KR101223990B1 - Composition for screening of TRPV4 activators or inhibitors, method for isolating TRPV4 positive or negative neurons, and method for screening TRPV4 activators or inhibitors, using DMAPP or its salt - Google Patents

Abstract

The present invention relates to a TRPV4 activator comprising DMAPP, and more particularly, to a TRPV4 activator or activity inhibitor screening method using DMAPP. DMAPP of the present invention acts specifically on TRPV4, so that it is possible to isolate only sensory neurons expressing TRPV4, and thus can be usefully used for research on TRPV4 mechanism of action and development of TRPV4 mechanism of analgesics.

Description

Composition for screening of TRP4 activators or inhibitors using DPMAP or salts thereof, methods for isolating TRP4 positive or negative neurons, and methods for screening TRP4 activators or inhibitors, and methods for screening of TRPV4 activators or inhibitors, methods for isolating TRPV4 positive or negative neurons, and method for screening TRPV4 activators or inhibitors, using DMAPP or its salt}

The present invention relates to a method for screening a composition for screening a TRPV4 activator or activity inhibitor using DMAPP or a salt thereof, and a TRPV4 positive or negative neuron, and a method for screening a TRPV4 activator or activity inhibitor.

Research in human physiology and genetics has revealed the presence of TRPV4 (transient receptor potential vanilloid 4) in humans and is expected to play a vital role in the survival of various tissues. In particular, the TRPV4 is expressed in sensory neurons that sense external stimuli and belongs to the thermoTRP group (temperature-sensitive transient receptor potential ion channels), which is a nociceptor group that detects temperature and pain-induced noxious stimuli.

TRPV4 has been shown to mediate pain during pain, in particular by mechanical pressure stimulation. By clarifying the function of nociceptor TRPV4, it is expected to reveal the mechanism of nociceptive body and to achieve the goal of pain relief through the development of TRPV4 modulator. In order to identify the TRPV4 function and develop the regulatory drug, there is a demand for developing a specific active agent that can selectively induce TRPV4 activation without acting on other TRP receptors.

The characteristics of the TRPV4 receptor for understanding the underlying technology used in the development of the specific active agent of TRPV4 is as follows. TRPV4 is an ion channel through which its cation migrates inside sensory neurons, thereby changing the membrane current. An action voltage is generated by the change of the cell membrane current, and this voltage signal is ultimately transmitted to the brain to sense pain.

As a technique for measuring the activation of TRPV4, first, a patch clamp electrophysiology technique capable of amplifying cell membrane currents and measuring the change thereof, and second, intracellular cells focused on TRPV4 transporting cations such as calcium ions. There is a technique for measuring calcium concentration change. The first technique is superior to the second in terms of measurement accuracy, and the second technique is complementary because it has the advantage of allowing higher speed measurement than the first technique. In addition, the TRPV4 activation measurement techniques described above can be implemented only when supported by animal neuron culture technology, cell line culture technology, TRPV4 DNA management and transfection technology. That is, various TRPV4 specific activator candidates may be administered to TRPV4 overexpressing cells to measure TRPV4 activation and to determine whether the activator is active and its potency.

TRPV4 specific activator is an essential technique for measuring TRPV4 activation in the future development of TRPV4 modulator. Accordingly, the present inventors prepared a cell line expressing several TRP and compared the reaction to the treatment of chemicals known as DMAPP (dimethylallyl pyrophosphate) and TRP activator, the present invention by confirming that the DMAPP specifically activates only TRPV4 Was completed.

It is an object of the present invention to provide a composition for screening an activator or activity inhibitor of TRPV4.

Another object of the present invention is to provide a method for isolating TRPV4 positive and negative neurons.

Another object of the present invention is to provide a method for screening a TRPV4 activator or an activity inhibitor using an activator that is specifically active against TRPV4.

In order to achieve the above object, the present invention provides a composition for screening a transient receptor potential vanilloid 4 (TRPV4) activator or activity inhibitor comprising DMAPP or a salt thereof.

In addition,

1) culturing the neurons isolated from the subject and treating DMAPP or salts thereof;

2) measuring the TRPV4 activity of the neurons treated in step 1); And

3) comparing the measurement of step 2) with the measurement of TRPV4 activity of a neuron untreated with DMAPP or a salt thereof to provide a method of isolating TRPV4 positive neurons comprising selecting neurons that exhibited a TRPV4 positive response.

In addition,

1) culturing the neurons isolated from the subject and then sequentially or reversely treating DMAPP or its salts and nonspecific thermoTRPV activators;

2) measuring calcium ion channel activity of the neurons treated in step 1), respectively; And

3) Measurements of step 2) were compared to measurements of calcium ion channel activity of neurons that had not been treated with DMAPP or its salts and non-specific thermoTRP activators to identify neurons that showed positive response to non-specific thermoTRP activators but negative to DMAPP or salts thereof. It provides a method for separating TRPV4 negative neurons comprising the step of screening.

In addition,

1) treating TRPV4 positive neurons with DMAPP or salts thereof and TRPV4 activity inhibitor candidates;

2) treating TRPV4 negative neurons with the TRPV4 activity inhibitor candidate and nonspecific thermoTRP activator;

3) measuring calcium ion channel activity of TRPV4 positive neurons and TRPV4 negative neurons treated in steps 1) and 2), respectively; And

4) Inhibit calcium ion channel activity of TRPV4 positive neurons treated with DMAPP or its salts and TRPV4 activity inhibitor candidates by comparing each measurement measured in step 3) with the activity measurements of DMRP or its salt-treated TRPV4 positive neurons And selecting candidates that do not affect calcium ion channel activity of TRPV4 negative neurons treated with the TRPV4 activity inhibitor candidates and the non-specific thermoTRP activator.

In addition,

1) preparing a transformant in which a plasmid comprising a polynucleotide encoding TRPV4 is transduced into a host cell;

2) treating the transformant with DMAPP or a salt thereof and a TRPV4 activity inhibitor candidate;

3) treating TRPV4 negative neurons with the TRPV4 activity inhibitor candidate and nonspecific thermoTRP activator;

4) measuring TRPV4 calcium ion channel activity of the transformants and TRPV4 negative neurons treated in steps 2) and 3), respectively; And

5) inhibiting calcium ion channel activity of transformants treated with DMAPP or its salts and TRPV4 activity inhibitor candidates by comparing each measurement in step 4) to the TRPV4 activity measurements of the transformants treated with DMAPP or its salts alone; It provides a TRPV4 activity inhibitor screening method comprising the step of selecting candidates that do not affect the calcium ion channel activity of the TRPV4 negative neuron treated with the TRPV4 activity inhibitor candidate and non-specific thermoTRP activator.

In addition,

1) administering DMAPP or a salt thereof and a TRPV4 activity inhibitor candidate to a subject;

2) measuring the invasive behavioral induction of the subject treated in step 1); And

3) providing a method of screening a TRPV4 activity inhibitor comprising comparing a measurement of step 2) with a measurement of invasive behavior of a subject treated with DMAPP or a salt thereof to screen for a candidate causing an invasive behavior of the candidate. do.

In addition,

1) treating TRPV4 positive cells with DMAPP or a salt thereof and a TRPV4 activator candidate test compound, respectively;

2) measuring TRPV4 activity of the treated TRPV4 positive cells of step 1), respectively; And

3) A method for screening a TRPV4 activator comprising comparing the respective measurements of step 2) and selecting a test compound having a higher activity value than the TRPV4 activity measurement of TRPV4 positive cells treated with DMAPP or salts thereof.

Also,

1) preparing a transformant in which a plasmid comprising a polynucleotide encoding TRPV4 is transduced into a host cell;

2) treating the transformant with DMAPP or a salt thereof and a TRPV4 activator candidate test compound;

3) measuring the TRPV4 activity of the treated transformants of step 2), respectively; And

4) A method for screening a TRPV4 activator comprising comparing the respective measurements of step 3) and selecting a test compound having a higher activity than that of the TRPV4 activity of a TRPV4 transformant treated with DMAPP or a salt thereof.

DMAPP or a salt thereof of the present invention specifically acts only on TRPV4 among several TRPs, which are important receptors for pain transmission, so that 1. an active agent or an inhibitor of TRPV4 can be screened and 2. sensory neurons expressing TRPV4 It can be used to study the mechanism of TRPV4 action and to develop TRPV4 action analgesic.

