JP3772188B2 - Screening method for therapeutic agents for neurodegenerative diseases or genetic brain diseases - Google Patents

Description

【図面の簡単な説明】
【図１】ムスカリン受容体刺激によりＫＣＮＱタンパク質がリン酸化のため抑制されて、神経細胞膜興奮の上昇に至るまでのシグナル伝達機構を説明する模式図である。
【図２】ＫＣＮＱとＡＫＡＰとＰＫＣとの存在状態を説明するために、ＫＣＮＱのＣ末端側を抜き出した模式図である。図２ａは、ＫＣＮＱに結合するＡＫＡＰがさらにＰＫＣとが複合体を形成している状態であり、図２ｂは、そのＡＫＡＰからＰＫＣが解離した状態である。
【図３】図３ａは、Ｏｘｏ−Ｍ濃度の段階的な増加に伴うＭ電流強度の標準化平均値の時間変化を示す図である。(白丸)はＰＫＣ阻害剤未添加のＳＣＧ神経細胞、(黒丸)はＰＫＣ阻害剤未添加のＡＫＡＰ(ΔＡ)（１−１４３）で形質転換したＳＣＧ神経細胞、(菱形)は１.２μＭケレリスリン添加ＳＣＧ神経細胞、(黒四角)は１００ｎＭビスインドリルマレイミド添加ＳＣＧ神経細胞、(白三角)は１００ｎＭカルフォスチンＣ添加ＳＣＧ神経細胞、(黒逆三角)は１０μＭサフィゴール添加ＳＣＧ神経細胞を示す。図３ｂは、ＰＫＣ阻害剤添加又は未添加のＳＣＧ細胞における、Ｍ電流抑制に対するＯｘｏ−Ｍの阻害の濃度依存性を示す図である。
【図４】図４ａは、Ｏｘｏ−Ｍの添加又は非添加でのＦＬＡＧ標識ＫＣＮＱ２チャンネルのリン酸化を調べた実験結果である。図４ｂは、ＫＣＮＱのセリン残基がリン酸化されることを示す実験結果である。図４ｃ上は、ＫＣＮＱへの放射性標識リン酸の取り込みをオートラジオグラフィーによって分析した結果であり、図４ｃ下は、ＫＣＮＱチャンネルのリン酸化は、ムスカリン阻害剤のピレンゼピンによってブロックされることを示す図である。図４ｃ中、*は標準誤差を表し、Ｐ＜０．０５である。図４ｄは、Ｏｘｏ−Ｍによるリン酸化の促進に対するＰＫＣ阻害剤の抑制効果を示す図である。*, **は標準誤差を表し、それぞれＰ＜０．０５ 及びＰ＜０．０１である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a screening method for therapeutic agents for neurodegenerative diseases such as anti-dementia drugs and antiepileptic drugs, or therapeutic agents for inherited brain diseases.
[0002]
[Prior art]
When acetylcholine, a neurotransmitter, activates muscarinic receptors, slow excitatory post-synaptic excitation (hereinafter referred to as sEPSP) and an increase in action potential frequency lasting from several seconds to several minutes occur in sympathetic ganglia and central nerve cells. . This sEPSP is caused by activation of a muscarinic receptor, which decreases the ion permeability of a potassium ion channel (hereinafter referred to as KCNQ) protein called M and suppresses M current. This series of signal transduction mechanisms leaves some unclear points.
[0003]
So far, several compounds such as diacylglycerol and inositol triphosphate, which are products of phospholipase C, have been listed as candidates for substances that play a role as second messengers in this increase in nerve excitation by acetylcholine. It has not been confirmed. Moreover, some hypotheses existed in the mechanism itself of nerve excitation in the cranial nerve (see, for example, Non-Patent Document 1).
[0004]
By the way, Alzheimer's disease, which is a dementia caused by cranial nervous system disorders, is caused by degeneration of cholinergic nerves. Therefore, an inhibitor of degrading enzyme (acetylcholinesterase) is clinically used as an anti-dementia agent in order to increase the acetylcholine concentration in the brain. Has been used. In recent years, a relationship between benign familial epilepsy, which is a hereditary brain disease, and KCNQ protein has been shown (Non-patent Document 2).
[0005]
[Non-Patent Document 1]
NV Marrion, Control of M-current, Annu. Rev. Physiol. 59, 483-504 (1997)
[0006]
[Non-Patent Document 2]
TJJentsch, Neuronal KCNQ potassium channels: physiology and role in didease. Nat. Rev. Neuurosci. 1, 21-30 (2000)
[0007]
[Problems to be solved by the invention]
KCNQ is attracting attention because it is thought to cause various diseases by its genetic abnormality, but the signal transduction pathway from muscarinic receptor to KCNQ suppression still remains unclear. In addition, although research on neurological treatments is ongoing, the development of fundamental therapies and drugs for dementia symptoms such as Alzheimer's disease and epilepsy has not been achieved, and new methods that differ from existing methods Development of a treatment method has been desired.
