CN116891523A

CN116891523A - TRPM3 truncate, cell line containing same and application thereof

Info

Publication number: CN116891523A
Application number: CN202310592892.8A
Authority: CN
Inventors: 张进; 李健; 曾博; 胡晗; 张雨婷; 李元朔; 冼翠玲
Original assignee: Shenzhen Jingdan Biomedical Technology Co ltd
Current assignee: Shenzhen Jingdan Biomedical Technology Co ltd
Priority date: 2023-05-24
Filing date: 2023-05-24
Publication date: 2023-10-17
Anticipated expiration: 2043-05-24
Also published as: CN116891523B

Abstract

The application provides a TRPM3 truncated body, a cell line containing the same and application thereof. The TRPM3 truncate comprises an amino acid sequence shown as SEQ ID NO. 5. The present application also provides a cell line comprising the TRPM3 truncations. The present application also provides a method of screening for a compound that interacts with TRPM3, comprising i) expressing the TRPM3 truncations on a cell membrane, contacting the compound with the TRPM3 truncations, and recording channel currents using whole cell patch clamp; or ii) contacting the compound with the cell line, and recording the channel current using whole cell patch clamp. Compared with wild type human TRPM3, the TRPM3 truncate provided by the application has obviously improved stability and yield, and the whole-cell patch clamp can record larger continuous and stable specific channel current without changing a conserved functional region.

Description

TRPM3 truncate, cell line containing same and application thereof

Technical Field

The application belongs to the technical field of biology, and in particular relates to a TRPM3 truncated body, a cell line containing the same and application thereof.

Background

Pain is an unresolved global medical problem, the most common symptom for which patients seek medical care ^[1] . In contrast to this high medical need, the available drugs for treating pain are often inadequate. In fact, about half of chronic pain patients feedback on existing prescription drugs is insufficient to relieve pain, and opioid-based pain therapies have serious side effects, including tolerance and addiction. To address this unmet clinical need, it is of particular importance to continually seek new pain targets and develop new analgesics that are effective but non-addictive treatments ^[2，3] . The cell bodies of nociceptors are located in the dorsal root ganglion (Dorsal root ganglia, DRG) that governs the body and the trigeminal ganglion (Trigeminal ganglion, TG) that governs the face, and express stimuli-sensitive molecules such as cell surface receptors and ion channels in the peripheral nerve endings of their neurons (classified as small diameter unmyelinated C fibers and myelinated medium diameter aδ fibers). Their activation can lead to sodium and calcium influx, depolarization and action potential generation, thereby regulating the information sent to the central nervous system, where TRP channels play a vital role as primary molecular sensors of noxious stimuli.

TRP channels consist of four subunits forming either homologous or heterologous channels. The general structure of each subunit consists of 6 transmembrane helix topologies (S1-S6), where S4 corresponds to a voltage sensor-like domain capable of sensing changes in intracellular ion concentration ^[4-6] . The pores of the channel are formed by alpha helical loops between the S5 and S6 subunits. Based on sequence similarity, the TRP channel family is subdivided into TRPC, TRPV, TRPM, TRPP, TRPML and TRPA6 subfamilies ^[7] . On the one hand Transient Receptor Potential (TRP) ion channels are involved in heterogeneous physiological and pathological processes, including temperature sensing ^[8] Visceral nociception ^[9] Pro-inflammatory cytokine production ^[10] Dopaminergic neuronal death ^[11] Neurogenic inflammation ^[12] Hardening of glomeruli ^[13，14] And cancer ^[15] Etc., functions are very diverse, on the other hand, targeting TRP ion channel design drugs will not have a significant off-target effect, which makes them very ideal drug targets, since the high variability of the length and the domains contained at the N-/C-terminus (both cytoplasmic) results in very low homology between TRP ion channels, typically only 10-25%.

The TRPM3 gene, located at 9q21.12-13, is the largest gene on chromosome 9, and spans 870kb, including 30 exons ^[16] . As with most other members of the TRP family, TRPM3 is a non-selective cation channel. However, TRPM3 proteins have several unique features that distinguish them from other members of this diverse family. TRPM3 has many different splice variants and it has been proposed that the encoded protein has at least 23 splice variants ^[17] Some variants (e.g., TRPM3 alpha 1) are selective for monovalent cations, while others (e.g., TRPM3 alpha 2) are highly selective for divalent cations ^[18] . TRPM3 is expressed primarily in nociceptive neurons, pancreatic beta cells, kidneys and vascular muscle layers, and is also distributed in various parts of the brain, ovaries, prostate, odontoblasts, adipocytes, oral mucosa, ciliary body and retinal pigment epithelium ^[19 ^-24] 。