1 is a diagram showing the TRPV4 specific activity of DMAPP.
1A is a diagram showing an increase in intracellular calcium influx (n = 5) by TRPV4 activated by 10 μM DMAPP treatment in Fluo-3 calcium imaging experiments using TRPV4-transformed HEK293T cells.
FIG. 1B is a diagram showing the current-voltage relationship of TRPV4 in response to 10 μM DMAPP, 10 μM 4α-PDD (4a-Phorbol 12-13-didecanoate) treatment.
1C is a diagram showing the current-voltage relationship of TRPV4 in response to 10 μM DMAPP and 10 μM DMAPP + 30 μM RN-1734 treatment.
1D is a diagram showing the quantitative relationship (Dose-response curve) of TRPV4 activity by the response to DMAPP determined by Fluo-3 calcium imaging experiment. EC50 of DMAPP determined by Hill-plot method was 2.5 μM.
Figure 1e is a diagram showing the response of several TRP transformed cell lines to DMAPP treatment. Only TRPV4 cell line showed increased calcium influx.
Figure 2 is a diagram showing the response of cultured mouse spinal muscle ganglion neurons by treatment with TRPV4 activator.
Figure 2a is a representative diagram of the pharmacological response of neurons in a low osmotic environment (stimulation to activate 10 μM DMAPP, 10 μM 4α-PDD (4a-Phorbol 12-13-didecanoate), and TRPV4 in Fura-2 calcium imaging experiments) 219 mOsm / kg: marked hypo) shows that the calcium influx of neurons appeared. KCl depolarization was conducted in the middle of the experiment to confirm that the electrical properties of the neurons were maintained. The calcium influx reaction also showed that the neurons maintained their normal electrical properties. N means the number of neurons.
FIG. 2B shows the current-voltage relationship of spinal cord ganglion neurons in response to 10 μM DMAPP, 10 μM or 4α-Phorbol 12-13-didecanoate (4a-Phorbol 12-13-didecanoate) treatment.
Figure 3 shows that DMAPP causes pain in inflammatory conditions pretreated with 100 ng prostaglandin E2.
Figure 3a shows that the increase in the number of pain behavior (flinch) acutely occurred by administration of 3 mM DMAPP, and this response is suppressed when co-administration of ruthenium red (RR), a general inhibitor of TRP pathway activity.
Figure 3b is a diagram showing the total number of pain behaviors of Figure 3a for 10 minutes.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for screening a TRPV4 activator or activity inhibitor comprising dimethylallyl pyrophosphate (DMAPP) and salts thereof.

The DMAPP or a salt thereof plays a role in promoting the activity of TRPV4. In one embodiment of the present invention, it was confirmed through calcium imaging experiments that DMAPP increases its activity in TRPV4 overexpressing cells (see FIG. 1A). In addition, the effect of 4α-PDD (4α-Phorbol 12-13-didecanoate), known as the DMAPP and TRPV4 activators, on TRPV4 was confirmed by a whole-cell voltage clamp test, a kind of patch clamp technique. It has been shown to promote (see FIG. 1C). That is, all cells that responded to 4α-PDD also responded to DMAPP. In addition, the currents in response to DMAPP and 4α-PDD exhibited outward rectifying, which is typical of TRPV4-mediated currents (see FIG. 1B).

In addition, the DMAPP or a salt thereof serves to specifically activate TRPV4. In one embodiment of the present invention, TRP1 (Transient receptor potential cation channel, subfamily A, member 1), TRPV1, TRPV2, TRPV3 and TRPM8 (Transient receptor potential cation channel, subfamily M) among TRP known to be expressed in spinal muscle ganglion sensory neurons , activity was measured by adding DMAPP to the transformed cell line expressing member 8). In other words, DMAPP is only active against TRPV4.

In addition, DMAPP-induced calcium influx in spinal cord ganglion neurons in an embodiment of the present invention was shown in neurons induced by calcium influx by 4α-PDD (see FIG. 2A), and in 4α-PDD-sensitive spinal muscle ganglion neurons. The DMAPP-induced currents of showed an outward rectification, which is typical of the TRPV4-mediated currents (see FIG. 2B).

In addition, by measuring the pain behavioral response intensity by administering the DMAPP to the animal, it is possible to confirm whether the TRPV4 activity is extrapolated to the actual behavioral response, and through this, the related pain of TRPV4 can be determined. That is, DMAPP can be used as a standard material of TRPV4 activity candidates in the screening of TRPV4 activators, and can be usefully used to develop TRPV4 activity inhibitors activated by DMAPP. In addition, the DMAPP can be applied to transform into higher active or active inhibitors through its chemical processing.

DMAPP according to the present invention can be used in the form of salts and includes all salts, hydrates and solvates prepared by conventional methods. As the acceptable salt, addition salts formed by free acid are useful. Suitable free acids can be used organic and inorganic acids, inorganic acids can be hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid, etc. As organic acids citric acid, acetic acid, lactic acid, tartariac acid, maleic acid, Fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galluxuronic acid, embonic acid, glutamic acid or aspartic acid Etc. may be used, but the present invention is not limited thereto.

In addition,

1) culturing the neurons isolated from the subject and treating DMAPP or salts thereof;

2) measuring the TRPV4 activity of the neurons treated in step 1); And

3) comparing the measurement of step 2) with the measurement of TRPV4 activity of a neuron untreated with DMAPP or a salt thereof to provide a method of isolating TRPV4 positive neurons comprising selecting neurons that exhibited a TRPV4 positive response.

The subject is a vertebrate, preferably a vertebrate except human, and more preferably a mammal except human. Specifically, they are experimental animals such as rats, rabbits, guinea pigs, hamsters, dogs, and cats, and most preferably are apes, such as chimpanzees or gorillas.

The DMAPP or salt thereof is preferably treated at a concentration of 1 to 100 μM, more preferably at a concentration of 1 to 10 μM. In an embodiment of the invention the EC50 (effective concentration 50%: 50% effect concentration) of TAPPV of DMAPP was 2.5 μM and the maximum effective amount was approximately 100 μM. This suggests that DMAPP has an active effect on TRPV4 over a range of micromolar concentrations (see FIG. 1D).

In addition, the measurement of TRPV4 activity of step 2) is not limited to this, but the whole cell voltage clamp technique which can amplify the cell membrane current and measure the change, and the cells of which TRPV4 is focused on the transfer of cations such as calcium ions It can be performed by calcium imaging, a technique for measuring calcium concentration change in the body.

By analyzing the results of DMAPP-induced calcium influx and outward rectification, not only TRPV4 activity inhibitors and active agents can be selected, but also TRPV4 positive neurons can be pharmacologically isolated using a novel TRPV4 activator, DMAPP or a salt thereof.

In one embodiment of the present invention, DMAPP exhibits specific activity only to TRPV4, and thus may be usefully used to isolate only TRPV4 positive cells from sensory neurons, thus making nociceptive properties of the sensory neurons (eg, thermal, chemical and mechanical). Identification of sensitization to stimuli and discrimination of particularly vulnerable diseases (eg inflammatory, neuropathic and adverse drug reactions).

In addition,

1) culturing the neurons isolated from the subject and then sequentially or reversely treating DMAPP or its salts or nonspecific thermoTRP activators;

2) measuring calcium ion channel activity of the neurons treated in step 1), respectively; And

3) The measurements of step 2) were compared to measurements of calcium ion channel activity of neurons that had not been treated with DMAPP or its salts and non-specific thermoTRP activators, but positive for non-specific thermoTRP activators but negative for DMAPP or its salts. It provides a method for separating TRPV4 negative neurons comprising the step of.

The DMAPP or salt thereof is preferably treated at a concentration of 1 to 100 μM, more preferably at a concentration of 1 to 10 μM.

The non-specific thermoTRP activator of step 1) is preferably one or two or more substances selected from the group consisting of 2-APB, capsaicin, cinnamic aldehyde, probeneside, camphor and menthol, but is not limited thereto.

In addition, the measurement of calcium ion channel activity of the neurons of step 2) can be performed by calcium imaging, which is not limited thereto, but a whole cell voltage clamp technique and intracellular calcium concentration change technique.

In addition,

1) treating TRPV4 positive neurons with DMAPP or salts thereof and TRPV4 activity inhibitor candidates;

2) treating TRPV4 negative neurons with the TRPV4 activity inhibitor candidate and nonspecific thermoTRP activator;

3) measuring calcium ion channel activity of TRPV4 positive neurons and TRPV4 negative neurons treated in steps 1) and 2), respectively; And

4) Inhibit calcium ion channel activity of TRPV4 positive neurons treated with DMAPP or its salts and TRPV4 activity inhibitor candidates by comparing each measurement measured in step 3) with the activity measurements of DMRP or its salt-treated TRPV4 positive neurons And selecting candidates that do not affect calcium ion channel activity of TRPV4 negative neurons treated with the TRPV4 activity inhibitor candidates and the non-specific thermoTRP activator.

The TRPV4 positive neurons and TRPV4 negative neurons can be separated by the separation method of positive and negative neurons described above.

The DMAPP or salt thereof is preferably treated at a concentration of 1 to 100 μM, more preferably at a concentration of 1 to 10 μM.

The non-specific thermoTRP activator of step 2) is preferably one or two or more substances selected from the group consisting of 2-APB, capsaicin, cinnamic aldehyde, probeneside, camphor and menthol, but is not limited thereto.

In addition, the measurement of calcium ion channel activity of the neuron of step 3) may be performed by calcium imaging, which is not limited thereto, but a whole cell voltage clamp technique and a technique for measuring intracellular calcium concentration change.

Candidate of step 1) is one or two selected from the group consisting of natural compounds, synthetic compounds, RNA, DNA, polypeptides, enzymes, proteins, ligands, antibodies, antigens, metabolites of bacteria or fungi and bioactive molecules It is preferable that the above materials are not limited thereto.

In addition,

1) preparing a transformant in which a plasmid comprising a polynucleotide encoding TRPV4 is transduced into a host cell;

2) treating the transformant with DMAPP or a salt thereof and a TRPV4 activity inhibitor candidate;

3) treating TRPV4 negative neurons with the TRPV4 activity inhibitor candidate and nonspecific thermoTRP activator;

4) measuring TRPV4 calcium ion channel activity of the transformants and TRPV4 negative neurons treated in steps 2) and 3), respectively; And

5) inhibiting calcium ion channel activity of transformants treated with DMAPP or its salts and TRPV4 activity inhibitor candidates by comparing each measurement in step 4) to the TRPV4 activity measurements of the transformants treated with DMAPP or its salts alone; It provides a TRPV4 activity inhibitor screening method comprising the step of selecting candidates that do not affect the calcium ion channel activity of the TRPV4 negative neuron treated with the TRPV4 activity inhibitor candidate and non-specific thermoTRP activator.

