon hillock. The action potential is characterized by the selective inflow of sodium ions through sodium channels into the cell interior. One speaks of a depolarization. The sodium ion inflow is affected by a time-delayed potassium ion Outflow through potassium channels counter (repolarization), so that the resting potential is set again. The action potential propagates along the axon down to the synaptic end button. This is the area of the neuron where the cell contacts target cells. The contact point is also known as a synapse. However, the cell membrane of the neuron and the target cell are not in direct contact, but are separated by a fluid-filled area, the synaptic cleft. The electrical signal (action potential) cannot be passed on via the synaptic gap. To transmit signals from the neuron to the target cell, the electrical signal must therefore be translated into a chemical signal. This is achieved in that the action potential causes the release of messenger substances, the so-called neurotransmitters, from the synaptic end button. After being released into the synaptic gap, the neurotransmitters diffuse to the surface of the target cell and bind there to high-affinity binding proteins, the so-called neurotransmitter receptors, which are specific to the respective neurotransmitter.

A distinction is usually made between two groups of neurotransmitter receptors: The first group combines those receptors that can themselves form ion channels (ionotropic receptors). These include the acetylcholine and glutamate receptors, which, when activated, depolarize the target cell and trigger a new action potential. Inhibitory receptors, such as glycine and γ-aminobutyric acid (GABA) -

Receptors hyperpolarize the target cell and therefore counteract the triggering of a new action potential. The second group of neurotransmitter receptors does not itself form any ion channels. The activation of these receptors is triggered by the binding of the specific ligand (= neurotransmitter) and leads to a change in the concentration of intracellular messenger substances, the so-called "second messenger" molecules. The receptors are therefore also called metabotropic receptors. Members of this receptor family have one thing in common Structural characteristic of seven hydrophobic sections in the primary structure (amino acid sequence) that span the cell membrane The change in the intracellular messenger concentration is achieved by the receptors interacting with GTP-binding (G) proteins. This interaction activates and stimulates the G proteins or inhibit downstream target enzymes, for example activation of the adenylate cyclase leads to an increase in the intracellular concentration of cyclic adenosine 3 ', 5'-monophosphate (cAMP). Another way is to activate a phospholip ase C and the resulting synthesis of inositol-l, 4,5-triphosphate (IP ₃ ) and diacylglycerol (DAG).

As a consequence of the regulation of different messenger pathways by the neurotransmitter receptors, there can be a change (modulation) in the properties of ion channels, enzymes and even gene activation. The metabotropic receptors thus intervene in the regulation of cellular activity and can decisively influence the plasticity of neuronal circuits. In the nervous system, modulation of synaptic plasticity is believed to play a crucial role in connection with learning performance and in memory formation. Many of the neurotransmitter / receptor systems known today have been preserved in the course of evolution. However, some only occur in certain animal species. An example of the differentiation of neurotransmitters can be found in the group of biogenic amines. The group of biogenic amines includes the neurotransmitters adrenaline, dopamine, histamine, noradrenaline, octopamine, serotonin and tyramine.

Dopamine, histamine and serotonin have been clearly demonstrated in both vertebrates and invertebrates as neurotransmitters. In contrast, the neurotransmitters adrenaline and noradrenaline are only found in vertebrates (Venter, JC et al .: Evolution of neurotransmitter receptor Systems ", Progress in Neurobiology 30, 1988, 105-169). The tasks of adrenaline / noradrenaline are in insects and many other invertebrates mainly taken over by octopamine.

In evertebrates, octopamine acts as a neurotransmitter, neurohormone and also as a neuromodulator and controls or modulates many biochemical reactions, motor skills and behavior. For example, it is described that octopamine and others. regulates metabolism and energy balance. In addition, octopamine modulates the animals' biorhythms and is crucial for learning and memory building. Only low octopamine concentrations are found in vertebrates. However, it is unclear whether the substance has neuroactive activity.

Octopamine receptors have also only been written in evertebrates. These proteins belong to the large gene family of the G protein-coupled receptors. Based on pharmacological binding studies and the investigation of the activated intracellular messenger pathways, two classes of octopamine receptors are distinguished (Evans, PD and Robb, S .: Octopamine receptor subtypes and their modes of action; Neurochemical Research 18, 1993, 869-874). One receptor class - octopamine 1 (OCTl) receptors - causes an increase in the intracellular Ca ²⁺ concentration - presumably induced via the intracellular messenger IP ₃ . The second class of receptors - octopamine 2 (OCT2) receptors - stimulates adenylate cyclase and there is an increase in the intracellular cAMP concentration. The physiologically effective concentration of octopamine is in the range from 10 " ⁸ to 10" ¹⁰ M.

