A method for detecting activity of a trpv4 channel mutant, a detection kit and application thereof

Abstract

The application provides a detection method and kit for TRPV4 channel mutant activity and application, and the kit comprises the following products: a host cell naturally lacking endogenous TRPV4 expression; an expression vector for expressing an exogenous TRPV4 gene or a mutant thereof; a ratio type calcium ion fluorescent indicator; and a TRPV4 channel specific agonist. The application has the beneficial effects that: the technology significantly improves the detection accuracy by selecting HeLa cells naturally lacking endogenous TRPV4, a low-concentration agonist of an nM level, and a ratio type indicator Fura-2-AM; the detection efficiency is greatly improved by optimizing PEI transfection, increasing the number of analyzable cells, and increasing the temperature to 28 DEG C; the stability of the results is ensured by detecting the background and peak calcium concentration; and finally, the stable, efficient and accurate determination of the TRPV4 mutant function is realized, which has important scientific research and application value.

Description

Technical Field

[0001] This application relates to the field of calcium ion channel detection technology, specifically to a method, detection kit, and application for detecting TRPV4 channel mutant activity. Background Technology

[0002] TRPV4 (transient receptor potential vanillin receptor 4) is a non-selective cation channel that responds to calcium ions (Ca²⁺). + TRPV4 is highly permeable and widely expressed in various tissues, playing a crucial role in bone development and homeostasis. In recent years, with the widespread adoption of gene testing technology, studies have revealed a close association between functional mutations in the TRPV4 gene and a series of rare inherited skeletal dysplasia diseases. Therefore, TRPV4 gene testing plays an increasingly important role in the accurate diagnosis, differential diagnosis, genetic counseling, and prognostic assessment of hereditary bone diseases. However, functional assessment of identified mutations of unknown significance in clinical laboratories remains a significant challenge hindering the establishment of a comprehensive clinical diagnostic system. Therefore, developing a simple, efficient, and diagnostically robust testing method is essential for improving the diagnostic rate of TRPV4-related hereditary bone diseases.

[0003] Currently, there are three main methods for studying ion channel-related scientific issues in this field: structural biology, electrophysiology, and imaging methods. Structural biology methods cannot detect changes in ion channel activity and require highly skilled equipment and personnel. Electrophysiological methods require specialized experimental equipment and personnel training or reserves. Currently, there is no imaging solution that can accurately evaluate calcium transport capacity between TRPV4 mutants and wild-type cells. Existing techniques often use HEK293 cells for TRPV4 activity detection; however, HEK293 cells express endogenous TRPV4, which interferes with the transgenic wild-type and mutant TRPV4, making the detection results difficult to interpret. Furthermore, conventional transfection efficiency is low, and the use of strong calcium indicators or incorrect combinations of fluorescent proteins often leads to unreliable results. High concentrations of agonists (usually above 1 µM) can also easily produce non-specific effects. Summary of the Invention

[0004] To address the aforementioned technical limitations, this application proposes a method, reagent kit, and application for detecting TRPV4 channel mutant activity. This method overcomes the deficiencies and defects mentioned in the background art, providing a feasible technique for clinical and laboratory use, and can accurately, efficiently, and stably detect TRPV4 channel mutant activity.

[0005] To achieve the above objectives, this application adopts the following technical solution: The first inventive point of this application is to provide a kit for detecting the activity of TRPV4 channel mutants, the kit comprising the following articles: (a) A host cell naturally lacking endogenous TRPV4 expression; (b) Expression vectors used to express exogenous TRPV4 genes or their mutants; (c) Ratio-type calcium ion fluorescent indicator; (d) TRPV4 channel-specific agonists.

[0006] Human source TRPV4 The CDS sequence of the cDNA is shown in SEQ ID No. 1 (NM 021625.5):

[0007] All TRPV4 mutants listed in the OMIM database are detectable by this kit. See the link for details: Entry Search - TRPV4 - OMIM - (OMIM.ORG) .

[0008] Optionally, in the above-mentioned kit, the host cell is selected as HeLa cells; the expression vector carries an mCherry fluorescent protein label; the ratio-type calcium ion fluorescent indicator is selected as Fura-2-AM; and the agonist is selected as GSK1016790A (usually referred to as GSK101; in addition to this agonist, hypotonic treatment and arachidonic acid can also be used as alternative agonist methods, but GSK101 is more commonly used).