The host cell of step 1) is not limited thereto, but is preferably a cell line that can be used for calcium channel activity research and high efficiency inhibitor search such as HEK cell line, CHO cell line, HeLa cell line, and RBL-2H3 cell line.

DMAPP of the step 2) or a salt thereof performs the action of specifically activating only TRPV4. In an embodiment of the present invention only the transforming cell lines expressing TRPV4 in thermoTRP known to be expressed in sensory neurons showed activity by DMAPP (see FIG. 1E).

The TRPV4 negative neurons can be isolated by the method of isolating negative neurons described above.

The DMAPP or a salt thereof is preferably treated at a concentration of 1 to 100 μM.

The non-specific thermoTRP activator of step 3) is preferably, but not limited to, one or two or more substances selected from the group consisting of 2-APB, capsaicin, cinnamic aldehyde, probeneside, camphor and menthol.

In addition, the measurement of TRPV4 calcium ion channel activity of step 4) may be performed by calcium imaging, which is not limited thereto, but a whole cell voltage clamp technique and a technique for measuring intracellular calcium concentration change.

In addition,

1) administering DMAPP or a salt thereof and a TRPV4 activity inhibitor candidate to a subject;

2) measuring the invasive behavioral induction of the subject treated in step 1); And

3) A method for screening a TRPV4 activity inhibitor comprising the step of selecting candidates that inhibit acute pain behaviors by comparing the measurements of step 2) with those of the invasive behavior-induced measurement of DMAPP or a salt-treated subject.

The subject is a vertebrate, preferably a vertebrate except human, and more preferably a mammal except human. Specifically, they are experimental animals such as rats, rabbits, guinea pigs, hamsters, dogs, and cats, and most preferably are apes, such as chimpanzees or gorillas.

The method of administration in step 1) is not particularly limited, but parenteral administration is preferred, and intradermal administration is most preferred.

DMAPP or its salt of step 2) is preferably administered at a concentration of 1 to 10 mM, but is not limited thereto.

The measurement of the invasive behavioral induction of step 3) may be performed by, but not limited to, analysis of pain behavior (eg, paw lick / flick) by inflammatory sensitization induction.

The inflammatory sensitization may be caused by administering one or two or more substances selected from the group consisting of prostaglandin E2, carrageenan, or CFA (complete Freund's adjuvant) prior to injection of the DMAPP or salts thereof.

TRPV4 functions as a pain sensor expressed in thinly myelinated and unmyelinated fibers of sensory nerve fibers composed of sensory neurons. In an embodiment of the present invention, it was observed whether the DMAPP treatment can actually cause nocifensive behavior. As a result of DMAPP administration in rats inflamed by prostaglandin E2 in the hind paw, a significant number of flinch behavior was observed. It was confirmed that the increase in the number of pain behaviors when the administration of ruthenium red which is an inhibitor of TRP (see Figs. 3a to 3b). It can be seen that through the treatment of DMAPP or its salts can be screened for pain inhibitors related to TRPV4.

In addition,

1) treating TRPV4 positive cells with DMAPP or a salt thereof and a TRPV4 activator candidate test compound, respectively;

2) measuring TRPV4 activity of the treated TRPV4 positive cells of step 1), respectively; And

3) A method for screening a TRPV4 activator comprising comparing the respective measurements of step 2) and selecting a test compound having a higher activity value than the TRPV4 activity measurement of TRPV4 positive cells treated with DMAPP or salts thereof.

In addition, the present invention,

1) preparing a transformant in which a plasmid comprising a polynucleotide encoding TRPV4 is transduced into a host cell;

2) treating the transformants with DMAPP or a salt thereof and a TRPV4 activator candidate test compound, respectively;

3) measuring the TRPV4 activity of the treated transformants of step 2), respectively; And

4) TRPV4 activator screening method comprising the step of screening a test compound having a higher activity value than the measurement value of TRPV4 activity of DMRP or a salt-treated TRPV4 transformant by comparing each measurement of step 3).

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example 1 TRPV Transformed Cell Line Sample

HEK293T cell lines (ATCC CRL-11268) were prepared using rTRPV1 (SEQ ID NO: 1), rTRPV2 (SEQ ID NO: 2), mTRPV3 (SEQ ID NO: 3), rTRPV4 (SEQ ID NO: 4), mTRPM8 (SEQ ID NO: 5), and mTRPA1 (SEQ ID NO: 6) Transformation was transiently carried out using plasmid DNA.

Specifically, the HEK293T cell line was prepared with Fugene6 (Roche) with 3 μg of rTRPV1, rTRPV2, mTRPV3, rTRPV4, mTRPM8 and mTRPA1 plasmid DNA and 600 ng / well of pCDNA3 (Invitrogen Corp., USA; green fluorescent protein cDNA), respectively, per 35 dish. Diagnostics Corp., USA), and were transformed according to the manufacturer's protocol. The transformed cells were incubated in DMEM / F12 medium containing 10% FBS, 1% penicillin / streptomycin in a CO 2 incubator for 24 hours and then plated on a poly-L-lysine coated glass cover slip, followed by 10 Further incubation for 24 h.

Example 2 Spinal Cord Muscle Ganglion Neuron Sample

1.5 mg / ml collagenase / dispase (collagenase) after isolated and scavenging Dorsal root ganglia of adult ICR mice (Hallym Laboratory Animal Research Institute, Korea) in cold PBS (Phosphate-buffered saline) / dispase; Roche Diagnostics Corp., USA) was treated at 37 ° C. for 45 minutes and 0.25% trypsin (Invitrogen Corp., USA) was treated for 15 minutes. Spinal cord ganglion neurons prepared by the above method were prepared using DMEM / containing 10% FBS (Fetal bovine / dirum), 1% penicillin / streptomycin and 5 ng / ml 2.5S NGF (Nerve growth factor, Invitrogen Corp., USA). After plating on a poly-L-lysine coated glass cover slip in F12 medium, it was incubated in a CO 2 incubator for 48-72 hours.

The experimental results of all the examples below were statistically analyzed using a two-tailed, unpaired Student's t-test, and presented in the form of mean ± S.E.M. In addition, ** P <0.01 and * P <0.05.

Experimental Example 1 Investigation of TRPV4's Response to DMAPP (Dimethylallyl pyrophosphate) and 4α-PDD (4a-Phorbol 12-13-didecanoate)

<1-1> DMAPP and 4α-PDD Treatment

The TRPV4 transgenic cell line prepared by the method of Example 1 was treated with 10 μM DMAPP (Dimethylallyl pyrophosphate; Sigma-Aldrich, USA) and 10 μM 4α-PDD (4a-Phorbol 12-13-didecanoate; Sigma-Aldrich, USA) It was. The stock solution of the chemicals was prepared using water, DMSO or ethanol and diluted with test solution immediately before use.

<1-2> Whole Cell Voltage Clamp Experiment

According to the method of Bandell M et al. (Neuron 41: 849-857,2004) recording of whole-cell voltage clamp, which is a kind of patch clamp technique, on the transformed cells treated by the method of Experimental Example 1-1. Was performed.

Specifically, an extracellular solution (140 mM NaCl, 5 mM KCl, 2 mM CaCl 2, 1 mM MgCl 2, 10 mMHEPES) and titrated to pH 7.4 using NaOH and a pipette solution (140 mM CsCl, 5 mM EGTA, 10 mM HEPES, 2.0 mM MgATP, 0.2 mM NaGTP and titrated to pH 7.2 with CsOH). In addition, the experiment was repeated without the intersweep period by repeatedly repeating the conditions of voltage ramp for 325 seconds from -60 ㎷, -80 ㎷ to +80 +, and return to -60 ㎳ for 250 ㎳. The experiment was repeated five times.

As a result, a significant increase in current was generated upon treatment with DMAPP as shown in FIG. 1B, and the cells responded comparably with 4α-PDD treatment, also known as TRPV4 activator. That is, all the cells responding to DMAPP also responded to 4α-PDD. The currents in response to DMAPP and 4α-PDD exhibited outward rectifying which is typical of TRPV4-related currents. In addition, as shown in FIG. 1C, the increase in current by DMAPP was reduced by simultaneous treatment of RN-1734, known as a TRPV4 inhibitor. In other words, DMAPP can be used to search for TRPV4 inhibitors.

Experimental Example 2 DMAPP Specific and Concentration-dependent Response of TRPV4

<2-1> DMAPP Processing

TRPV4 transgenic cell lines prepared by the method of Example 1 were treated with 10 μM DMAPP.

<2-2> DMAPP concentration

The TRPV4 transgenic cell line prepared by the method of Example 1 was treated with DMAPP while gradually increasing the concentration from 0.01 to 30 μM.

<2-3> Intracellular Calcium Level Change Using Calcium Imaging

Calcium imaging was performed on the transformed cells treated by the method of Experimental Example 2-1.