[0008]
The present invention has been proposed in view of such conventional circumstances, and efficiently selects an anti-dementia drug and an antiepileptic drug that control the ion permeability of potassium ion channel protein by a mechanism different from the conventional one. It is an object of the present invention to provide a screening method for a therapeutic agent for neurodegenerative disease or a genetic brain disease.
[0009]
[Means for Solving the Problems]
By the way, as a result of studies conducted over many years in order to elucidate the mechanism of neuronal excitability caused by acetylcholine, the present inventors have recently found that AKAP and proteins bound to AKAP have been introduced into the signal transduction mechanism from muscarinic receptor to KCNQ. We have succeeded in elucidating for the first time that a kinase is involved. The inventors have found that this mechanism can be applied to the development of new drugs, and have completed the present invention.
[0010]
That is, the screening method for a therapeutic agent for neurodegenerative disease or a genetic brain disease according to the present invention comprises a muscarinic receptor and AKAP150 When KCNQ2 And a cell expressing muscarinic receptor agonist Protein kinase C inhibitor And a step of measuring a current intensity generated in the cell; Have Measured current intensity Is compared with the current intensity generated in the cells when a muscarinic receptor and a protein kinase C control site target inhibitor are administered to the cells, and the protein kinase C inhibitor is classified as the protein kinase C control site target inhibitor. To determine whether or not It is characterized by.
[0011]
Further, the screening method for the therapeutic agent for neurodegenerative disease or the genetic brain disease according to the present invention comprises a muscarinic receptor and AKAP150 When KCNQ2 And cells expressing muscarinic receptor agonist, radiolabeled phosphate and Protein kinase C inhibitor And a step of administering KCNQ2 Measuring the radioactivity of the radiolabeled phosphate bound to Have Measured radioactivity above From the above, it is determined whether or not the protein kinase C inhibitor is a specific inhibitor for AKAP150-binding protein kinase C. It is characterized by that.
[0012]
In normal nervous tissue, activation occurs through the mechanism of muscarinic receptor → phospholipase C → inositol-3-phosphate / calcium ion → A-kinase anchor protein, protein kinase C, and potassium ion channel (KCNQ) protein is phosphorylated. As a result, it was clarified that M current suppression occurs in the cell membrane system and the excitement of the nerve cell membrane increases.
[0013]
Already, anti-dementia drugs that act directly on KCNQ ion channels and suppress KCNQ ion permeability, and conversely, antiepileptic drugs that increase KCNQ ion permeability have been developed.
[0014]
In contrast, the present invention targets protein kinase C, which is involved in the signal transduction mechanism from muscarinic receptor to KCNQ inherent in the living body, and inhibits or promotes the activity of protein kinase C, thereby allowing KCNQ ion permeability. It is possible to screen for substances that indirectly regulate. The most characteristic feature is that only the activity of protein kinase C of the A-kinase anchor protein-binding type can be effectively measured, not the activity of protein kinase C that is present free in the cell.
[0015]
The complex of A-kinase anchor protein and protein kinase C according to the present invention is bound to KCNQ via the A-kinase anchor protein, and the binding of the A-kinase anchor protein to the protein kinase C The protein kinase C can be dissociated by a compound or peptide that regulates sex.
[0016]
For example, when a potassium ion channel protein is expressed in a cell together with a compound that weakens the binding between the A-kinase anchor protein and the protein kinase C, the protein kinase C is released, and the potassium ion channel protein is phosphorylated. Oxidizes to suppress M current and produces slow EPSP. In addition, the second and subsequent reactions to acetylcholine are lost. That is, this complex is absolutely necessary when acetylcholine activates muscarinic receptors. For this reason, if the complex is actively present in the cell and a state that occurs even with a weak dissociation stimulus is created, it has an anti-dementia effect, while if the complex is eliminated or dissociation does not occur, the reaction stops. It can be used as an antiepileptic drug.
[0017]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the screening method for the therapeutic agent for neurodegenerative disease or the genetic brain disease of the present invention will be described in detail.
[0018]
As a result of intensive studies over many years, the present inventors, as shown in FIG. 1, acetylcholine, which is a neurotransmitter, activates a muscarinic receptor and ion permeates a potassium ion channel (hereinafter referred to as KCNQ) protein. We succeeded in elucidating the whole mechanism of ion channel control in nerve cells to reduce sex. In particular, in FIG. 1, protein kinase C (hereinafter sometimes referred to as PKC) bound to an A-kinase anchor protein (hereinafter sometimes referred to as AKAP) has a calcium ion concentration or inositol triphosphate. The mechanism of dissociation from AKAP due to an increase or the like, and the dissociated PKC becomes a second messenger to phosphorylate KCNQ to decrease ion permeability was newly elucidated. That is, activation of the muscarinic receptor → phospholipase C → inositol-3-phosphate / calcium ion → AKAP → PKC leads to phosphorylation of KCNQ protein, resulting in suppression of M current in the cell membrane system, and sEPSP It became clear that it caused an increase in the excitement of the nerve cell membrane.