[0009] The expression transfection reagent was PEI (Hieff Trans® Polyethylenimine Linear (PEI) MW40000 (rapid lysis)), and Lipofectamine™ 3000 transfection reagent was selected as a control (L300015, Invetrogen).

[0010] The second inventive point of this application is to provide a method for detecting TRPV4 channel mutant activity, using the aforementioned kit, and the detection method includes the following steps: (1) Provide a host cell line that is naturally lacking in endogenous TRPV4 expression; (2) Transform the expression vector carrying the exogenous TRPV4 gene or its mutant into the host cell to obtain recombinant cells expressing wild-type or mutant TRPV4; (3) Load the recombinant cells with a ratio-type calcium ion fluorescent indicator; (4) At a preset ambient temperature, in the presence of a TRPV4 channel-specific agonist, the changes in calcium ion concentration in recombinant cells expressing wild-type and mutant TRPV4 were detected and compared.

[0011] Optionally, in the above detection method, the host cell in step (1) is a HeLa cell.

[0012] Optionally, in the detection method described above, the transfection in step (2) is achieved by PEI-mediated transfection, which is carried out in a complete culture medium and no medium change is performed after transfection; preferably, the ratio of plasmid to PEI is 1:6; the expression vector carries an mCherry fluorescent protein label for labeling TRPV4 expression.

[0013] Optionally, in the detection method described above, the ratio-type calcium ion fluorescent indicator in step (3) is selected as Fura-2-AM.

[0014] Optionally, in the above detection method, the preset ambient temperature in step (4) is 26-30 ℃, preferably 28 ℃; the agonist is GSK101, and its concentration is 9-11 nM, preferably 10 nM.

[0015] Optionally, in the above detection method, the calcium ion concentration change detected in step (4) includes the background calcium concentration and the peak calcium concentration after agonist stimulation.

[0016] The third inventive point of this application is to provide the application of the above-described reagent kit or detection method, including any one of the following: 1) Application in constructing animal models of TRPV4-related hereditary bone diseases; 2) Application in screening TRPV4 channel activity modulators; 3) Application in screening TRPV4 channel mutant candidate drugs.

[0017] Optionally, the animal model is used for scientific research.

[0018] Compared with the prior art, this application has the following beneficial effects: The technology in this application is more accurate, more efficient, more stable, and more reliable; specifically, it is reflected in: 1. More accurate: This is reflected in three aspects: 1) HeLa cells that are naturally lacking in endogenous TRPV4 were selected to eliminate background interference; 2) Using low-concentration agonists at the nM level reduces the risk of non-specific activation of other calcium channels by the drug; 3) The use of the ratio indicator Fura-2-AM eliminated various interfering factors such as intercellular differences and instrument fluctuations, allowing for objective comparison of the activities of wild-type and different mutant TRPV4 expressed in different cells; 2. Greater efficiency: This is reflected in three aspects: 1) The optimized PEI transfection method increases transgenic efficiency to over 70%, thereby increasing the number of usable cells; 2) High transfection efficiency increases the number of cells that can be analyzed in each imaging field, thereby increasing the amount of sample collected per unit time; 3) Increasing the ambient temperature to 28 ℃ lowered the effective concentration of the agonist and accelerated the reaction kinetics, resulting in faster data acquisition and making data from each cell available for analysis; 3. More stable: This application's technology fully opens the detection window by detecting two indicators: the baseline calcium concentration and the peak calcium concentration after agonist stimulation. This allows the curves of different functional mutations to be distinguished within a relatively large range of differences, resulting in more stable differences between cells and groups. 4. More reliable: Stable, efficient and accurate detection methods are applied to the determination of TRPV4 mutation function, assisting in decision-making for the construction of TRPV4-related hereditary bone disease animal models, screening of TRPV4 channel activity regulators and clinical scientific research, which is of great significance for TRPV4-related bone disease research. Attached Figure Description

[0019] Figure 1 This is a graph comparing the efficiency of two cell transfection methods in one embodiment of this application; wherein, Figure 1 A. HeLa cells were transfected using the widely used transfection reagent (Lipofectamine™ 3000) and according to the manufacturer's recommended transfection procedure. After 12 hours, the cell membrane was stained with Cell Mask PM DeepRed dye. After washing off the staining solution, the cells were imaged using a fluorescence confocal microscope. Figure 1 B is a parallel experiment. HeLa cells were transfected using the transfection reagent (PEI) of this application, the reagent formulation (plasmid: reagent = 1:6) and the process flow. Then, the red fluorescence (cyan pseudocolor) carried by TRPV4 and the far-infrared fluorescence (purple pseudocolor) of the cell membrane dye were analyzed, and the two channels were merged using ImageJ software. Figure 1 C represents the statistical results of the proportion of double-positive cells in two channels under five random fields of view counted manually, and Student's t test was performed. *, p<0.05.