Specifically, the bath solution containing 0.02% pluronic acid (Invitrogen Corp., USA) (bath solution, 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1mMMgCl2, 10mMHEPES containing a pH using NaOH Titrated to 7.4), the Fluo-3AM or Fura-2AM (5 μM; Sigma aldrich, USA) was injected into the transformed cells treated by the method of Experimental Example 3-1 at 37 ° C. for 1 hour. Calcium imaging was then performed using an LSM5 Pascal confocal microscope (Carl Zeiss, Germany), and time-lapse image (488) using Carl Zeiss ratio tool software (Carl Zeiss, Germany). Excitation / 514 release) was stored every 3 seconds. An average numerical curve of the calcium influx reaction was prepared by Hill plot (Kd value: 2.5 μM).

As a result, the Fluo-3 calcium imaging experiment also showed a TRPV4-specific reaction result as in FIG. 1B (see FIG. 1A).

In addition, as shown in FIG. 1D, the EC50 (effective concentration 50%: 50% effect concentration) for TRPV4 of DMAPP was 2.5 μM and the maximum effective amount shown was approximately 100 μM. This suggests that DMAPP has an active effect on TRPV4 in the range of micromolar concentrations.

Experimental Example 3 Response to DMAPP by TRP Transgenic Cell Line

TRPA1, TRPV1, TRPV2, TRPV3, TRPV4 and TRPM8 transformed cell lines and untransformed HEK cell lines (control) prepared by the method of Example 1 were treated with 10 μM DMAPP. Thereafter, calcium imaging was performed on the transformed cells treated by the above method in the same manner as in Experimental Example 2-3.

As a result, only TRPV4 among the five TRP known to be expressed in the dorsal root ganglion neurons, as shown in Figure 1e showed activity by DMAPP.

Experimental Example 4 Response to DMAPP in Spinal Cord Muscle Ganglion Neurons

Spinal dorsal ganglion neurons treated by the method of Example 2 were treated with 10 μM of DMAPP and 10 μM of known TRPV4 specific activator 4α-PDD, followed by calcium imaging in the same manner as in Experimental Example 2-3 or Example 1-. Whole cell voltage clamp experiments were performed according to the method of 2.

As a result, as shown in FIG. 2a, a group of calcium-induced neurons was found in response to DMAPP and 4α-PDD. That is, 4α-PDD sensitive neurons responded to DMAPP. In addition, as shown in FIG. 2B, DMAPP-induced currents in 4α-PDD-sensitive spinal muscle ganglion neurons showed outward rectification, which is typical of TRPV4-mediated currents.

Experimental Example 5 Acute Pain Behavior (flinch)

<5-1> Inflammatory sensitization

To observe the inflammatory sensitization response by DMAPP, 10 μl of prostaglandin E2 (100 ng; Sigma aldrich, USA) was injected 30 minutes prior to the DMAPP injection into the sole of the right hind paw. In addition, mice were purified for 1 hour prior to the experiment to acclimate to the experimental environment. The mice were injected intravenously in the right hind paw.

<5-2> Acute Pain Behavior Analysis

The number of hindpaw flinching behaviors was measured for 10 minutes according to the method of Bang S et al. (Br J Pharmacol. 2010, 161 (3): 707-20). Statistical data were analyzed using a two-tailed, unpaired Student's t-test and presented in the form of mean ± SEM (*** p <0.001, ** p <0.01, * p <0.05 and ND, p> 0.05).

As a result, when DMAPP was injected into the hind paw, no significant change in behavior was found in normal rats, but behavior changed over time in rats that were locally inflamed with prostaglandin E2 for 30 minutes (Fig. 3a, FIG. 3b), no effect was observed in rats administered with excipients only or ruthenium red only. In other words, the results suggest that DMAPP can induce pain under inflammatory conditions. Coadministration of DMAPP and ruthenium red significantly reduced pain behavior, demonstrating that TRPV4 blockers could be detected through behavioral responses induced by DMAPP.