Since octopamine is only detected as a neurotransmitter in invertebrates, it would be of great interest to develop specific pharmaceuticals (ligands) that only bind to octopamine receptors. Since these receptors are not found in vertebrates, such ligands could be used as insecticides without fear of side effects in vertebrates.

So far, pharmacological studies of the neurotransmitter receptors could only be carried out on tissue homogenates. This procedure has the disadvantage that the homogenate consists of a mixture of different receptors or subtypes, i.e. the receptor populations are present side by side or mixed (Roeder, T. and Nathanson, JA: Characterization of insect neuronal octopamine receptors; Neurochemical Research 18, 1992 , 921-925; Hiripi, L. et al .: Characterization of ty ramine and octopamine receptors in the insect (Locusta migratoria migratorioides) brain; Brain Research 633, 1994, 119-126). In addition, the preparation of such tissue homogenates is relatively time-consuming and labor-intensive and sometimes requires the use of protein chemical cleaning and separation processes. A similar problem also applies to the quantification of the intracellular messengers cAMP and IP ₃ . The determination of the corresponding concentrations is labor-intensive, time-consuming and often error-prone because different messenger pathways can be activated simultaneously in the tissue preparations.

Furthermore, the characterization of the receptors has so far mainly been carried out using the method of radioligand binding. For this purpose, tissue homogenates are incubated with different, radioactively labeled compounds and the specifically bound radioactivity is measured after several filtration and washing steps. The classification of the receptors is only possible if a variety of different substances have been tested to determine whether and which substance is suitable to displace the radioactively labeled compound from the receptor protein (competition experiments). This procedure is also labor-intensive and, due to the use of the radio-labeled compounds, is not harmless for environmental and health reasons (radiation exposure). The use of a detection method that does not require the use of radioactive material is therefore desirable and advantageous.

It is therefore an object of the invention to provide materials with which a simple method for examining the properties of neurotransmitter receptors is made possible. This object is achieved according to the invention by a method for producing octopamine receptor-producing cells, in which at least one gene coding for an octopamine receptor is transformed into a host cell. For this purpose, octopamine receptor genes are preferably cloned from the fruit fly Drosophila melanogaster. After cloning or isolation, in particular genes with the SEQ ID no. 1 and / or 3 specified nucleotide sequences or with one for which under SEQ ID No. 2 and / or 4 indicated amino acid sequence and / or their allele variations coding nucleotide sequence available. These genes can be used primarily for the production of octopamine receptor type 1 and / or 2 (OCT1 or 2) producing cells.

Using a DNA probe, for example from a cDNA clone that codes for a Drosophila dopamine receptor (Gotzes, F. et al .: Primary structure and functional characterization of a Drosophila dopamine receptor with high homology to human Dl / 5 receptors; Receptors & Channels 2, 1994, 131-141), can be genomic and cDNA libraries under degraded

Stringency conditions are hybridized. Sub-fragments of a genomic DNA clone can then be used to hybridize a cDNA library which has been produced, for example, from adult head mRNA, with high stringency. The two cDNA clones obtainable in this way are identical in the 5 'region, but differ immediately behind the sequence section which codes for the transmembrane region 5. Studies of the gene structure show that the mRNAs which code for the receptors are written off from the gene by so-called "alternative splicing". For the stable expression of neurotransmitter receptor genes in host cells, these are incorporated into gene constructs, for example into the plasmid pcDNAIneo (Invitrogen, 9351 NV Leek, The Netherlands). The constructs can then in particular in a cell line, preferably in the human kidney cell line HEK293, with the calcium phosphate method (Chen, C. and Okayama, H .: High-efficiency transformation of mammalian cells by plasmid DNA; Molecular and Cellular Biology 7 , 1987, 2745-2752). Cell clones that have taken up the DNA stably can be selected by adding the antibiotic Geneticin (G418 sulfate). The DNA molecules that code for the above-mentioned splice variants can also be integrated into the genome of the host cell independently of one another.

In addition to using the cell line mentioned (HEK 293), it is also conceivable to introduce the DNA which codes for a receptor into other cells, such as, for example, COS, NIH 3T3 or Drosophila S2 cells. This can be done in a stable form, as described above, or alternatively by transient transfection. The disadvantage of transient transfection, however, is that the receptors only last for a short time, i.e. can be examined up to approx. 48 h after the transfection. After that, the progressive cell division means that the expression constructs are only present in the cells in small numbers of copies. This results in a lower expression rate or synthesis rate of the receptor proteins.