[0020] Figure 2 This is a comparative diagram showing the effects of HeLa cells and HEK293 cells on TRPV4 function identification in one embodiment of this application; wherein, Figure 2 A is a graph showing the change in cytoplasmic calcium ion concentration over time in HEK293 cells, control HeLa cells, and TRPV4-overexpressing HeLa cells detected by the ratio indicator Fura-2-AM. At the beginning of the experiment, the cells were in extracellular solution (ECS) containing 2 mM extracellular calcium. At the time points indicated by the arrows, the cells were treated with the TRPV4-specific agonist GSK101. After 400 s, the extracellular solution was replaced with extracellular solution containing 1 mM EGTA to block TRPV4-mediated extracellular calcium influx into the cells. Statistical analysis was performed on the cellular baseline calcium signal concentration and the peak concentration after agonist addition, as shown in [data missing]. Figure 2 B and Figure 2 In C; ANOVA is used for statistical analysis among multiple groups, ****, p<0.0001.

[0021] Figure 3 This is a comparison graph showing the response of TRPV4 to the agonist GSK101 at different ambient temperatures (22 ℃ and 28 ℃) in one embodiment of this application; wherein, Figure 3 A shows the curves of calcium ion concentration over time in HeLa cells overexpressing TRPV4, as indicated by Fura2 at two ambient temperatures: 22 ℃ (blue) and 28 ℃ (red). At the time points indicated by the arrows, cells were treated with a very low concentration (10 nM) of the TRPV4-specific agonist GSK101. Statistical analysis was performed on the baseline calcium signal concentration and the rate of increase in calcium concentration after agonist addition, as shown in [data missing]. Figure 3 B and Figure 3 In C; the Student t-test is used for inter-group statistical analysis, *, p<0.5; ****, p<0.0001.

[0022] Figure 4 This is a diagram illustrating the effect of the detection method of this application in distinguishing between wild-type TRPV4 and functionally enhanced mutants in one embodiment of this application; wherein, Figure 4 A shows the change in calcium ion concentration over time in HeLa cells overexpressing wild-type (WT) and functionally enhanced mutant (Mutant) TRPV4 at an ambient temperature of 28 °C, as indicated by Fura2; at the time points indicated by the arrows, cells were treated with a very low concentration (10 nM) of the TRPV4-specific agonist GSK101. Statistical analysis was performed on the baseline calcium signal concentration and the peak calcium concentration after agonist addition, as shown in [data missing]. Figure 4 B and Figure 4 In C; the Student t-test is used for inter-group statistical analysis, ****, p<0.0001. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this application clearer, a more detailed description is provided below. However, it should be understood that the description herein is merely for explaining this application and is not intended to limit its scope.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. All reagents and instruments used herein are commercially available, and the characterization methods involved can be found in relevant descriptions in the prior art, and will not be repeated here.

[0025] The source of the biological products used in this application: 1. HeLa cells: A cell line derived from the HeLa cell line at ATCC (CTCC-001-0006, Mason cells). HeLa cells grow as a monolayer adherent in culture dishes. Under a microscope, the cells are densely spread out with irregularly extending edges, forming a "paving stone" pattern. Their morphology is highly heterogeneous; in the same culture system, round, spindle-shaped, polygonal, or irregularly shaped cells can be observed. 2. Expression vector: Derived from Addgene, named TPC1-mCherry, sequence number Plasmid #135182. After modification, the TPC1 DNA sequence was digested with enzymes and replaced with the coding sequence of TRPV4 shown in SEQ ID No. 1. The full-length sequence of TPC1-mCherry is shown in SEQ ID No. 2.

[0026] SEQ ID No. 2:

[0027] 3. Ratio-type calcium ion fluorescent indicator: derived from ThermoFisher, catalog number F1221.