<110> Korea University Research and Business Foundation <120> Composition for screening of TRPV4 activators or inhibitors,          method for isolating TRPV4 positive or negative neurons, and          method for screening TRPV4 activators or inhibitors, using DMAPP          or its salt <130> HY101453 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 2847 <212> DNA <213> rTRPV1 <400> 1 cagctccaag gcacttgctc catttggggt gtgcctgcac ctagctggtt gcaaattggg 60 ccacagagga tctggaaagg atggaacaac gggctagctt agactcagag gagtctgagt 120 ccccacccca agagaactcc tgcctggacc ctccagacag agaccctaac tgcaagccac 180 ctccagtcaa gccccacatc ttcactacca ggagtcgtac ccggcttttt gggaagggtg 240 actcggagga ggcctctccc ctggactgcc cttatgagga aggcgggctg gcttcctgcc 300 ctatcatcac tgtcagctct gttctaacta tccagaggcc tggggatgga cctgccagtg 360 tcaggccgtc atcccaggac tccgtctccg ctggtgagaa gcccccgagg ctctatgatc 420 gcaggagcat cttcgatgct gtggctcaga gtaactgcca ggagctggag agcctgctgc 480 ccttcctgca gaggagcaag aagcgcctga ctgacagcga gttcaaagac ccagagacag 540 gaaagacctg tctgctaaaa gccatgctca atctgcacaa tgggcagaat gacaccatcg 600 ctctgctcct ggacgttgcc cggaagacag acagcctgaa gcagtttgtc aatgccagct 660 acacagacag ctactacaag ggccagacag cactgcacat tgccattgaa cggcggaaca 720 tgacgctggt gaccctcttg gtggagaatg gagcagatgt ccaggctgcg gctaacgggg 780 acttcttcaa gaaaaccaaa gggaggcctg gcttctactt tggtgagctg cccctgtccc 840 tggctgcgtg caccaaccag ctggccattg tgaagttcct gctgcagaac tcctggcagc 900 ctgcagacat cagcgcccgg gactcagtgg gcaacacggt gcttcatgcc ctggtggagg 960 tggcagataa cacagttgac aacaccaagt tcgtgacaag catgtacaac gagatcttga 1020 tcctgggggc caaactccac cccacgctga agctggaaga gatcaccaac aggaaggggc 1080 tcacgccact ggctctggct gctagcagtg ggaagatcgg ggtcttggcc tacattctcc 1140 agagggagat ccatgaaccc gagtgccgac acctatccag gaagttcacc gaatgggcct 1200 atgggccagt gcactcctcc ctttatgacc tgtcctgcat tgacacctgt gaaaagaact 1260 cggttctgga ggtgatcgct tacagcagca gtgagacccc taaccgtcat gacatgcttc 1320 tcgtggaacc cttgaaccga ctcctacagg acaagtggga cagatttgtc aagcgcatct 1380 tctacttcaa cttcttcgtc tactgcttgt atatgatcat cttcaccgcg gctgcctact 1440 atcggcctgt ggaaggcttg cccccctata agctgaaaaa caccgttggg gactatttcc 1500 gagtcaccgg agagatcttg tctgtgtcag gaggagtcta cttcttcttc cgagggattc 1560 aatatttcct gcagaggcga ccatccctca agagtttgtt tgtggacagc tacagtgaga 1620 tacttttctt tgtacagtcg ctgttcatgc tggtgtctgt ggtactgtac ttcagccaac 1680 gcaaggagta tgtggcttcc atggtgttct ccctggccat gggctggacc aacatgctct 1740 actatacccg aggattccag cagatgggca tctatgctgt catgattgag aagatgatcc 1800 tcagagacct gtgccggttt atgttcgtct acctcgtgtt cttgtttgga ttttccacag 1860 ctgtggtgac actgattgag gatgggaaga ataactctct gcctatggag tccacaccac 1920 acaagtgccg ggggtctgcc tgcaagccag gtaactctta caacagcctg tattccacat 1980 gtctggagct gttcaagttc accatcggca tgggcgacct ggagttcact gagaactacg 2040 acttcaaggc tgtcttcatc atcctgttac tggcctatgt gattctcacc tacatccttc 2100 tgctcaacat gctcattgct ctcatgggtg agaccgtcaa caagattgca caagagagca 2160 agaacatctg gaagctgcag agagccatca ccatcctgga tacagagaag agcttcctga 2220 agtgcatgag gaaggccttc cgctctggca agctgctgca ggtggggttc actcctgacg 2280 gcaaggatga ctaccggtgg tgtttcaggg tggacgaggt aaactggact acctggaaca 2340 ccaatgtggg tatcatcaac gaggacccag gcaactgtga gggcgtcaag cgcaccctga 2400 gcttctccct gaggtcaggc cgagtttcag ggagaaactg gaagaacttt gccctggttc 2460 cccttctgag ggatgcaagc actcgagata gacatgccac ccagcaggaa gaagttcaac 2520 tgaagcatta tacgggatcc cttaagccag aggatgctga ggttttcaag gattccatgg 2580 tcccagggga gaaataatgg acactatgca gggatcaatg cggggtcttt gggtggtctg 2640 cttagggaac cagcagggtt gacgttatct gggtccactc tgtgcctgcc taggcacatt 2700 cctaggactt cggcgggcct gctgtgggaa ctgggaggtg tgtgggaatt gagatgtgta 2760 tccaaccatg atctccaaac atttggcttt caactcttta tggactttat taaacagagt 2820 gaatggcaaa tctctacttg gacacat 2847 <210> 2 <211> 2768 <212> DNA <213> rTRPV2 <400> 2 ctgctctgtc cactgtgtga gacgaacagg tggagggtgg acgacgcaga gaaagctcgg 60 agcgggccgc ggaggttccc acagccccat tactgtcagc gttgagccgc acccctccgg 120 gccgcacttc ctctctcagt ccccgctgcc ggagagcccc gctaggctcg gtgatcctag 180 cctgcagttt gccgccgcta caccttggct tcagcctgcg ggcccctctc catcaccttc 240 tccaggtccc agccaggcct gcccctgcgg tatgagagag gaaccttaac atctccatct 300 ctacagaggt ttcagctgta aggagcatcc tcctctctca ggatgacttc agcctccagc 360 cccccagctt tcaggctgga gacttccgat ggagatgaag agggcaatgc tgaggtgaac 420 aaggggaagc aggaaccgcc ccccatggag tcaccattcc agagggagga ccggaattcc 480 tcccctcaga tcaaagtgaa cctcaacttc ataaagagac ctcctaaaaa cacttctgct 540 cccagccagc aggagccaga tcggtttgac cgtgaccgac tcttcagtgt ggtctcccgg 600 ggtgtccccg aggaactgac tggactgcta gaatacctgc gctggaacag caagtacctc 660 actgactctg catacacaga aggctccact ggaaagacgt gcctgatgaa ggctgtgctg 720 aaccttcagg atggggtcaa tgcctgcatc atgccgctgc tgcagattga caaggattcc 780 ggcaatccca agcccctcgt caatgcccag tgcatcgatg agttctacca aggccacagt 840 gcgctgcaca tcgccataga gaagaggagc ctgcagtgcg tgaagctgct ggtagagaat 900 ggagcggatg ttcacctccg agcctgtggc cgcttcttcc aaaagcacca aggaacttgt 960 ttctattttg gagagctacc tctttctctg gctgcgtgca ccaagcagtg ggatgtggtg 1020 acctacctcc tggagaaccc acaccagccg gccagcctgg aggccaccga ctccctgggc 1080 aacacagtcc tgcatgctct ggtaatgatt gcagataact cgcctgagaa cagtgccctg 1140 gtgatccaca tgtacgacgg gcttctacaa atgggggcgc gcctctgccc cactgtgcag 1200 cttgaggaaa tctccaacca ccaaggcctc acacccctga aactagccgc caaggaaggc 1260 aaaatcgaga ttttcaggca cattctgcag cgggaattct caggaccgta ccagcccctt 1320 tcccgaaagt ttactgagtg gtgttacggt cctgtgcggg tatcgctgta cgacctgtcc 1380 tctgtggaca gctgggaaaa gaactcggtg ctggagatca tcgcttttca ttgcaagagc 1440 ccgaaccggc accgcatggt ggttttagaa ccactgaaca agcttctgca ggagaaatgg 1500 gatcggctcg tctcaagatt cttcttcaac ttcgcctgct acttggtcta catgttcatc 1560 ttcaccgtcg ttgcctacca ccagccttcc ctggatcagc cagccatccc ctcatcaaaa 1620 gcgacttttg gggaatccat gctgctgctg ggccacattc tgatcctgct tgggggtatt 1680 tacctcttac tgggccagct gtggtacttt tggcggcggc gcctgttcat ctggatctca 1740 ttcatggaca gctactttga aatcctcttt ctccttcagg ctctgctcac agtgctgtcc 1800 caggtgctgc gcttcatgga gactgaatgg tacctacccc tgctagtgtt atccctagtg 1860 ctgggctggc tgaacctgct ttactacaca cggggctttc agcacacagg catctacagt 1920 gtcatgatcc agaaggtcat ccttcgagac ctgctccgtt tcctgctggt ctacctggtc 1980 ttccttttcg gctttgctgt agccctagta agcttgagca gagaggcccg aagtcccaaa 2040 gcccctgaag ataacaactc cacagtgacg gaacagccca cggtgggcca ggaggaggag 2100 ccagctccat atcggagcat tctggatgcc tccctagagc tgttcaagtt caccattggt 2160 atgggggagc tggctttcca ggaacagctg cgttttcgtg gggtggtcct gctgttgctg 2220 ttggcctacg tccttctcac ctacgtcctg ctgctcaaca tgctcattgc tctcatgagc 2280 gaaactgtca accacgttgc tgacaacagc tggagcatct ggaagttgca gaaagccatc 2340 tctgtcttgg agatggagaa tggttactgg tggtgccgga ggaagaaaca tcgtgaaggg 2400 aggctgctga aagtcggcac caggggggat ggtacccctg atgagcgctg gtgcttcagg 2460 gtggaggaag taaattgggt tgcttgggag aagactcttc ccaccttatc tgaggatcca 2520 tcagggccag gcatcactgg taataaaaag aacccaacct ctaaaccggg gaagaacagt 2580 gcctcagagg aagaccatct gccccttcag gtcctccagt ccccctgatg gcccagatgc 2640 agcagcaggc tggcaggatg gagtagggaa tcttcccagc cacaccagag gctactgagt 2700 tttggtggaa atataaatat ttttttgcat aaccaaaaaa aaaaaaaaaa aaaaaaaaaa 2760 aaaaaagg 2768 <210> 3 <211> 2440 <212> DNA <213> mTRPV3 <400> 3 gatctcaagg caaggactgc caccaccatc tggaacctgc cagcatatgc cttaggctcc 60 agcaatgaat gcccactcca aggagatggc gcccctcatg ggcaaaagaa ccacggcacc 120 tggcgggaac cctgttgtac tgacagagaa gaggccagca gatctcaccc ccaccaagaa 180 gagtgcacac ttcttcctgg agatagaagg atttgagccc aaccccacgg tcaccaagac 240 ctctccaccc atcttctcca agccgatgga ctccaacatc cggcagtgcc tctctggcaa 300 ctgtgatgac atggactctc cccagtctcc tcaggatgat gtgacagaga ccccatccaa 360 tcccaacagt ccgagcgcaa acctggccaa ggaagaacag aggcagaaga agaagcgact 420 gaagaagcgc atcttcgcgg ctgtgtccga gggctgcgtg gaggagctgc gggaactcct 480 acaggatctg caggacctct gcaggaggcg ccgcggcctg gatgtgcctg acttcctcat 540 gcacaagctg acagcctcag acaccgggaa gacctgcctg atgaaggctt tgctcaacat 600 caatcccaac accaaagaga tcgtgcggat