Another way of expressing the receptor proteins is, for example, using the baculovirus system. For this, the DNA only has to be cloned into an appropriate expression plasmid. The cloning of octopamine receptor genes and their stable expression makes it possible for the first time to examine individual, structurally precisely defined neurotransmitter receptor subtypes. Since the cells according to the invention produce approximately the same amounts of receptors, a further disadvantage of using tissue homogenates is overcome, namely the fluctuation in the receptor density, which can be extremely variable from experiment to experiment. In addition, in particular stable cell lines are grown under constant and easy to control conditions with relatively little effort. This offers the possibility of automating the multiplication process: for example, the cells can be grown in fermenters.

Such cells transformed with neurotransmitter receptor genes can then be used in a simple method for examining the properties of neurotransmitter receptors, in which a ligand to be examined is allowed to act on these cells and the effect is determined by determining intracellular messenger substances. For example, after the neurotransmitter or ligand has been bound, cloned octopamine receptors activate a cell-specific G protein and subsequently a phospholipase. This enzyme (PLC) cleaves a membrane-based substrate into two intracellular messenger substances: IP3 and DAG. The IP3 binds to an intracellular receptor, which enables the passage of Ca ²⁺ ions from the cell compartments into the cytoplasm of the cell. The increase in the cytoplasmic Ca ²⁺ ion concentration can be registered with the aid of a Ca ²⁺ sensitive indicator, the fluorescent dye FURA-2. The intracellular Ca ²⁺ ion concentration is well calibrated and the change in concentration quantifiable. The relative change in the Ca ²⁺ ion concentration is therefore a measure of whether the ligand tested activates the receptor, ie acts as an agonist, or alternatively in the presence of octopamine, which inhibits receptor activity, ie acts as an antagonist. This procedure enables the investigation of a large number of substances (screening) with high throughput numbers. Important information about the properties of the ligands tested is thus available in a relatively short time, for example whether a ligand is agonistic or antagonistic and what affinity and specificity the ligand has for the receptor. Since the measurement is carried out on intact cells, all the labor-intensive steps required to produce tissue homogenates are also eliminated. The measurement method, the so-called Ca ²⁺ imaging, is established (Roe, MW et al .: Assessment of FURA-2 for measurements of cytosolic free calcium; Cell Calcium 11, 1990, 63-75) and can also be used for screening methods be automated.

However, the method according to the invention can also be transferred to other receptors, for example those which bring about the production of the messenger substance cAMP. The increase in the intracellular cAMP concentration can be registered by ion channels that open depending on the cAMP and allow the influx of Ca ²⁺ ions. The cyclic nucleotide-controlled ion channels have these properties. The increase in cAMP concentrations thus also causes an increase in the intracellular Ca ²⁺ concentration, which can be measured using the Ca ²⁺ imaging method. In contrast to the signaling pathway mediated by the octopamine receptor, however, the Ca ^{2+ flows} in this second variant from the extracellular area into the cell interior. The method according to the invention can therefore be applied, inter alia, to all other receptor systems which bring about an increase in the intracellular messenger substances cAMP / cGMP and IP3. Coupling paths can also be recorded, as a result of which the messenger concentration - for example of cAMP / cGMP - drops.

In summary, the methods according to the invention and the cells according to the invention offer i.a. following advantages:

1. The availability of the receptors in a stably expressed form offers the possibility of examining defined, homogeneous receptor preparations. The cells can be cultivated in small dishes, so that many substances can be tested in parallel. This option is particularly important for screening procedures in the pharmaceutical industry.

2. The examination method according to the invention offers a great time advantage. The change in the intracellular Ca ²⁺ concentration can be registered and quantified within a few minutes.

3. The process can be largely automated and many work steps can be carried out mechanically.

The production method according to the invention of cells producing octopamine receptor and the one according to the invention

Examination methods are explained in more detail using the following exemplary embodiment: Embodiment

1. Isolation of the octopamine prescription gene

The method of low-stringent hybridization of gene libraries was used to isolate the receptor gene. A Hindlll / Nrul restriction fragment of the Drosophila dopamine Dl receptor (DmDop [-126]; Gotzes, F. and Baumann, A .: Functional properties of Drosophila dopamine Dl receptors are not altered by the size of the N-terminus ; Biochemical Biophysical Research Communications 222, 1996, 121-126). For this purpose, the plasmid DNA was cleaved as follows:

DmDopl [-126] (3 µg) 3.0 µl

Restriction buffer (10 x) 3.0 ul H ₂ 0 22.0 ul

Hindlll (lOU / μl) 1.0 μl

Nrul (lOU / μl) 1.0 μl

The incubation was carried out at 37 ° C. for 90 min. The mixture was then mixed with 6 μl stop buffer (5 ×) (100 mM EDTA, 20% (w / v) Ficoll 400, 0.01% (w / v) bromophenol blue , 0.01% (w / v) Xylencyanol) added, applied to a 0.75% agarose gel (with ethidium bromide) and separated electrophoretically. The -1000 bp HindIII / Nrul fragment was cut out of the agarose gel under UV light and the DNA from the agarose piece was centrifuged (Heery, DM et al .: A simple method for subcloning DNA fragments from gel slices; Trends in Genetics 6, 1990, 173). 100 ng of the purified fragment were radioactively labeled with [32p] dCTP using the "Megaprime DNA Labeling System" from Amersham.