[0019]
By the way, it is known that abnormalities in the KCNQ gene are involved in benign familial epilepsy and hearing loss. Moreover, it has been experimentally shown that by controlling the ion permeability of KCNQ, it is possible to control neurodegenerative diseases including Alzheimer's disease accompanied by dementia and inherited brain diseases such as epilepsy. That is, efforts have been made to develop an anti-dementia drug that acts directly on KCNQ and suppresses KCNQ ion permeability, and conversely, an antiepileptic drug that increases KCNQ ion permeability.
[0020]
As described above, the present inventors say that PKC dissociated from a triple complex of AKAP and PKC bound to KCNQ (hereinafter sometimes simply referred to as a complex) controls the ion permeability of KCNQ. And found new findings. What is important here is that it was found that PKC dissociated from the complex in contact with KCNQ acts on nearby KCNQ, not PKC widely present in the cytoplasm. From this, it is considered important to regulate the ion permeability of KCNQ by inhibiting or promoting the activity of PKC dissociated from the complex.
[0021]
That is, a drug targeting PKC dissociated from the complex has a mechanism different from that of an anti-dementia drug that directly acts on KCNQ as described above, but the same KCNQ as an anti-dementia drug of a type that directly acts on KCNQ. It is thought that the ion permeability can be reduced. Similarly, a PKC inactive agent targeting PKC dissociated from the complex is a type that acts directly on KCNQ, although it is different from the antiepileptic drugs that act directly on KCNQ as described above. It is considered possible to increase the KCNQ ion permeability in the same manner as other antiepileptic drugs.
[0022]
Therefore, the present inventors have proposed a method of regulating the dissociation itself from the complex, or screening a regulatory substance targeting the PKC after the dissociation. First, a first example of a screening method for a therapeutic drug for neurodegenerative disease or a genetic brain disease according to the present invention will be described.
[0023]
In the first example, first, a muscarinic receptor agonist and a test substance are administered in the presence of cells expressing muscarinic receptors, AKAP, and KCNQ.
[0024]
Examples of muscarinic receptors include, but are not particularly limited to, muscarinic m1 receptors.
[0025]
AKAP is a group of proteins that are functionally defined as proteins that originally bind only to protein kinase A, but there is also AKAP that serves as a scaffold for calmodulin, PKC, and calcineurin. Examples of AKAP that integrates a plurality of enzymes at a post-synaptic site in a nerve cell include rat-derived AKAP150 represented by SEQ ID NO: 1 and human AKAP79.
[0026]
KCNQ protein is an ion channel protein involved in ion permeability in nerve cells. Specific examples of the KCNQ protein include KCNQ2 represented by SEQ ID NO: 2, KCNQ3 represented by SEQ ID NO: 3, and the like.
[0027]
As the cells expressing muscarinic receptor, AKAP, and KCNQ protein used here, for example, nerve cells such as upper cervical ganglion cells and central nerve cells that originally express these proteins are used. be able to. More specifically, rat SCG (superior cervical ganglion) cells can be used. Also, Chinese hamster ovary cells transformed by introducing an expression vector into which a gene encoding a protein such as AKAP or KCNQ described above has been introduced can be used.
[0028]
As a muscarinic receptor agonist, for example, a compound capable of activating a muscarinic receptor such as acetylcholine, muscarin, oxotremorine-M and the like can be used.
[0029]
Next, the current value of M current suppression is measured using, for example, an electrophysiological technique, and the intensity change is observed. Thereby, the operation | movement condition of KCNQ in a cell membrane can be observed.
[0030]
Then, the measured current intensity of the cell is compared with the current intensity of the cell when the test substance, which is a control, is not administered.
[0031]
Here, the interaction between AKAP and PKC will be described in detail. As shown in FIG. 2a, PKC is mainly composed of two parts, a catalytic active site and a control site, and forms a complex by binding to the vicinity of the N-terminus of AKAP at the catalytic active site. Conceivable. AKAP is considered to be triple-complexed with KCNQ in the vicinity of its N-terminus. Note that the PKC in a complex-formed state has its enzyme activity hidden (masked), and does not phosphorylate KCNQ.
[0032]
When the muscarinic receptor agonist activates the muscarinic receptor, the catalytically active site of PKC is dissociated from AKAP by, for example, dissociation promoting action by inositol triphosphate, calcium ion or the like, as shown in FIG. 2b. By dissociating from the complex, the enzyme activity of this PKC appears in the table and is activated under the influence of activation, and phosphorylates the most recent KCNQ. As a result, the ion permeability of KCNQ is reduced and M current is suppressed.