[0028] 4. TRPV4 channel-specific agonist: derived from Sigma, catalog number G0798-10MG.

[0029] To further understand this application, the following detailed description is provided in conjunction with the preferred embodiments.

[0030] Example 1 First, this application protects a kit for detecting TRPV4 channel mutant activity, comprising the following products: (a) Select a HeLa cell line that is naturally deficient in endogenous TRPV4 expression as the host cell; (b) The expression vector used to express the exogenous TRPV4 gene or its mutants was selected as a PEI plasmid and carried an mCherry fluorescent protein marker. (c) Ratio-type calcium ion fluorescent indicator, Fura-2-AM was selected; (d) TRPV4 channel-specific agonist, selected as GSK101.

[0031] This application also protects a detection method for detecting TRPV4 channel mutant activity using the aforementioned kit, comprising the following steps: (1) Provide a host cell line that naturally lacks endogenous TRPV4 expression, namely HeLa cells; (2) The expression vector carrying the exogenous TRPV4 gene or its mutant was transferred into the host cells to obtain recombinant cells expressing wild-type or mutant TRPV4; the transfer was carried out by PEI-mediated transfection, the transfection was performed in complete culture medium and no medium change was performed after transfection; the ratio of plasmid to PEI was 1:6; the expression vector carried an mCherry fluorescent protein label for labeling TRPV4 expression; (3) Load the recombinant cells with the ratio-type calcium ion fluorescent indicator Fura-2-AM; The specific steps are as follows: (1) Sow HeLa cells at an appropriate density (approximately 80% density) in a 35 mm confocal dish; (2) Remove Fura2-AM from -20 ℃; Dissolve in 26 µl of DMSO, dispense 2 µl per vial, and store at -20 °C; (4) Mix 2 µl of Fura-2-AM with 2 µl of 10% (w / v) F127 pluronic acid thoroughly; (5) Use 1 ml of 2 mM Ca 2+ ECS was dissolved to prepare a working solution, and adherent cells were then treated with 2 mM Ca2+. 2+ After washing with ECS once, add 1 mL of staining working solution; (6) The adherent cells were treated with 2 mM Ca 2+ After rinsing once with ECS, add 1 mL of staining working solution; (7) Incubate at room temperature in the dark for 30 min for loading cells; (8) Use 2 mM Ca 2+ Rinse twice with ECS, then add 1 ml of 2 mM Ca 2+ ECS is ready for imaging; (9) Select transfected positive cells with red fluorescence and excite Fura-2 fluorescence alternately at 340 nm / 380 nm, collecting the fluorescence signal at 510 nm. Sample at 3 s intervals and perform time-lapse photography (exposure time can be optimized according to microscope configuration). Stimulate with 10 nM GSK101 at 60 s. The fluorescence ratio of 340 nm / 380 nm before 60 s is taken as the background calcium concentration, and the maximum ratio after 60 s is taken as the peak calcium concentration.

[0032] 2 mM Ca 2+ ECS solution formulation: 140 mM NaCl, 5 KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM Mglucose, and 20 mM HEPES, pH = 7.4.

[0033] mCherry-labeled TRPV4 combined with the Fura2 ratio-based calcium indicator yields reliable results. Since Dr. Roger Tsien invented the Fura2 ratio-based calcium indicator, all instructions and manuals for using this dye have emphasized its advantage in comparing differences in calcium concentration among different cells. However, numerous reports in the literature still use the intensity-based calcium concentration indicator Fluo4 for comparing calcium concentrations between different cells or cells transfected with different plasmids. The spectral properties of Fura2 mean that it can cross-pollinate with GFP; that is, when Fura2 is excited with a 380 nm excitation wavelength, GFP is also excited simultaneously, incorporating the GFP signal into channel 380. The higher the GFP intensity, the more signal is incorporated into channel 380, resulting in a lower measured Fura2 ratio. Therefore, experiments overexpressing TRPV4-GFP and its mutants combined with Fura2 detection contain flawed designs. Based on a deep understanding of these two principles, the inventors used mCherry-labeled TRPV4 and its mutants to avoid the risk of cross-pollination. (4) In the presence of the TRPV4 channel-specific agonist GSK101 (at a concentration of nM, 10 nM), at a preset ambient temperature of 26-30 ℃ (preferably 28 ℃), the changes in calcium ion concentration of recombinant cells expressing wild-type and mutant TRPV4 were detected and compared; the detected changes in calcium ion concentration included the background calcium concentration and the peak calcium concentration after agonist stimulation.