A gene library in which genomic DNA of the Drosophila wild-type strain CantonS is cloned in Charon 4A λ phages (Maniatis, T. et al: The isolation of structural genes from libraries of eucaryotic DNA; Cell 15, 1978, 687-701) was opened Plated agar plates. Filter prints were made from 15 plates, each of which had ~ 45,000 recombinant λ phages grown on them (Sambrook, J. et al: Molecular Cloning: A laboratory manual, 2 ^nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY , 1989). The membrane discs (Quibrane; Qiagen, Hilden) were prehybridized for 2 h at 65 ° C in a solution which had the following composition: 5 x SET (20 x SET = 3 M NaCl, 400 mM Tris-HCl pH 7.4, 20 mM EDTA), 5 x Denhardts (100 x Denhardts = 2% (w / v) bovine serum albumin, 2% (w / v) Ficoll400, 2% (w / v) polyvinylpyrrolidone), 0.1% (w / v ) SDS and 150 μg / ml autoclaved herring sperm DNA. This solution was then replaced with fresh

Hybridization solution was exchanged, which contained ~ 1 x 10 ^ cpm / ml of the radiolabelled sample. The first hybridization was carried out under high stringency for 16 h at 65 ° C. The non-specifically bound radioactivity was removed by two washing steps at 65 ° C in 1 x SET / 0.1% SDS for 30 minutes each. Specifically bound radioactivity was visualized by placing an X-ray film for 4-14 h at -80 ° C. The signals found correspond to gene sequences which are identical to the DmDopl gene. 33973

14

In order to obtain new gene sequences, the stringency was reduced in a second round of hybridization. The hybridization temperature was reduced from 65 ° C to 52 ° C. After the hybridization, the non-specifically bound radioactivity was removed in two washing steps at 52 ° C. in 2 × SET / 0.1% SDS for 25 minutes each. The specific signals were again made visible by placing an X-ray film. In comparison to the first round of hybridization, an additional signal was identified on the X-ray film.

The signal was assigned to a "plaque region" on the agar plate. This region was excised from the agar and eluted in phage buffer (Sambrook, J. et al., See above). After several rounds of separation, the hybridization signals could finally be assigned to individual plaque regions on the agar plate. The recombinant λ phage DNA was then isolated from small lysates

(Santos, MA: An improved method for the small scale preparation of bacteriophage DNA based on phage precipitation by zinc chloride; .Nucleic Acids Research 19, 1990, 5442). The DNA was digested with several single and double combinations of restriction enzymes, mixed with stop buffer (see above), applied to a 0.75% agarose gel (with ethidium bromide) and separated electrophoretically. The DNA was transferred from the agarose gel to a nylon membrane (Sambrook, J. et al., See above) and this Southern blot was hybridized again with the above-mentioned probe with low stringency. Several restriction fragments showed a specific hybridization signal. Some fragments were selected and isolated from the λ phage DNA using the above methods. The fragments were in suitable pBluescript plasmid vectors subcloned. The ligation approaches had the following composition:

pBluescript vector (50ng) 1.0 ul

DNA fragment (10 ng) 3.0 ul

Ligase buffer (10 x) 1.5 ul

H ₂ 0 8.5 ul T4 DNA ligase (1U) 1.0 ul

The incubation was carried out at room temperature for 3 h. The ligation mixture was transformed into transformation-competent cells of the bacterial strain XL1-Blue (Invitrogen). The cells were spread on LB agar plates containing ampicillin (Sambrook, J. et al, see above) and incubated at 37 ° C. overnight. Individual colonies were grown in 5 ml LB liquid cultures and the plasmid DNA was isolated by the alkaline lysis method (Sambrook, J. et al., See above). The correct incorporation of the DNA fragments was checked by restriction analyzes. Larger amounts of the plasmid subclones were purified from 50 ml liquid cultures. The nucleic acid sequence of the subcloned DNA fragments was determined by the chain termination method (Sanger, F. et al: DNA sequencing with chain-terminating inhibitors; Proceedings National Academy Sciences USA 74, 1977, 5463-5467). A comparison of the nucleic acid and derived amino acid sequences with those of the DmDopl receptor showed that the cloned genomic DNA contains gene sequences which code for two previously unknown members of the G protein-coupled receptor gene family. The new sequence information was used to synthesize oligonucleotides that were used in polymerase chain reactions (PCR). The 5 'oligonucleotide had the sequence: (# 1) 5'-GCCGTACTCGAGTTCATCAAC-3'. Two 3 'located