[0033]
By the way, PKC inhibitors are roughly classified into two types, active site target inhibitors and control site target inhibitors, depending on whether they act on the catalytic active site or control site of PKC. Of the PKC inhibitors, the active site target inhibitor is phosphorylated before reaching PKC, and thus cannot inhibit the receptor reaction. On the other hand, since the control site target inhibitor is in a state in which preparations for activity suppression are already prepared at the time of complex formation, the activity of PKC is suppressed even if PKC is released by receptor stimulation. In other words, the PKC regulatory site target inhibitor inhibits phosphorylation of KCNQ and does not change the ionic permeability, thus preventing M current suppression. This PKC control site target inhibitor causes the same effect on cells as a conventional anti-dementia drug, which decreases KCNQ ion permeability and prevents M current suppression.
[0034]
Therefore, by examining the degree of M current suppression, whether or not the test substance is a PKC control site inhibitor, that is, whether or not the test substance is an inhibitor specific for AKAP-binding PKC. Can be determined. Then, by selecting an inhibitor specific for AKAP-binding PKC, a therapeutic agent for neurodegenerative disease or a therapeutic agent for inherited brain disease can be screened. Specifically, a test substance that has relatively inhibited M-current suppression may be a candidate compound for antiepileptic drugs. Conversely, a test substance that can be expected to suppress M-current is an anti-dementia drug. Can be a candidate compound.
[0035]
As described above, according to the screening method of the first example of the present invention, it is possible to effectively measure not only the activity of PKC freely present in cells but the amount of PKC that is released from the AKAP binding type. A substance that indirectly regulates the ion permeability of KCNQ, that is, a novel anti-dementia drug or antiepileptic drug candidate compound can be efficiently selected.
[0036]
In addition, a second example of the screening method for a therapeutic drug for neurodegenerative disease or a genetic brain disease according to the present invention will be described.
[0037]
First, a cell in which a muscarinic receptor, AKAP, and KCNQ protein are expressed is prepared, and a muscarinic receptor agonist, a radiolabeled phosphate, and a test substance are administered to the cell and incubated for a predetermined time.
[0038]
As the cells expressing muscarinic receptor, AKAP, and KCNQ protein used here, for example, nerve cells such as upper cervical ganglion cells and central nerve cells that originally express these proteins are used. be able to. More specifically, rat SCG (superior cervical ganglion) cells can be used. Also, Chinese hamster ovary cells transformed by introducing an expression vector into which a gene encoding a protein such as AKAP or KCNQ described above has been introduced can be used.
[0039]
Moreover, as a muscarinic receptor, AKAP, and KCNQ protein, the thing similar to what was enumerated in the 1st example can be used.
[0040]
As radiolabeled phosphate, [ ³² P] -H _Three PO _Four Ordinary radiolabeled phosphoric acid such as can be used.
[0041]
Next, the radioactivity of the radiolabeled phosphate bound to the potassium ion channel protein is measured. Specifically, the cells are solubilized, KCNQ is separated by immunoprecipitation and electrophoresis, and the radioactivity after separation is measured by autoradiography or the like. Thereby, the phosphorylation amount of KCNQ is obtained.
[0042]
By the way, phosphorylation of KCNQ is a phenomenon caused by PKC dissociated from the complex as well as M current suppression.
[0043]
Therefore, by examining the degree of phosphorylation of KCNQ, whether or not the test substance is a PKC control site inhibitor, that is, whether the test substance changes the degree of dissociation of the complex, or AKAP-binding PKC It is possible to determine whether or not it is a specific inhibitor. Then, by selecting an inhibitor specific for AKAP-binding PKC, a therapeutic agent for neurodegenerative disease or a therapeutic agent for inherited brain disease can be screened. Specifically, a test substance having a relatively increased amount of phosphorylation may be a candidate compound for an anti-dementia drug, and conversely, a test substance having a low KCNQ phosphorylation level is an antiepileptic drug. Can be a candidate compound.
[0044]
As described above, according to the screening method of the second example of the present invention, it is possible to effectively measure only the activity of PKC of A-kinase anchor protein binding type, not the activity of PKC free to exist in cells. Therefore, a substance or peptide that indirectly regulates the ion permeability of KCNQ, that is, a novel anti-dementia drug or antiepileptic drug candidate compound can be efficiently selected.
[0045]
Further, the complex of A-kinase anchor protein and protein kinase C according to the present invention is a complex formed by binding KCNQ, A-kinase anchor protein and protein kinase C as shown in FIG. This complex can dissociate the protein kinase C by a compound that regulates the binding between the A-kinase anchor protein and the protein kinase C. Examples of the substance that regulates the binding property between the A-kinase anchor protein and the protein kinase C include inositol triphosphate and calcium ion.