tctgcttgcc ttcgctgagg agaacgacat 660 cctggacagg ttcatcaacg ctgagtacac ggaagaggcc tatgaagggc agacagcgct 720 gaacatcgcc atcgagcggc gccagggaga catcacagca gtgcttatag cagcgggtgc 780 tgacgtcaat gctcacgcca agggggtctt cttcaacccc aaataccagc atgaaggctt 840 ctattttggc gagacacccc tggctttggc agcgtgtact aaccagcctg agattgtgca 900 gctgctgatg gagaatgagc agacagacat cacttcccag gattcccggg gaaacaacat 960 cctgcacgcg ctggtgacag tggctgagga cttcaagact cagaatgact tcgttaagcg 1020 catgtatgac atgatcctgc tgaggagtgg caactgggag ctggagacca tgcgcaacaa 1080 cgatgggctc acaccactgc agctggctgc caagatgggc aaggctgaga tcctgaagta 1140 catcctcagc cgcgagatca aggagaagcc tctccggagc ttgtccagga agttcacgga 1200 ctgggcgtat gggcctgtgt catcctcact ctatgacctc accaatgtag acacaacgac 1260 ggataactct gtgctggaaa tcatcgtcta caacaccaac attgataacc gacatgagat 1320 gctgaccctg gagcctctgc atacgctgct acacacgaaa tggaagaaat ttgccaagta 1380 catgttcttc ttgtccttct gcttctattt cttctacaac atcaccctga cccttgtctc 1440 ttactaccgt cctcgggaag atgaggatct cccacacccc ttggccctga cacacaaaat 1500 gagttggctt cagctcctag ggaggatgtt tgtcctcatc tgggccacat gcatctctgt 1560 gaaagaaggc attgccattt tcctgctgag accctccgat cttcagtcca tcctgtcaga 1620 tgcctggttt cactttgtct tttttgtcca agctgtactt gtgatactgt ctgtattctt 1680 gtacttgttt gcctacaaag aatacctcgc ctgcctcgtg ctggccatgg ccctgggctg 1740 ggcgaacatg ctctactaca cgagaggctt ccagtctatg ggcatgtaca gcgtcatgat 1800 ccagaaggtc attttgcatg atgtcctcaa gttcttgttt gtttacatcc tgttcttact 1860 tggatttgga gtagcgctgg cctcactgat tgagaagtgc tccaaggaca aaaaggactg 1920 cagttcctat ggcagcttca gcgacgcggt gctggagctc ttcaagctca ccataggcct 1980 gggcgacctg aacatccagc agaactccac ctaccccatc ctctttctct tcctactcat 2040 cacctatgtc atcctcacct tcgtcctcct cctcaacatg ctcattgccc tgatggggga 2100 gacggtggag aacgtctcca aagaaagtga gcggatctgg cgcttgcaga gagccaggac 2160 catcttggag tttgagaaaa tgttaccaga atggctgaga agcagattcc gcatgggcga 2220 gctgtgcaaa gtagcagatg aggacttccg gctgtgtctg cggatcaacg aggtgaagtg 2280 gacggaatgg aaaacacacg tgtccttcct taatgaagac ccgggaccca taagacggac 2340 agcagattta aacaagattc aagattcttc caggagcaat agcaaaacca ccctctatgc 2400 gtttgatgaa ttagatgaat tcccagaaac gtcggtgtag 2440 <210> 4 <211> 3211 <212> DNA <213> rTRPV4 <400> 4 gggaggagga cgcggcggga tcaggaagcg gctgcgctgc gcccgcgtcc caagcaggcc 60 gagaagtcca aacagatctg ctcagggtcc agtatggcag atcctggtga tggcccccgt 120 gcagcgcctg gggatgtggc tgagccccct ggagacgaga gtggcacttc tggtggggag 180 gccttccccc tctcttccct ggccaacctg tttgagggag aggaaggctc ctcttctctt 240 tcaccagtgg atgctagccg ccctgctggc cccggggatg gacgtccaaa cctgcgtatg 300 aagttccagg gcgctttccg caagggggtt cccaacccca ttgacctgct ggagtccacc 360 ctgtatgagt cctcagtagt gcctgggccc aagaaagcgc ccatggattc gttgttcgac 420 tatggcactt accggcacca ccccagtgac aacaagagat ggaggaggaa ggtcgtagag 480 aagcagccac agagccccaa agctcccgcc ccccagccac cccccatcct caaagtcttc 540 aaccggccca tcctctttga catcgtgtcc cggggctcca ctgccgacct ggacggactg 600 ctctcctact tgctgaccca caagaagcgc ctgactgatg aggagttccg ggaaccatcc 660 acagggaaga cctgcctgcc caaggcactt ctgaacttaa gcaatggccg aaacgacacc 720 atcccagtgt tgctggacat tgcggaacgc acgggcaaca tgcgggagtt catcaactcg 780 cccttcagag acatctacta ccgagggcag acggcactgc acatcgccat tgaacggcgc 840 tgcaagcatt acgtggagct cctggtggcc cagggagccg atgtgcacgc gcaggcccga 900 gggcggttct tccagcccaa ggatgagggt ggctacttct actttgggga gctgcccttg 960 tccttggcag cctgcaccaa ccagccgcac atcgtcaact acctgacaga gaaccctcac 1020 aagaaagccg atatgaggcg acaggactcc agaggcaaca cggtgctcca cgcgctggtg 1080 gccatcgctg acaacacccg agagaacacc aagtttgtca ccaagatgta tgacctgttg 1140 cttctcaagt gctcccgcct cttcccagac agcaacctgg agactgtgct taacaatgac 1200 ggtctttcgc ccctcatgat ggctgccaag actggcaaga tcggggtctt tcagcacatc 1260 atccgacggg aggtgacaga tgaggacaca cggcacctgt ctcgcaagtt caaggactgg 1320 gcctacgggc ctgtgtattc ttctctctac gacctctcct ccctggatac gtgcggggag 1380 gaagtgtccg tgctggagat cctggtttac aacagcaaga tcgagaaccg ccatgagatg 1440 ctggctgtgg agcccattaa cgaactgctg agggacaagt ggcgtaagtt cggggccgtg 1500 tccttctaca tcaacgttgt ctcctatctg tgtgccatgg tcatcttcac cctcacagcc 1560 tactatcagc cactggaggg cacgccaccc tacccttacc gtaccacggt ggactacctg 1620 aggctggctg gtgaggtcat cacgctcctc acaggagtcc tgttcttctt taccagtatc 1680 aaagacttgt tcatgaagaa atgccctgga gtgaattctc tcttcgtcga tggctccttc 1740 cagttgctct acttcatcta ctcagtgctg gtggttgtgt ctgcggcgct ctacctggca 1800 gggatcgagg cctatctggc tgtgatggtc tttgccctgg tcctgggctg gatgaatgcc 1860 ctttacttca cccgtgggct gaagctgaca gggacctaca gcatcatgat tcagaagatc 1920 ctcttcaaag atctcttccg ctttctgctg gtctacctgc tttttatgat tggctatgcc 1980 tcagctctgg tcaccctcct gaatccgtgc accaacatga aggtctgtaa cgaggaccag 2040 agcaactgca cggtgccctc ataccccgcg tgccgggaca gcgagacctt cagcgccttc 2100 ctactggacc tcttcaagct caccatcggc atgggcgacc tggagatgct gagcagcgct 2160 aagtaccccg tggtcttcat tctcctgctg gttacctaca tcatcctcac cttcgtgctc 2220 ctgctgaaca tgctcatcgc cctcatgggt gagaccgtgg gccaggtgtc caaggagagc 2280 aagcacatct ggaagctgca gtgggccacc accatcctgg acatcgagcg ctccttccct 2340 gtgttcctga ggaaggcctt ccgctccgga gagatggtga cagtgggcaa gagctcggat 2400 ggcactccag accgcaggtg gtgcttcagg gtggacgagg tgaactggtc tcactggaac 2460 cagaacctgg gcatcattaa cgaggacccc ggcaagagcg agatctacca gtactatggc 2520 ttctcccata ccatggggcg cctccgcagg gatcgctggt cctcagtggt gccccgcgtg 2580 gtggagctga acaagaactc aggcacagat gaagtggtgg tccccctgga taacctaggg 2640 aaccccaact gtgacggcca ccagcaaggt tatgctccca agtggagggc ggaggacgca 2700 ccactgtagg ggccatgcca gggctggggt caatggccca ggcttggccc ttgctcccac 2760 ctacatttca gcatctgtcc tgtgtcttcc cacacccaca cgtgacctcg gaggtgaggg 2820 cctctgtgga gactctgggg aggccccagg accctctggt ccccacaaag acttttgctc 2880 ttatttctac tcctccccac atgggggacg gggctcctgg ccacctgtct cactcccatg 2940 gagtcaccta agccagctca gggcccctcc actcacaggg ctcaggcccc tgtccctctt 3000 gtgcactatt tattgctctc ctcaggaaaa tgacatcaca ggagtctacc tgcagctgga 3060 acctggccag ggctgaggct catgcaggga cactgcagcc ctgacccgct gcagatctga 3120 cctgctgcag cccgggctag ggtgggtctt ctgtactttg tagagatcgg ggctgttggt 3180 gctcaataaa tgtttgttta ttctcggtgg a 3211 <210> 5 <211> 3869 <212> DNA <213> mTRPM8 <400> 5 tcctccctcc tccagtgagc taagagacaa gcaggctctt tgaggagaga gaagctcttg 60 gctgattgag cagctccacg tcctggctgt cccggagctt gatacataga aaagactgac 120 ctcagataca cagagatcct tctgcttctg tctcccaagt gctgggatca caggcaagat 180 gtccttcgag ggagccaggc tcagcatgag gagccgcaga aatggtacta tgggcagcac 240 ccggaccctg tactccagtg tatctcggag cacagacgtg tcctacagtg acagtgattt 300 ggtgaatttt attcaggcaa attttaaaaa acgagaatgt gtcttcttta ccagagactc 360 caaggccatg gagaacatat gcaagtgtgg ttatgcccag agccagcaca tcgaaggcac 420 ccagatcaac caaaatgaga agtggaacta caaaaaacat accaaggagt ttccaacaga 480 cgccttcggg gacattcagt ttgagactct ggggaagaaa ggcaagtact tacgcttgtc 540 ctgtgacacc gactctgaaa ctctctacga actgctgacc cagcactggc acctcaaaac 600 acccaacctg gtcatttcag tgacgggtgg agccaaaaac tttgctttga agccacgcat 660 gcgcaagatc ttcagcaggc tgatttacat cgcacagtct aaaggtgcgt ggattctcac 720 tggaggcact cactacggcc tgatgaagta cataggcgag gtggtgagag acaacaccat 780 cagcaggaac tcagaagaga acatcgtggc cattggcatc gcagcatggg gcatggtctc 840 caacagggac accctcatca ggagctgtga tgatgaggga catttttcag ctcaatacat 900 catggatgac tttaccagag accctctata catcctggac aacaaccata cccacctgct 960 gcttgtggac aacggttgtc atggacaccc cacagtggaa gccaagctcc ggaatcagct 1020 ggaaaagtac atctctgagc gcaccagtca agattccaac tatggtggta agatccccat 1080 cgtgtgtttt gcccaaggag gtggaagaga gactctaaaa gccatcaaca cctctgtcaa 1140 aagcaagatc ccttgtgtgg tggtggaagg ctcggggcag attgctgatg tgatcgccag 1200 cctggtggag gtggaggatg ttttaacctc ttccatggtc aaagagaagc tggtacgctt 1260 tttaccacgc actgtgtccc ggctgcctga agaggaaatt gagagctgga tcaaatggct 1320 caaagaaatt cttgagagtt ctcacctact cacagtaatt aagatggaag aggctggaga 1380 tgagattgtg agcaacgcca tttcctatgc gctgtacaaa gccttcagca ctaatgagca 1440 agacaaggac aactggaatg gacagctgaa gcttctgctg gagtggaacc agttggacct 1500 tgccagtgat gagatcttca ccaatgatcg ccgctgggag tctgccgacc