Counter oligonucleotides had the sequence: (# 2) 5'-

GGCTGAATGCAGTGGTCG-3 'and (# 3): 5'-GGTCGACGCAGTCCTGGC-3'. PCR approaches were carried out with the oligonucleotide pairs # 1 / # 2 and # 1 / # 3. A 2nd strand cDNA preparation, which had been synthesized on Drosophila head mRNA as starting material, served as the template. The PCR approaches had the following composition:

2nd strand cDNA (25 ng) 1.0 ul oligonucleotide # 1 (10ng / base x ul) 1.0 ul

Oligonucleotide # 2 or # 3 (10ng / base x ul) 1.0 ul

Prime-Zyme buffer (10 x) 10.0 μl

Deoxynucleotides (20 mM each) 1.0 ul

H ₂ 0 85.0 μl Prime-Zyme (2 U; Biometra) 1.0 μl

The amplification was carried out with the following parameters:

1 cycle: 94 ° C, 2 min. 30 sec.

the following cycle sequence was then carried out 45 times:

94 ° C, 1 min. 56 ° C, 1 min. 72 ° C, 1 min. 30 sec. After amplification, the fragments were extracted twice with phenol / chloroform, then twice with chloroform and then precipitated by adding 0.1 vol. LiCl (3 M) and 3 vol. Ethanol (absolute). After centrifugation: Sigma centrifuge, 18,000 xg, 10 min., 4 ° C., the precipitate was dried and taken up in 10 μl TE buffer (10 mM Tris-HC1, pH 7.4; 1 mM EDTA). The fragments were phosphorylated:

DNA fragment 10.0 ul

Kinase buffer (10 x) 1.5 ul

ATP (10 mM) 0.5 ul

H ₂ 0 2.0 ul

Polynucleotide kinase (5 U / µl) 1.0 µl

Incubation was carried out at 37 ° C for 30 min. The enzyme was then inactivated at 65 ° C. for 10 minutes. The DNA was mixed with stop buffer (see above), applied to a 0.75% agarose gel (with ethidium bromide), separated electrophoretically and then isolated from the agarose gel using the centrifugation method (Heery, DM et al., See above). The purified fragments were ligated into an EcoRV cut, dephosphorylated pBluescript vector and then transformed into XL1-Blue cells (see above). The cells were spread on LB agar plates containing ampicillin (Sambrook, J. et al, see above) and incubated at 37 ° C. overnight. Individual colonies were grown in 5 ml LB liquid cultures and the plasmid DNA was isolated by the alkaline lysis method (Sambrook, J. et al., See above). The incorporation of the PCR fragments was carried out by a restriction analysis with the Restriction enzymes EcoRI and Hindill checked. One clone each, which contained the PCR fragment # 1 / # 2 or # 1 / # 3, was sequenced.

To isolate the complete cDNA clones, the PCR fragments from the subclones (see above) were cut out with EcoRI / HindIII, separated by gel electrophoresis and isolated from the agarose (see above). The fragments were radioactively labeled (see above) and used to hybridize an adult Drosophila head cDNA library. The library has already been used to isolate the DmDopl receptor gene (Gotzes, F. et al., See above). The cDNA molecules were cloned into λgtll phages. The library was hybridized with high stringency (see above). The DNA of the individual phages was prepared from small lysates (Santos, M.A., see above), cut with EcoRI and subcloned into a pBluescript vector which was also cut with EcoRI. The nucleic acid sequences of the recombinants were determined in an overlapping double-stranded manner using suitable subclones and gene-specific oligonucleotides according to the chain termination method (Sanger, F. et al., See above).

2. Production of a cell line which stably expresses the octopamine 1B receptor from Drosophila:

The octopamine receptor genes should be expressed in a human embryonic kidney cell line (HEK 293 cells). In order to achieve optimal expression, experience has shown that it makes sense to place the consensus sequence of the ribosome binding site of vertebrate gene sequences (CCACC, see Kozak, cf. M .: Compalition and analysis of sequences upstream from the translational start site in eukaryotic mRNAs; Nucleic Acids Research 12, 1984, 857-872). This is done using the polymerase chain reaction (PCR) with a so-called mutagenesis oligonucleotide. At the same time as the Kozak sequence is inserted, a suitable restriction site is inserted at the 5 'end of the amplified fragment. In the present case, this was the recognition sequence for the restriction enzyme EcoRI (GAATTC). The mutagenesis oligonucleotide had the sequence: 5'-CTCAGGAATTCCACCATGAATGAAACAGAGTGC-3 '. The counter oligonucleotide for the PCR has the sequence: 5'-CCATCCTCCGAGCTTGA-3 '. This sequence is complementary to nucleotides 1009-1025 of the open reading frame of the nucleic acid sequence of the Drosophila octopamine receptors.