[0046]
For example, when a potassium ion channel protein is expressed in a cell together with a compound that weakens the binding between the A-kinase anchor protein and the protein kinase C, the protein kinase C is released, and the potassium ion channel protein is phosphorylated. Oxidizes to suppress M current and produces slow EPSP. In addition, the second and subsequent reactions to acetylcholine are lost. That is, this complex is absolutely necessary when acetylcholine activates muscarinic receptors. For this reason, if the complex is actively present in the cell and a state that occurs even with a weak dissociation stimulus is created, it has an anti-dementia effect, while if the complex is eliminated or dissociation does not occur, the reaction stops. It can be used as an antiepileptic drug.
[0047]
【Example】
Next, examples of the present invention will be specifically described, but the present invention is not limited to these examples.
[0048]
Experiment 1. (Preparation of expression plasmids for KCNQ2 and AKAP150)
KCNQ2 and AKAP150 cDNAs were designed using LA-taq-DNA synthase (Takara) based on the respective gene data obtained from GenBank (SEQ ID NO: 2 and SEQ ID NO: 1), respectively. It was prepared by reverse transcriptase reaction using brain RNA as a template and DNA synthase chain reaction (RT-PCR). A mutant cDNA was prepared by a recombinant DNA synthase chain reaction. KCNQ2 cDNA was inserted into plasmid pZeoSV to prepare an expression vector. The AKAP cDNA was inserted into the plasmid pTracer-SV40 to obtain an expression vector for AKAP150. A glutathione-S-transferase (GST) fusion AKAP150 protein was prepared by incorporating the gene DNA into a pGEX vector.
[0049]
Experiment 2. (Cell culture, cDNA introduction and transformation)
Nerve cells were isolated from 2-3 week old rats and cultured according to a previous report (Delmas, P. et al., J Physiol 506, 319-329 (1998)). Next, the plasmid incorporating the above DNA was diluted to 100 μg / ml and pneumatically injected into the nucleus of SCG neurons. The transformed cells were stored in a cultured state. The GFP-Zeocin resistant gene fusion protein encoded by pTracer-SV40 was detected by its fluorescence for the patch clamp method. Human-muscarinic subtype m1 receptor-expressing Chinese hamster ovary cells (hereinafter sometimes referred to as CHOhm1 cells) were cultured in an α-Eagle culture medium supplemented with 1% calf serum. The cells were transplanted into the culture solution using a lipofectamine plus transducing agent (Invitrogen) or Fugene 6 transducing agent (Rossi Molecular Biochemical), and transformed on the first day. The plasmid used in the experiment was duplicate transformed with pTrace into which AKAP150-DNA was inserted or untreated pTracer. On the second day after transformation, transformed cells were selected. In order to examine the effect of the PKC inhibitor, an enzyme inhibitor was added to the cells at the following concentrations and cultured overnight. Specifically, 100 nM bis-indolylmaleimide I (Calbiochem) treated SCG neurons, 100 nM Calfostin C (Calbiochem) treated SCG neurons, 1.2 μM chelerythrine (Calbiochem) treated SCG neurons, or 10 μM Safgor (sigma) -treated SCG neurons.
[0050]
Experiment 3 (Electrophysiological measurement of nerve cell membrane)
The measurement of sEPSP in the separated cells was performed using an Axo Patch 200B patch clamp amplifier (Axon Instruments). The signal from the cells was removed at 2 kHz and filtered at 1 kHz. Current analysis and measurement was performed with peak lamp software. Fine currents from SCG neurons using the membrane perforated patch method according to previous reports (Selyanko, AA, Stansfeld, CE & Brown, DA, Proc R Soc Lond B Biol Sci 250, 119-125 (1992)) Was measured. Amphotericin B (0.1 mg / ml to 0.2 mg / ml) was dissolved in the following extracellular solution. The extracellular solution contains 130 mM potassium acetate, 15 mM potassium chloride, 3 mM magnesium chloride, 6 mM sodium chloride, 10 mM HEPES buffer (pH 7.3). When the intracellular solution was filled into a pipette, the resistance value was 2 to 3 MΩ. The internal solution contains 120 mM sodium chloride, 6 mM potassium chloride, 1.5 mM magnesium chloride, 2.5 mM calcium chloride, 11 mM glucose, 10 mM HEPES buffer (pH 7.4). The M current intensity was measured as an inactivation current generated by a test stimulus of 500 milliseconds from a holding current of −20 mV to −50 mV.
[0051]
The M current in CHOhm1 cells was measured in the whole cell membrane potential fixation mode of the patch fixation method. The internal resistance of the patch pipette electrode was 4-8 MΩ and filled with the following solution. That is, it is a solution consisting of 90 mM potassium citrate, 20 mM potassium chloride, 3 mM magnesium chloride, 1 mM calcium chloride, 3 mM EGTA, 40 mM HEPES buffer (pH 7.4). The external solution is 140 mM sodium chloride, 5 mM potassium chloride, 2 mM magnesium chloride, 1 mM calcium chloride, 10 mM HEPES buffer (pH 7.4). Also, connection resistance (90-95%) and total cell volume correction were used. Most of the transformed CHOhm1 cells showed a relatively small decay under these measurement conditions.