ttcaggaggt 1560 catgttcacg gctctcataa aggacagacc caagtttgtc cgcctctttc tggagaatgg 1620 cctgaatctg cagaagtttc tcaccaatga agtcctcaca gagctcttct ccacccactt 1680 cagcacccta gtgtaccgga atctgcagat cgccaagaac tcctacaatg acgcactcct 1740 cacctttgtc tggaagttgg tggcaaactt ccgtcgaagc ttctggaaag aggacagaag 1800 cagcagggag gacttggatg tggaactcca tgatgcatct ctcaccaccc ggcacccgct 1860 gcaagctctc ttcatctggg ccattcttca gaacaagaag gaactctcca aggtcatttg 1920 ggagcagacc aaaggctgta ctctggcagc cttgggggcc agcaagcttc tgaagaccct 1980 ggccaaagtt aagaatgata tcaacgctgc tggggaatcg gaggaactgg ccaatgaata 2040 tgagacccga gcagtggagt tgttcaccga gtgttacagc aatgatgaag acttggcaga 2100 acagctactg gtctactcct gcgaagcctg gggtgggagc aactgtctgg agctggcagt 2160 ggaggctaca gatcagcatt tcatcgctca gcctggggtc cagaatttcc tttctaagca 2220 atggtatgga gagatttccc gagacacgaa gaactggaag attatcctgt gtctattcat 2280 catcccctta gtgggctgtg gcctcgtatc atttaggaag aaacccattg acaagcacaa 2340 gaagctgctg tggtactatg tggccttctt cacgtcgccc ttcgtggtct tctcctggaa 2400 cgtggtcttc tacatcgcct tcctcctgct gtttgcctat gtgctgctca tggacttcca 2460 ctcagtgcca cacacccccg agctgatcct ctacgccctg gtcttcgtcc tcttctgtga 2520 tgaagtgagg cagtggtaca tgaacggagt gaattatttc accgacctat ggaacgttat 2580 ggacaccctg ggactcttct acttcatagc gggtattgta ttccggctcc actcttctaa 2640 taaaagctcg ttgtactctg ggcgcgtcat tttctgtctg gattacatta tattcacgct 2700 aaggctcatc cacattttca ccgtcagcag gaacttggga cccaagatta taatgctgca 2760 gcggatgctg atcgacgttt tcttcttcct gttcctcttt gctgtgtgga tggtggcctt 2820 tggcgtggcc agacagggga tcctaaggca aaatgaacag cgctggagat ggatcttccg 2880 ctctgtcatc tatgagccct acctggccat gtttggccag gttcccagtg acgtggatag 2940 taccacatat gacttctccc actgtacctt ctcgggaaat gagtccaagc cactgtgtgt 3000 ggagctggat gagcacaacc tgccccgctt ccctgagtgg atcaccattc cgctggtgtg 3060 catctacatg ctctccacca atatccttct ggtcaacctc ctggtcgcca tgtttggcta 3120 cacggtaggc attgtacagg agaacaacga ccaggtctgg aaattccagc ggtacttcct 3180 ggtgcaggag tactgcaacc gcctaaacat ccccttcccc ttcgttgtct tcgcttattt 3240 ctacatggtg gtgaagaagt gtttcaaatg ctgctgtaaa gagaagaata tggagtctaa 3300 tgcctgctgt ttcagaaatg aggacaatga gactttggcg tgggagggtg tcatgaagga 3360 gaattacctt gtcaagatca acacgaaagc caacgacaac tcagaggaga tgaggcatcg 3420 gtttagacaa ctggactcaa agcttaacga cctcaaaagt cttctgaaag agattgctaa 3480 taacatcaag taaggctggc gatgcttgtg gggagaaacc aaatcacaat gaggtcacag 3540 caaccacctg gatgtggagg ctcatgggac actgatggac agtactgcta atgacttcta 3600 aaggagacat tttcaggtcc ctgagcacag ggtggatgac tcttagtcac cctcaagggc 3660 ataggtcagg gagcaaagtg tacagaggac tttacacctg aagaggggtg caaaggacca 3720 tgttcttctg tgaaggtgcc tgtgttttct gcatctcaga gccttgtcct gatgctgagg 3780 gattaagtgt tgacactcct ttcccacgac tgtgactctg gccctgattt tatacttata 3840 ctgcaaaaaa aaaaaaaaaa aaaaaaaaa 3869 <210> 6 <211> 4263 <212> DNA <213> mTRPA1 <400> 6 gcgccagccg gcgtccaggt ggagtcaatg aagcgcggct tgaggaggat tctgctcccg 60 gaggaaagga aggaggtcca gggcgttgtc tatcgcggcg tcggggaaga catggactgc 120 tccaaggaat cctttaaggt ggacattgaa ggagatatgt gtagattaga agacttcatc 180 aagaaccgaa gaaaactaag caaatatgag gatgaaaatc tctgtcctct gcatcacgca 240 gcagcagaag gtcaagttga actgatggaa ctgatcatca atggttcttc gtgtgaagtg 300 ctgaatataa tggatggtta tggaaatacc ccactgcatt gtgctgcaga aaaaaatcaa 360 gttgaaagtg taaagtttct tctcagccaa ggagcaaatc caaacctccg aaatagaaac 420 atgatgtcac cccttcacat agctgtgcat ggcatgtaca acgaagtgat caaggtgttg 480 actgagcaca aggccactaa catcaattta gaaggagaga atgggaacac ggctttgatg 540 tccacgtgtg ccaaagacaa cagtgaagct ttgcaaattt tgttagaaaa aggagctaag 600 ctgtgtaaat caaataagtg gggagactac cctgtgcacc aggcagcatt ttcaggtgcc 660 aaaaaatgca tggaattaat cttagcatat ggtgaaaaga acggctacag cagggagact 720 cacattaatt ttgtgaatca caagaaagcc agccctctcc acctagcagt tcaaagcgga 780 gacttggaca tgattaagat gtgcctggac aacggtgcac acatcgacat gatggagaat 840 gccaaatgca tggccctcca ttttgctgca acccagggag ccactgacat cgttaagctc 900 atgatctcat cctataccgg aagtagtgat attgtgaatg cagttgatgg caatcaggag 960 accctgcttc acagagcctc gttatttgat caccatgacc tggcagaata cctaatatca 1020 gtgggagcag acatcaacag cactgattct gaaggacgct ctccacttat tttagcaaca 1080 gcttctgcat cctggaacat tgtgaatttg ctcctctgta aaggtgccaa agtagacata 1140 aaagatcatc ttggacgtaa ctttttgcat ttgactgtgc agcagcctta tggactaaga 1200 aatttgcggc ctgagtttat gcagatgcaa cacatcaaag agctggtgat ggatgaagac 1260 aatgacggat gcacacctct ccattatgcc tgtaggcagg gggttcctgt ctctgtaaat 1320 aacctccttg gcttcaatgt gtccattcat agcaaaagta aagataagaa gtcgcccctg 1380 cattttgcag ccagttatgg gcgcatcaat acatgtcaga gacttctgca agacataagt 1440 gatacgaggc ttttgaatga aggggatctc catgggatga cccctctcca cctggcagca 1500 aaaaatgggc atgataaagt cgttcaactc cttctgaaga aaggggcctt atttctcagt 1560 gaccacaatg gctggactgc tttgcatcac gcctccatgg gtgggtacac tcagaccatg 1620 aaggtcattc ttgatactaa cttgaaatgc acagaccgac tagatgaaga agggaacaca 1680 gcactccact ttgcagcacg ggaaggccat gccaaggctg ttgcaatgct tttgagctac 1740 aatgctgaca tcctcctgaa caagaagcaa gcttcctttc tgcatattgc cctgcacaat 1800 aagcgcaagg aagtggttct cacaaccatc agaaataaaa gatgggatga gtgtcttcaa 1860 gttttcactc ataattctcc aagcaatcga tgtccaatca tggagatggt agaatacctc 1920 cccgagtgca tgaaagttct tttagatttc tgcatgatac cttccacaga agacaagtcc 1980 tgtcaagact accatattga gtataatttc aagtatctcc aatgcccatt atccatgacc 2040 aaaaaagtag cacctaccca ggatgtggta tatgagcctc ttacaatcct caatgtcatg 2100 gtccaacata accgcataga actcctcaac caccctgtgt gtagggagta cttactcatg 2160 aaatggtgtg cctatggatt cagagcccat atgatgaacc taggatctta ttgtcttggt 2220 ctcataccca tgacccttct tgttgtcaaa atacagcctg gaatggcctt caattctact 2280 ggaataatca atggaactag tagtactcat gaggaaagaa tagacactct gaattcattt 2340 ccaataaaaa tatgtatgat tctagttttt ttatcaagta tatttggata ttgcaaagaa 2400 gtgatccaaa ttttccaaca gaaaaggaat tacttcctgg attacaacaa tgctctggaa 2460 tgggttatct atacaactag tatcatcttc gtgttgccct tgttcctcaa catcccagcg 2520 tatatgcagt ggcaatgtgg agcaatagcg atattcttct actggatgaa cttcctactg 2580 tatcttcaaa ggtttgagaa ctgtggaatt ttcattgtta tgttggaggt gatttttaaa 2640 acattgctga gatcgaccgg agtgtttatc ttcctcctac tggcttttgg cctcagcttt 2700 tatgttctcc tgaatttcca agatgccttc agcaccccat tgctttcctt aatccagaca 2760 ttcagtatga tgctaggaga catcaattat cgagatgcct tcctagaacc attgtttaga 2820 aatgagttgg catacccagt cctgaccttt gggcagctta ttgccttcac aatgtttgtc 2880 ccaattgttc tcatgaactt actgattggc ttggcggttg gggacattgc tgaggtccag 2940 aagcatgcgt cattgaagag gattgctatg caggtggaac ttcataccaa cttagaaaaa 3000 aagctgccac tctggtactt acgcaaagtg gatcagaggt ccaccatcgt gtatccaaat 3060 agacccaggc acggcaggat gctacggttt tttcattact ttcttaatat gcaagaaaca 3120 cgacaagaag taccaaacat tgacacatgc ttggaaatgg aaatattgaa acagaaatat 3180 cggctgaagg acctcacttc cctcttggaa aagcagcatg agctcatcaa actcatcatc 3240 cagaagatgg agatcatctc agagacagaa gatgaagata accattgctc tttccaagac 3300 aggttcaaga aggagaggct ggaacagatg cacagcaagt ggaattttgt cttaaacgca 3360 gttaagacta aaacacattg ttctattagc cacccggact tttagttctg tgtcttatgg 3420 gagtgggaga ctgctttaca tacttatttc agtgaatttc agtttggaaa agagcaaaga 3480 aacagaaagt tgactaacat tgctgcatgg agatcctagt tcctgcaacc tcacccatac 3540 atatgctcat atttcctgtc aattactatg tattgagaag atcctttctg acatgttcaa 3600 tttgaacatg aaggatagtc tctttcgagt gaataaaaac cagggttgtt ggaatgcata 3660 ttatggagga taagaattaa tgtaactatt aaggcagaac acaactacat aatacaagat 3720 gcatataatt ccaagtatta tatttaatct cctaccatgt taaaccttcc tgtgttataa 3780 cctgtctggg acactataat ctctgttcct actatgatta gatcatagtc tcaccctcct 3840 cgtcccatca cacatgacat cattttgagc cacatgacag aagtcctagt tagtagactg 3900 tgataagtat gaatgttaca atagaaatgt gttcccttag tgttcatcag ttgtgatggt 3960 ttaaatgaga aacgttgccc acagactcat acatttaaac ccttagtccc agttgttgct 4020 gctgcttagg ggggccacac agccttgctt gctctctcct ttctgagtgt ggagagaaat 4080 gtgatcagta agactcctgc tcctgctgcc atgctcttta ttccattatg gacttcttct 4140 gaaactgcaa gcagaaattc actgttcctt cctcaaattt cttttggtca tggtattata 4200 tcatagcaac agaaactaac ttatgtacca atggtcttaa taaagaataa agcctgtaca 4260 gtc 4263