The PCR was carried out in a 100 μl batch:

Receptor cDNA clone (in pBluescript, 10 ng) 1.0 ul mutagenesis oligonucleotide (10 ng / base x ul) 1.0 ul

Counter oligonucleotide (10 ng / base x ul) 1.0 ul

Prime-Zyme buffer (10 x) 10.0 μl

Deoxynucleotide (20 mM each) 1.0 ul

H ₂ 0 85.0 μl Prime-Zyme (2 U; Biometra) 1.0 μl

The amplification was carried out with the following parameters: 1 cycle: 94 ° C, 2 min. 30 sec.

The following cycle sequence was then carried out 25 times:

94 ° C, 45 sec. 54 ° C, 45 sec. 72 ° C, 45 sec.

After amplification, the fragment was extracted twice with phenol / chloroform, then twice with chloroform and then precipitated by adding 0.1 vol. LiCl (3 M) and 3 vol. Ethanol (absolute). After centrifugation (Sigma centrifuge, 18,000 x g, 10 min., 4 ° C.) the precipitate was dried and taken up in 10 μl TE buffer (10 mM Tris-HC1, pH 7.4; 1 mM EDTA).

The fragment was cut with the restriction enzymes EcoRI (5 'end) and Nael (cleaves at position 426):

PCR fragment 10.0 ul

Restriction buffer (10 x) 2.5 ul

H ₂ 0 11.5 ul

EcoRI (10 U / μl) 0.5 μl

Nael (10 U / μl) 0.5 μl

The incubation was carried out at 37 ° C. for 90 min. The mixture was then mixed with 5 μl stop buffer (5 ×) (100 mM EDTA, 20% (w / v) Ficoll 400, 0.01% (w / v) Bromophenol blue, 0.01% (w / v) xylene cyanol) added, applied to a 1.5% agarose gel (with ethidium bromide) and separated electrophoretically. The -440 bp EcoRI / ael fragment was cut out of the agarose gel under UV light and the DNA from the piece of agarose was centrifuged (Heery, DM et al .: A simple method for subcloning DNA fragments from gel slices; Trends in Genetics 6, 1990, 173). In parallel to the restriction of the PCR fragment, the complete cDNA clone (in pBluescript) was also restricted with EcoRI and Nael. The cleavage products were separated in a 0.75% agarose gel (with ethidium bromide) and the = 2000 bp fragment, which contains the 3 'region of the octopamine receptor gene, was also isolated using the centrifugation method (see above).

The isolated fragments were then ligated into an EcoRI cut, dephosphorylated vector (pcDNAI, Invitrogen):

pcDNAI (50 ng) 1.0 ul

PCR fragment (10 ng) 3.0 ul

3 'fragment (40 ng) 5.0 ul

Ligase buffer (10 x) 1.5 ul

T4 DNA ligase (1 U) 1.0 ul

The incubation was carried out at room temperature for 3 h. The ligation approach was carried out in transformation-competent cells of the bacterial strain MC1061-P3 (Invitrogen) transformed. The cells were plated on ampicillin containing LB agar plates (Sambrook, J. et al: Molecular cloning: A laboratory manual, 2 ^nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and plated overnight at 37 ° C incubated. Individual colonies were grown in 5 ml LB liquid cultures and the plasmid DNA was isolated by the alkaline lysis method (Sambrook, J. et al, see above). The correct incorporation of the receptor-coding cDNA was checked by a restriction analysis with the restriction enzyme BamHI. This creates two BamHI fragments of 684 bp and 1395 bp in size. The nucleic acid sequence of the PCR-amplified region was additionally determined from two clones into which the cDNA was correctly incorporated. After this test, larger amounts of the pcDNAI construct (pcDmOCTIB) were purified from 250 ml liquid cultures.