[0052]
Experiment 4. (Concentration dependence of M current suppression in SCG neurons by PKC inhibitor)
First, various nerve cells were refluxed with oxotremorine-M (hereinafter referred to as Oxo-M), which is a muscarinic acetylcholine receptor stimulant (agonist) at different concentrations in stages, and M current was measured. . After normalizing the results, each experimental value was averaged, and the change in the value was plotted against the time change when the Oxo-M concentration was increased stepwise. The result is shown in FIG. Cell preparations were untreated SCG neurons (control cell samples), SCG neurons transformed with AKAP (ΔA) (1-143), 100 nM bis-indolylmaleimide I (Calbiochem) (hereinafter referred to as bis) treatment. SCG neurons, 100 nM calfostin C (calbiochem) (hereinafter referred to as cal) treated SCG neurons, 1.2 μM chelerythrine (calbiochem) (hereinafter referred to as che), or 10 μM safigor (sigma) (hereinafter referred to as saf) Note.) Treated SCG neurons.
[0053]
Experiment 5.
The result of the Hill-plot of the concentration-dependent inhibition of Oxo-M of the M-current value obtained with each nerve cell preparation is shown in FIG. 3b. The cells used were untreated SCG neurons (control cell preparation), SCG neurons transformed with AKAP (ΔA) (1-143), 100 nM bis-treated SCG neurons, 100 nM cal-treated SCG neurons. 2 μM che-treated neurons or 10 μM saf-treated SCG neurons. As a result, in Saf or Cal-treated cells that are AKAP (ΔA) and PKC-regulated site target inhibitors, the PKC-active site targeted inhibitor Bis or Che-treated cells and untreated cells are M-currents. It turned out that the shape of the Oxo-M dose-dependent curve of inhibition changed and shifted to the high concentration side, and the inhibitory concentration increased by 50% (it was difficult to suppress).
[0054]
Experiment 6. (Effects of various commercially available PKC inhibitors on PKC phosphorylation of potassium channel KCNQ2 protein in Oxo-M treated cells)
48 hours after CHOhm1 cells were transformed with FLAG-labeled KCNQ2, 0.5 Ci / ml radioactive phosphate [ ³² P] -H _Three PO _Four (Amersham Pharmacia Biotech) was added and left for 4 hours. Some of them were added with the PKC inhibitor used in Experiment 5. Thereafter, treatment with 10 μM Oxo-M for 3 minutes was performed. Cells were then scraped and transferred to 700 μl of ice-cold TNE solution. The TNE solution is composed of 150 mM sodium chloride, 2 mM EDTA, 1% nonidet P-40 (manufactured by Nacalai), 50 mM sodium fluoride, 1 mM sodium vanadate, 1 mM dithiothreitol, and a protease inhibitor mixture.
[0055]
Cell solubilization was carried out in TNE solution for 20 minutes with gentle shaking at 4 ° C. The solubilized cell solution was centrifuged and the supernatant was collected and purified by passing through 30 μl Protein G Sepharose 4 (Amersham Pharmacia Biotech). The supernatant was reacted with 4 μg of M2-anti-FLAG antibody for 2 hours, and then protein G-bound beads were added and treated at 4 ° C. for 1 hour. The immunoreacted precipitate was washed 6 times with TNE buffer. After performing 6% SDS-polyacrylamide gel electrophoresis, ³² P] -H _Three PO _Four The KCNQ2 labeled with was detected by the phosphoric acid image method using BAS1000 manufactured by Fuji Film. On the other hand, among the immunoprecipitated proteins, the phosphorylated protein was confirmed to be KCNQ2 protein by Western blotting using an anti-FLAG antibody (FIG. 4a).
[0056]
Experiment 7. (Of phosphorylated KCNQ2 protein ³² Analysis of P-labeled amino acids)
[0057]
The analysis of phosphorylated amino acids was performed by blotting the sample protein on SDS-polyacrylamide gel on a PDVF membrane, cutting out the membrane of the protein-bound portion, hydrolyzing it by treating in 6N-hydrochloric acid at 110 ° C. for 90 minutes, It was separated by electrophoresis using cellulose (support) thin-layer chromatographic paper, ³² Amino acids labeled with P] -phosphate were detected. as a result,[ ³² From the migration position of the P] -labeled amino acid, it was identified as phosphorylated serine (FIG. 4b).
[0058]
Experiment 8. (Promoting effect of Oxo-M on phosphorylation of KCNQ2 protein) CHOhm1 cells were simultaneously transformed with FLAG-labeled KCNQ2 protein and AKAP150 protein or AKAP (ΔA) protein. After 48 hours, 0.5 Ci / ml radioactive phosphate in phosphoric acid-free solution [ ³² P] -H _Three PO _Four (Amersham Pharmacia Biotech) was added and left for 4 hours. Thereafter, treatment with 10 μM Oxo-M for 3 minutes was performed. Thereafter, phosphorylation of KCNQ2 protein was analyzed by the method of Experiment 6.