[0034] Example 2 High-efficiency cell transfection methods: This embodiment optimizes the transfection method to achieve higher transfection and expression efficiency.

[0035] The inventors tried various transfection reagents and methods and finally found that using PEI as a transfection reagent, transfecting in complete culture medium (instead of the recommended serum-reduced culture medium), and having a plasmid to reagent ratio of 1:6, without changing the medium after transfection, can significantly improve transfection efficiency.

[0036] The transfection process is as follows: (1) Spread cells evenly in a 3.5 cm confocal dish and transfect after 24 h; (2) Prepare two 1.5 ml EP tubes, label them with PEI and the name of the plasmid to be transfected, respectively, for later use; (3) Take out Opti-MEM and put 50 µl of Opti-MEM into each of the two tubes; (4) Add 6 μL of PEI solution to the PEI tube, mix well, and let stand for 2 min; (5) Add 1 µg of plasmid to the tube to be transfected, mix well, and let stand for 2 min; (6) Mix the two tubes of reagents into a MIX and let stand for 30 min; (7) Take 107 μl of MIX and add it to a 3.5 cm confocal culture dish. Perform the operation 24 h later.

[0037] Parallel experiments compared the method of this application with the widely used Lipofectamine™ 3000 transfection reagent (according to the manufacturer's recommended procedure). Results are as follows: Figure 1 As shown, Figure 1 A represents the result of Lipofectamine™ 3000 transfection. Figure 1 B represents the PEI transfection results of this application. The cell membrane was stained with Cell Mask PM DeepRed dye, and co-localization analysis was performed using the red fluorescence carried by TRPV4. Figure 1 The results show that the transfection efficiency (proportion of double-positive cells) of the method described in this application is approximately 70%, which is nearly 20% higher than that of the Lipofectamine™ 3000 method (p<0.05). This result demonstrates that the transfection method described in this application can efficiently introduce the exogenous TRPV4 gene into HeLa cells, laying the foundation for subsequent high-throughput detection.

[0038] Example 3 Advantages of HeLa cells as a model for TRPV4 function research: This embodiment verifies that ATCC-derived HeLa cells (CTCC-001-0006, Mason cells) naturally lack endogenous TRPV4 expression, making them an ideal cellular tool for studying the function of exogenous TRPV4.

[0039] The inventors used the ratio indicator Fura2 to detect calcium ion concentrations in HEK293 cells, control HeLa cells, and TRPV4-overexpressing HeLa cells. At the start of the experiment, cells were placed in extracellular fluid containing 2 mM extracellular calcium, and at the time points indicated by the arrows, they were treated with the TRPV4-specific agonist GSK101 (1 μM). Results are as follows: Figure 2 As shown. Compared with HEK293 cells, control HeLa cells exhibited a lower background calcium concentration ( Figure 2 (A and 2B). More importantly, HEK293 cells showed a weak calcium response after treatment with the agonist GSK101, while control HeLa cells showed no response at all, indicating a lack of endogenous functional TRPV4 expression. However, when exogenous TRPV4 was overexpressed in HeLa cells, not only was the baseline calcium concentration significantly increased ( Figure 2(A and 2B), and the processing of GSK101 produced a violent response ( Figure 2 (A and 2C). This result demonstrates that the clean endogenous background of HeLa cells provides a more accurate platform for studying the function of exogenous TRPV4, enabling a clear distinction between baseline activity and agonist-induced activity.

[0040] Example 4 The effect of ambient temperature on TRPV4 activity and its optimization: This embodiment found that ambient temperature is a key factor affecting TRPV4 activity and optimized it.

[0041] The inventors used Fura-2-AM to detect the response of TRPV4-overexpressing HeLa cells to a low concentration (10 nM) of GSK101 at ambient temperatures of 22 ℃ and 28 ℃. The results are as follows: Figure 3 As shown. Under 28 ℃ conditions, HeLa cells overexpressing TRPV4 not only exhibited higher background calcium concentrations ( Figure 3 B), and the rate of increase in calcium concentration after the addition of the agonist was significantly faster than that in cells at 22°C. Figure 3 A and Figure 3 (C) This indicates that raising the ambient temperature to 28 °C significantly enhances the sensitivity of TRPV4 to agonists, allowing researchers to obtain rapid and stable activation curves using extremely low concentrations of agonists (nM levels). Therefore, this application specifies 28 °C as the standard temperature for conducting this experiment, which helps reduce drug nonspecific effects and improve experimental efficiency.