Claims (26)

A composition for screening a TRPV4 activator or activity inhibitor comprising DMAPP or a salt thereof,
The DMAPP or a salt thereof selectively screens a TRPV4 activator or activity inhibitor, characterized in that to promote the activity of TRPV4.
delete 1) culturing the neurons isolated from the subject and treating DMAPP or salts thereof;
2) measuring the TRPV4 activity of the neurons treated in step 1); And
3) A method of isolating TRPV4-positive neurons comprising the step of selecting the neurons that showed a TRPV4-positive response by comparing the measurements of step 2) with those of DMRP-treated neurons.
The separation method according to claim 3, wherein the DMAPP or salt thereof of step 1) is treated at a concentration of 1 to 100 µM.
The method of claim 3, wherein the measurement of TRPV4 activity in step 2) is performed by whole cell voltage clamp technique and calcium imaging.
1) culturing the neurons isolated from the subject and then treating the isolated neurons with DMAPP or its salts and nonspecific thermoTRP activators;
2) measuring calcium ion channel activity of the neurons treated in step 1), respectively; And
3) Each measurement of step 2) was compared with the measurements of calcium ion channel activity of DMAPP or its salts and the neurons that had not been treated with the nonspecific thermoTRP activator to select neurons that showed a positive response to the nonspecific thermoTRP activator but negative to the DMAPP. Isolation method of TRPV4 negative neuron comprising the step of.
The method of claim 6, wherein the DMAPP or salt thereof of step 1) is treated at a concentration of 1 to 100 μM.
The method of claim 6, wherein the non-specific thermoTRP activator of step 1) is characterized in that one or two or more substances selected from the group consisting of 2-APB, capsaicin, cinnamic aldehyde, probeneside, camphor and menthol Way.
The method of claim 6, wherein the calcium ion channel activity measurement of step 2) is performed by whole cell voltage clamp technique and calcium imaging.
1) treating TRPV4 positive neurons with DMAPP or a salt thereof, and a TRPV4 activity inhibitor candidate;
2) treating TRPV4 negative neurons with the TRPV4 activity inhibitor candidate and nonspecific thermoTRP activator;
3) measuring calcium ion channel activity of TRPV4 positive neurons and TRPV4 negative neurons treated in steps 1) and 2), respectively; And
4) Inhibition of calcium ion channel activity of TRPV4-positive neurons treated with DMAPP or its salts and TRPV4 activity inhibitor candidates by comparing each measurement measured in step 3) with that of DMRP or its salt-treated TRPV4 positive neurons However, the TRPV4 activity inhibitor screening method comprising the step of selecting a candidate that does not affect the calcium ion channel activity of the TRPV4 negative neurons treated with the TRPV4 activity inhibitor candidate and non-specific thermoTRP activator.
The method of claim 10, wherein the TRPV4 positive neurons are isolated by the method of claim 3 TRPV4 activity inhibitor screening method.
The method of claim 10, wherein the TRPV4 negative neurons are isolated by the method of claim 6 TRPV4 activity inhibitor screening method.
The method of claim 10, wherein the DMAPP or salt thereof in step 1) is treated with a concentration of 1 to 100 μM TRPV4 activity inhibitor screening method.
The method of claim 10, wherein the nonspecific thermoTRP activator is one or two or more substances selected from the group consisting of 2-APB, capsaicin, cinnamic aldehyde, probeneside, camphor, and menthol. .
The method of claim 10, wherein the measurement of TRPV4 calcium ion channel activity of step 3) is performed by whole cell voltage clamp technique and calcium imaging.
1) preparing a transformant in which a plasmid comprising a polynucleotide encoding TRPV4 is transduced into a host cell;
2) treating the transformant with DMAPP or a salt thereof, and a TRPV4 activity inhibitor candidate;
3) treating TRPV4 negative neurons with the TRPV4 activity inhibitor candidate and nonspecific thermoTRP activator;
4) measuring TRPV4 calcium ion channel activity of the transformants and TRPV4 negative neurons treated in steps 2) and 3), respectively; And
5) inhibiting calcium ion channel activity of transformants treated with DMAPP or its salts and TRPV4 activity inhibitor candidates by comparing each measurement in step 4) to the TRPV4 activity measurements of the transformants treated with DMAPP or its salts alone; And screening for candidates that do not affect calcium ion channel activity of TRPV4 negative neurons treated with the TRPV4 activity inhibitor candidates and non-specific thermoTRP activators.
The method of claim 16, wherein the TRPV4 negative neurons are isolated by the method of claim 6 TRPV4 activity inhibitor screening method.
The method of claim 16, wherein the DMAPP or salt thereof in step 1) is treated with a concentration of 1 to 100 μM TRPV4 activity inhibitor screening method.
17. The method of claim 16, wherein the non-specific thermoTRP activator is one or two or more substances selected from the group consisting of 2-APB, capsaicin, cinnamic aldehyde, probeneside, camphor and menthol.
The method of claim 16, wherein the measurement of calcium ion channel activity of step 2) is performed by whole cell voltage clamp technique and calcium imaging.
1) administering DMAPP or a salt thereof, and a TRPV4 activity inhibitor candidate to a subject;
2) measuring the invasive behavioral induction of the subject treated in step 1); And
3) TRPV4 activity modulator screening method comprising the step of selecting a candidate substance causing acute pain behavior by comparing the measurement of step 2) with the invasive behavior-induced measurement of DMAPP or a salt-treated subject.
The method of claim 21, wherein the DMAPP or salt thereof in step 1) is administered at a concentration of 1 to 10 mM TRPV4 activity inhibitor screening method.
22. The method according to claim 21, wherein the measurement of invasive behavioral induction of step 2) is performed by pain behavioral analysis by inducing inflammatory sensitization.
24. The method according to claim 23, wherein the inflammatory sensitization is caused by administering one or two or more substances selected from the group consisting of prostaglandin E2, carrageenan or CFA prior to administration of the DMAPP or salts thereof. .
1) treating TRPV4 positive cells with DMAPP or a salt thereof and a TRPV4 activator candidate test compound, respectively;
2) measuring TRPV4 activity of the treated TRPV4 positive cells of step 1), respectively; And
3) TRPV4 activator screening method comprising comparing the respective measurements of step 2) and selecting a test compound having a higher activity value than the TRPV4 activity measurement of TRPV4 positive cells treated only with DMAPP or salts thereof.
1) preparing a transformant in which a plasmid comprising a polynucleotide encoding TRPV4 is transduced into a host cell;
2) treating the transformants with DMAPP or a salt thereof and a TRPV4 activator candidate test compound, respectively;
3) measuring the TRPV4 activity of the treated transformants of step 2), respectively; And
4) TRPV4 activator screening method comprising the step of selecting a test compound having a higher activity value than the measurement value of TRPV4 activity of DMAPP or a salt-treated TRPV4 transformant by comparing each measurement of step 3).

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