[0059]
As a result, in the cells expressing AKAP150 protein, phosphorylation was induced by muscarinic stimulation, whereas in the cells expressing AKAP (ΔA) mutant protein, the phosphorylation effect by Oxo-M was suppressed. This is because the action of phosphorylation of Oxo-M was suppressed as a result of blocking the formation of the complex between AKAP150 protein and PKC by the AKAP (ΔA) mutant protein. (Fig. 4c)
[0060]
Experiment 9 (Effect of PKC inhibitor on promotion of phosphorylation of KCNQ2 protein by Oxo-M)
CHOhm1 cells transformed with FLAG-labeled KCNQ2 protein were treated with various commercially available PKC inhibitors used in Experiment 5, and then phosphorylation of KCNQ2 protein was examined. After the treated cells were destroyed, the amount of phosphorylation was determined by analyzing the KCNQ2 protein separated by immunoprecipitation and electrophoresis using the phosphate image method. The result is shown in FIG. As shown in FIG. 4d, phosphorylation increases 1.5-fold when Oxo-M is added to the control cell preparation. On the other hand, the amount of phosphorylation only slightly decreases in the presence of Bis and Che, but Cal and Saf show a strong inhibitory effect on phosphorylation promotion. Together with the results of the previous experiment 5, it is shown that the four commercially available PKC inhibitors are divided into two groups, that is, the action mechanism of the inhibitors is different. Cal and Saf, which are referred to as control site target inhibitors, are already in the state of inhibiting PKC in the AKAP binding state. Therefore, even when released and the enzyme active site is exposed, PKC does not act and phosphorylation of KCNQ2 protein While oxidation does not occur, Bis and Che, which are active site targeted inhibitors, only slightly suppress the action of Oxo-M.
[0061]
Here, an outline of the results of Experiments 1 to 9 will be described below. First, construct an expression vector into which cDNA of KCNQ2, AKAP150, AKAP's KCNQ2-binding domain-deficient protein (AKAP (ΔA) (1-143)) or AKAP mutant protein was inserted, and then expressed in rat SCG cells Vectors were injected to prepare transformed cells. Further, a plasmid in which AKAP150-DNA was inserted was introduced into CHOhm1 cells for transformation.
[0062]
Next, the change in the intensity of the M current due to the action of Oxo-M on the muscarinic receptor was measured by an electrophysiological technique using SCG cells, and the operating state of the potassium ion channel in the nerve cell membrane was observed. The M current shows a concentration-dependent change in Oxo-M. Further, when cells transformed with the gene DNA of AKAP (ΔA) (1-143) are used, the concentration dependence of M current on Oxo-M is changed. After the addition of a PKC inhibitor to SCG neurons, the M-current suppression of Oxo-M was measured. As a result, there were two types, one acting like AKAP and the other acting like AKAP (ΔA) (1-143). Divided into groups. The former is an inhibitor referred to as a PKC active site target inhibitor, and the latter is an inhibitor referred to as a PKC regulatory site target inhibitor. In the former, Hill plot against Oxo-M concentration shows 50% inhibition of M current at low concentration of Oxo-M, whereas in the latter, 50% inhibition was observed at high concentration, and these inhibitors inhibit PKC. It was shown that the style was different. Therefore, by measuring the M current suppression with respect to the Oxo-M concentration, it is possible to determine the action of the drug on nerve cells (whether it is a binding type or not).
[0063]
By the way, when a cell obtained by transforming CHOhm1 cells with KCNQ2 is used as a reaction system, phosphorylation of KCNQ2 is promoted by Oxo-M. Moreover, the effect of Oxo-M is antagonized by pirenzepine, which is an m1 receptor inhibitor. In cells transformed with AKAP (ΔA) at the same time, the promotion by Oxo-M was offset, indicating that AKAP is involved in the phosphorylation reaction. Analysis of the phosphorylated amino acid of KCNQ2 revealed serine phosphorylation, suggesting that phosphorylation proceeded by the catalytic action of PKC.
[0064]
In addition, after adding a PKC inhibitor to CHOhm1 cells transformed with KCNQ2, the amount of phosphate incorporated into KCNQ2 is increased by 1.5 times in the control due to the effect of addition of Oxo-M. Inhibitors reduce phosphate uptake. On the other hand, the effect of Oxo-M is not changed in the active site targeted inhibitor. This suggests that only bound PKC is involved in the phosphorylation reaction. Inhibition of the ion permeability of KCNQ2 by AKAP-binding PKC is exactly the action of acetylcholine, and activates (excites) neural activity. Based on the results of this inhibitor, an agent equivalent to the intracellular action of acetylcholine can be used as an anti-dementia drug and positioned as a PKC control site target activator or phosphorylation promoter. On the other hand, the PKC control site target inhibitor is an antiepileptic agent and can be screened by this method.