[0042] Example 5 Distinguishing between wild-type and pathogenic mutant TRPV4 using the method of this invention: This embodiment applies the complete method established by this invention to functionally identify clinically discovered enhanced TRPV4 mutants. Following the method in Example 1, expression vectors carrying mCherry-labeled wild-type (WT) and enhanced TRPV4 mutants were transfected into HeLa cells. Under the conditions of Example 3, changes in calcium ion concentration before and after stimulation with 10 nM GSK101 were detected using Fura-2-AM at 28 °C. The results are as follows. Figure 4 As shown. Compared with cells expressing WTTRPV4, cells expressing Mutant TRPV4 exhibited significantly higher basal calcium concentrations ( Figure 4 A and 4B). However, after the addition of the agonist GSK101, the peak calcium concentration in the Mutant group was significantly lower than that in the WT group ( Figure 4(A and 4C). This result precisely reveals the characteristics of this type of functionally enhanced mutant: the channel is in a state of sustained activation (elevated basal calcium), but is insensitive to agonists, exhibiting a state of self-inhibition. The method in this application can effectively distinguish pathogenic mutations from wild-type mutations based on two indicators: basal calcium and peak calcium, providing an accurate means of functional assessment for mutations of unclear clinical significance.

[0043] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A kit for detecting the activity of TRPV4 channel mutants, characterized in that, The kit includes the following products: (a) A host cell naturally lacking endogenous TRPV4 expression; (b) Expression vectors used to express exogenous TRPV4 genes or their mutants; (c) Ratio-type calcium ion fluorescent indicator; (d) TRPV4 channel-specific agonists.

2. The reagent kit according to claim 1, characterized in that, The host cell was selected as HeLa cells; the expression vector carried an mCherry fluorescent protein label; the ratio-type calcium ion fluorescent indicator was selected as Fura-2-AM; and the agonist was selected as GSK101.

3. A method for detecting TRPV4 channel mutant activity, characterized in that, The detection method, performed using the kit described in claim 1 or 2, includes the following steps: (1) Provide a host cell line that is naturally lacking in endogenous TRPV4 expression; (2) Transform the expression vector carrying the exogenous TRPV4 gene or its mutant into the host cell to obtain recombinant cells expressing wild-type or mutant TRPV4; (3) Load the recombinant cells with a ratio-type calcium ion fluorescent indicator; (4) In the presence of a TRPV4 channel-specific agonist, at a preset ambient temperature, the changes in calcium ion concentration in recombinant cells expressing wild-type and mutant TRPV4 were detected and compared.

4. The detection method according to claim 3, characterized in that, The host cell mentioned in step (1) is a HeLa cell.

5. The detection method according to claim 3, characterized in that, The transfection described in step (2) is achieved by PEI-mediated transfection, which is carried out in a complete culture medium and no medium change is performed after transfection; preferably, the ratio of plasmid to PEI is 1:6; the expression vector carries an mCherry fluorescent protein label for labeling TRPV4 expression.

6. The detection method according to claim 3, characterized in that, The ratio-type calcium ion fluorescent indicator mentioned in step (3) is Fura-2-AM.

7. The detection method according to claim 3, characterized in that, The preset ambient temperature in step (4) is 26-30 ℃, preferably 28 ℃; the agonist is GSK101, and its concentration is 9-11 nM, preferably 10 nM.

8. The detection method according to claim 3, characterized in that, The changes in calcium ion concentration detected in step (4) include the background calcium concentration and the peak calcium concentration after agonist stimulation.

9. The application of the kit according to claim 1 or 2 or the detection method according to any one of claims 3-8, characterized in that, Includes any one of the following: 1) Application in constructing animal models of TRPV4-related hereditary bone diseases; 2) Application in screening TRPV4 channel activity modulators; 3) Application in screening TRPV4 channel mutant candidate drugs.

10. The application according to claim 9, characterized in that, The animal model is used for scientific research.

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