For the production of the cell line, which stably expresses the receptor, the gene fragment was cut out of the pcDNAI construct (pcDmOCTIB) again. The restriction enzymes Hindlll and Xbal were used for this. These enzymes cut only in the vector sequence (in the area of the multiple cloning site, MCS), but not within the receptor sequence. The cDNA fragment was isolated as described above and then ligated into a pcDNAINeo vector (Invitrogen), which was also cut in HindIII / Xbal. The ligation batch was transformed into MC1061-P3 bacterial cells and spread on LB agar plates containing ampicillin. Individual colonies were grown in 5 ml LB liquid cultures and the plasmid DNA was isolated by the alkaline lysis method (Sambrook, J. et al., See above). The correct incorporation of the cDNA fragment was restricted by a restriction with the Restriction enzymes Hindlll and Xbal checked. The construct is referred to below as pcNeoDmOCTIB.

Initial evidence that the cloned gene sequences encode functional receptors was obtained after transient transfections of HEK 293 cells with the pcDNAI construct (pcDmOCTIB). For this purpose = 2 x 10 ⁵ cells were sown on a 5 cm (diameter) Petri dish and the following day with = 10 μg of the plasmid construct using the calcium phosphate method (Chen, C. and Okayama, H: High-efficiency transformation of mammalian cells by plasmid DNA; Molecular and Cellular Biology 7, 1987, 2745-2752). The day after the transfection, the precipitate was removed with PBS / EDTA and the cells were detached from the dish by trypsin treatment. Different volumes of this cell suspension were sown on glass plates, which had previously been coated with poly-L-lysine, and incubated in the incubator for a further 20 h.

To measure the change in the intracellular Ca ²⁺ ion concentration, the cells were loaded with the fluorescent dye FURA-2 1 h before the experiment (Frings, S. et al: Profoundly different calcium permeation and blockage determine the specific function of distinct cyclic nucleotide-gated channels; Neuron 15, 1995, 169-179). The measurement was then carried out in the Ca ²⁺ imaging apparatus. For this purpose, a glass plate was transferred to the measuring chamber and washed with PBS. To activate the receptors, the PBS was exchanged for a PBS solution using a perfusion device defined concentration of the ligand to be tested, in the present case 1 μM octopamine. By binding the octopamine, an enzyme cascade is activated and the messenger substance IP3 is formed. Subsequently, Ca ²⁺ ions are released from intracellular stores. The change in Ca ²⁺ ion concentration was recorded with the imaging system. In the procedure described so far, however, not all cells reacted to the addition of the ligand with a change in the Ca ²⁺ ion concentration, because only a small percentage of the cells had taken up the plasmid and expressed the receptor. To avoid this variability, cell lines were produced in which all cells express the receptors stably, ie permanently.

In the first steps, the methodical approach is comparable to the transient transfection procedure. However, the pcNeoDmOCTIB construct was used for the transfection. The neomycin (neo) resistance gene is still present on this plasmid, the expression of which enables the selection of cells which have taken up the plasmid, since they are resistant to the addition of the antibiotic G418 (geneticin). Cells that have not taken up a plasmid die after a few days when G418 is added to the medium. As in the transient transfection, = 10 μg of the pcDNAINeoDmOCTlB construct were transfected with the calcium-phosphate method (Chen, C. and Okayama, H., see above) in approx. 2 x 10 ⁵ cells in a 5 cm Petri dish. 20 h after the transfection, the precipitate was removed with PBS / EDTA and the cells were detached by a trypsin treatment. From this cell suspension, 2 × 10 ⁴ cells were placed on five 9 cm Petri dishes sown. After 20 h, the medium was suctioned off and exchanged for fresh medium which contained 1 mg / ml G418. After approximately 12-14 days, in which the medium was changed with G418 every 3 days, most of the cells which had not taken up any plasmid had died. A total of 50 cell clones which were G418-resistant were isolated from the five Petri dishes. The clones were transferred individually to microtiter plates and further propagated. To check the receptor expression, a small part of the cells were sown on glass plates (see above) and the reaction of the cells to the addition of octopamine was measured using the Ca ²⁺ imaging method. Of the 50 antibiotic-resistant cell lines, one cell clone stably expressed the octopamine receptor. This line was expanded and some of the cells were frozen in liquid nitrogen.

About 10 different cell lines were produced in the manner described above, which stably express different receptors or ion channels. The integration of the construct DNA was not targeted, but random, i.e. anywhere in the genome of the host cell. However, it was found that the integration site of the foreign DNA in the genome of the host cell had no serious influence on the expression of the receptor proteins.

3. Investigation of the properties of neurotransmitter receptors

The pharmacological properties of the cloned octopamine receptors were determined on the stable cell lines. For this purpose, cells were sown on poly-L-lysine-coated flakes (see above) and transferred to the Ca2 ⁺ imaging apparatus. The cells reacted to the addition of 1 μM concentrations of the neurotransmitters tyramine and octopamine with a significant increase in the intracellular Ca2 ⁺ concentration. The Ca2 ⁺ was not released continuously but oscillatingly. No increase in the intracellular Ca2 ⁺ concentration was observed when incubated with the neurotransmitters dopamine and serotonin.