[0065]
From the above experimental results, by examining the drug concentration for inhibiting the M current or the inhibitory or promoting action of the drug on the phosphorylation of KCNQ2 protein, a substance that indirectly regulates the ion permeability of KCNQ can be screened. Drugs that promote the action of the potassium ion channel KCNQ2 or KCNQ3 in nerve cells and increase potassium ion permeability are expected to be antiepileptic drugs, whereas drugs that decrease permeability are expected to be anti-dementia drugs. . The development of such a PKC inhibitor is extremely important in developing a novel treatment method for dementia symptoms and epilepsy.
[0066]
【The invention's effect】
As described above, according to the present invention, a compound capable of controlling the ion permeability of potassium ion channel protein can be efficiently selected by a mechanism different from conventional anti-dementia drugs and antiepileptic drugs. Can contribute to the development of anti-dementia drugs and antiepileptic drugs.
[0067]
[Sequence Listing]

[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a schematic diagram illustrating a signal transduction mechanism from the suppression of KCNQ protein due to phosphorylation by muscarinic receptor stimulation leading to an increase in neuronal membrane excitation.
FIG. 2 is a schematic diagram in which the C-terminal side of KCNQ is extracted in order to explain the existence state of KCNQ, AKAP, and PKC. FIG. 2a shows a state where AKAP binding to KCNQ further forms a complex with PKC, and FIG. 2b shows a state where PKC is dissociated from the AKAP.
FIG. 3a is a diagram showing a time change of a standardized average value of M current intensity with a gradual increase in Oxo-M concentration. (White circles) are SCG neurons without PKC inhibitor added, (black circles) are SCG neurons transformed with AKAP (ΔA) (1-143) without PKC inhibitors, (diamonds) are 1.2 μM chelerythrine added SCG neurons, (black squares) are SCG neurons added with 100 nM bisindolylmaleimide, (white triangles) are SCG neurons added with 100 nM calfostin C, and (black inverted triangles) are SCG neurons added with 10 μM safigor. FIG. 3b is a graph showing the concentration dependence of inhibition of Oxo-M with respect to M current suppression in SCG cells with or without a PKC inhibitor.
FIG. 4a shows the results of experiments examining phosphorylation of FLAG-labeled KCNQ2 channels with or without the addition of Oxo-M. FIG. 4b is an experimental result showing that the serine residue of KCNQ is phosphorylated. Fig. 4c shows the result of autoradiography analysis of the incorporation of radiolabeled phosphate into KCNQ, and Fig. 4c shows that KCNQ channel phosphorylation is blocked by the muscarinic inhibitor pirenzepine. It is. In FIG. 4c, * represents standard error and P <0.05. FIG. 4d is a diagram showing the inhibitory effect of a PKC inhibitor on the promotion of phosphorylation by Oxo-M. * And ** represent standard errors, P <0.05 and P <0.01, respectively.

Claims (6)

Administering a muscarinic receptor agonist and a protein kinase C inhibitor to a cell in which a muscarinic receptor, AKAP150 and KCNQ2 are expressed;
Measuring the current intensity generated in the cell ,
The measured current intensity is compared with the current intensity generated in the cell when a muscarinic receptor and a protein kinase C control site target inhibitor are administered to the cell, and the protein kinase C inhibitor is controlled by the protein kinase C control. A screening method for a therapeutic agent for a neurodegenerative disease or a hereditary brain disease, characterized by discriminating whether it is classified as a site-targeted inhibitor .   2. The screening method for a therapeutic agent for neurodegenerative disease or a genetic brain disease according to claim 1, wherein the muscarinic receptor agonist is oxotremorine-M. 2. The neurodegenerative disease treatment according to claim 1, wherein the cell is an SCG cell or a Chinese hamster ovary cell transformed with the AKAP150 gene represented by SEQ ID NO: 1 and the KCNQ2 gene represented by SEQ ID NO: 2. A screening method for drugs or drugs for inherited brain diseases. Administering a muscarinic receptor agonist, a radiolabeled phosphate and a protein kinase C inhibitor to a cell in which a muscarinic receptor, AKAP150 and KCNQ2 are expressed;
Measuring the radioactivity of the radiolabeled phosphate bound to the KCNQ2 .
A therapeutic agent for neurodegenerative disease or hereditary brain disease characterized by determining whether or not the protein kinase C inhibitor is a specific inhibitor for AKAP150-binding protein kinase C from the measured radioactivity A screening method for therapeutic drugs.   5. The screening method for a therapeutic agent for neurodegenerative disease or a genetic brain disease according to claim 4, wherein the muscarinic receptor agonist is oxotremorine-M. The neurodegenerative disease treatment according to claim 4, wherein the cells are SCG cells or Chinese hamster ovary cells transformed with the AKAP150 gene represented by SEQ ID NO: 1 and the KCNQ2 gene represented by SEQ ID NO: 2. A screening method for drugs or drugs for inherited brain diseases.

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