It was examined whether lower concentrations of the neurotransmitters octopamine and tyramine can also trigger the cellular response. It was found that a concentration of 10 ^~ 9

Octopamine causes a significant increase in the intracellular Ca2 ⁺ concentration. The reaction to the incubation takes place with

10 "6 M octopamine, however, slower. With increasing concentration of the neurotransmitter, the cellular response becomes faster and less

At concentrations of 10 ^~ 'M octopamine, the oscillation of the Ca2 ⁺ concentration is observed. In contrast to octopamine, a response of the cells to tyramine can only be observed from concentrations> 10 "^ M.

The reaction of the cells to octopamine is inhibited by various ligands depending on the concentration. For example, there is no increase in the intracellular Ca2 ⁺ concentration when the cells are incubated with octopamine and 25 phentolamine (= antagonist) in a concentration of 10 ^~ 'M each. The incubation with the antagonist can be started both before and after the addition of the octopamine. In both

In certain cases, the effect of octopamine is competed for and the

There is no increase in the intracellular Ca2 ⁺ concentration. This competitive effect decreases when the antagonist is added in a lower concentration, ie the increase in the intracellular Ca2 ⁺ concentration occurs again, for example when 10 "- ⁷ M octopamine and

10 "8 M phentolamine can be used.

Claims

claims

1. A method for producing octopamine receptor-producing cells, in which at least one gene coding for an octopamine receptor is transformed into a host cell.

2. The method according to claim 1 for the production of octopamine receptor type 1 and / or octopamine receptor type 2 producing

Cells.

3. The method according to claim 1 or 2, characterized in that the gene comes from Drosophila melanogaster.

4. The method according to any one of the preceding claims, characterized by an octopamine receptor gene with one for the SEQ ID no. 2 indicated amino acid sequence and / or their allele variations coding nucleotide sequence.

5. The method according to claim 4, characterized by the octopamine receptor gene with the nucleotide sequence according to SEQ ID

No.1 or an essentially equivalent DNA sequence.

6. The method according to any one of the preceding claims, characterized by an octopamine receptor gene with one for the SEQ ID no. 4 indicated amino acid sequence and / or their allele variations coding nucleotide sequence.

7. The method according to claim 6, characterized by the octopamine receptor gene with the nucleotide sequence according to SEQ ID No.3 or an essentially equivalent DNA sequence.

8. The method according to any one of the preceding claims, characterized in that the gene is incorporated into a gene construct for the transformation.

9. The method according to any one of the preceding claims, characterized in that the host cell is a cell line.

10. The method according to claim 9, characterized by the cell line HEK293.

11. Octopamine receptor genes with one for those under SEQ ID No. 2 indicated amino acid sequence and / or their allele variations coding nucleotide sequence.

12. Octopamine receptor gene according to claim 11 with the nucleotide sequence according to SEQ ID No. 1 or a DNA sequence having essentially the same effect.

13. Octopamine receptor genes with one for those under SEQ ID No. 4 indicated amino acid sequence and / or their allele variations encoding nucleotide sequence.

14. Octopamine receptor gene according to claim 13 with the nucleotide sequence according to SEQ ID No. 3 or a DNA sequence having essentially the same effect.

15. Octopamine receptor with an amino acid sequence according to SEQ ID

No. 2 or an essentially equivalent amino acid sequence.

16. Octopamine receptor with an amino acid sequence according to SEQ ID No. 4 or an essentially equivalent

Amino acid sequence.

17. Gene construct containing an octopamine receptor gene.

18. A gene construct according to claim 17, containing an octopamine

Receptor gene according to one of claims 11 to 14.

19. Transformed cell containing an octopamine receptor gene.

20. Transformed cells according to claim 19, containing an octopamine receptor gene according to one of claims 11 to 14.

21. Transformed cell according to one of claims 19 or 20, containing a gene construct according to one of claims 17 or 18.

22. Transformed cell according to one of claims 19 to 21, characterized in that it is a cell line.

23. Transformed cell according to claim 22, characterized by the cell line HEK293.

24. A method for examining the properties of neurotransmitter receptors, in which a ligand is allowed to act on cells transformed by neurotransmitter receptor genes and the effect is determined by determining the change in the intracellular Ca ²⁺ ion concentration.

25. The method according to claim 24 for examining the properties of octopamine receptors.

26. The method according to claim 25 for examining the properties of octopamine receptor type 1 and / or 2.

27. The method according to any one of claims 24 to 26, wherein the change in the intracellular Ca ²⁺ ion concentration by means of

Ca ²⁺ imaging method is determined.