There is also a great deal of work on TRPM3 at presentCan be reported by research. It is pointed out that TRPM3 plays a role in the detection of deleterious heat and in heat-related inflammation ^[25] The temperature rise increases cytoplasmic Ca by activating TRPM3 ^2+[26] . TRPM3 is considered as an alternative drug target for the treatment of induced and spontaneous neuropathic pain, involved in the development of acute noxious thermosensory and inflammatory hyperalgesia ^[27，28] . Recently, it has been demonstrated in the literature that the Gprotein βγ subunit released by Gi/o, gs or Gq-coupled receptors has an inhibitory effect on TRPM3 ^[29，30] . Inhibition of TRPM3 by Gi-coupled GABA-B receptor mediated by the G protein βγ subunit is considered as a regulatory mechanism of nociceptive response in mouse Dorsal Root Ganglion (DRG) neurons ^[31] And is considered as the main mechanism of action of opioids in analgesic treatment ^[32] . Furthermore, pharmacological inhibition of TRPM3 reduces pain in various preclinical models in mice and rats ^[33-35] Again, the potential of TRPM3 as a novel analgesic drug target is emphasized.

At present, no human TRPM3 protein purification data and related drug screening application for channel function detection are found at home and abroad. In order to develop targeted drug molecules against human TRPM3, cloning engineering and screening methods for TRPM3 are needed.

Disclosure of Invention

Since TRPM3 has a clinical pharmaceutically valuable molecule requiring an inhibitor, but currently lacks an engineered human TRPM3 useful for drug screening, the prior art can transfect murine trpm3α2 sequences on HEK293T cells and record currents using patch clamp, but if a new drug to be targeted for TRPM3 screening uses human sequences more appropriate, we found that it is difficult to record stable currents using human sequence transfection into HEK293T cells with reference to many previous techniques. In order to solve the problem that wild-type human TRPM3 is difficult to use for drug screening, the application provides a TRPM3 truncated body, a cell line containing the same and application thereof.

Specifically, the first aspect of the present application provides a TRPM3 truncate comprising an amino acid sequence as shown in SEQ ID NO. 5.

The TRPM3 truncations according to the first aspect of the present application are further linked to a6 xhis tag, MBP protein and/or HRV3C protease cleavage site.

Preferably, said ligation occurs at the N-terminus of the TRPM3 truncations described above, and/or between said 6 xhis tag, said MBP protein and said HRV3C protease cleavage site by means of a linker.

More preferably, the MBP protein comprises an amino acid sequence as shown in SEQ ID NO. 9, and the TRPM3 truncate comprises an amino acid sequence as shown in SEQ ID NO. 4.

In a second aspect, the present application provides a cell line comprising a TRPM3 truncate according to the first aspect of the present application.

Preferably, the cell line is selected from 293T cells.

In a third aspect the present application provides a method of screening a compound that interacts with TRPM3 comprising i) expressing a TRPM3 truncate according to the first aspect of the present application on a cell membrane, contacting said compound with said TRPM3 truncate, recording channel current using whole cell patch clamp; or ii) contacting the compound with a cell line according to the second aspect of the application, and recording the channel current using whole cell patch clamp.

Preferably, the compounds are TRPM3 agonists and/or inhibitors.

In a fourth aspect, the present application provides an isolated nucleic acid encoding a TRPM3 truncate according to the first aspect of the present application.

Preferably, the MBP protein comprises a nucleotide sequence as shown in SEQ ID NO. 10, and the TRPM3 truncate comprises a nucleotide sequence as shown in SEQ ID NO. 7 or 8.

In a fifth aspect, the application provides a recombinant expression vector comprising a promoter and a nucleic acid according to the fourth aspect of the application.

Preferably, the backbone plasmid of the recombinant expression vector is a pEGBacmam vector or a PCDH vector.

In a sixth aspect the application provides a plasmid combination comprising a recombinant expression vector according to the fifth aspect of the application.

Preferably, the plasmid combination further comprises a second plasmid encoding a gag gene, a pol gene, a rev gene, and a third plasmid encoding a vsvg gene; more preferably, the backbone plasmid of the second plasmid is a psPAX2 vector and the backbone plasmid of the third plasmid is a pMD2G vector.

Wherein the gag gene encodes the major structural protein of the virus; the pol gene encodes a virus-specific enzyme; the rev gene encodes a regulator that regulates the expression of the gag and pol genes. The vsvg gene encodes an envelope protein required for viral packaging.

In a seventh aspect, the application provides a recombinant baculovirus comprising a nucleic acid as described in the fourth aspect of the application; preferably, the recombinant baculovirus is produced by infecting insect cells with a recombinant expression vector according to the fifth aspect of the application.

In an eighth aspect, the application provides a recombinant lentivirus comprising a nucleic acid as set forth in the fourth aspect of the application; preferably, the recombinant lentivirus is produced by transfecting mammalian cells with a plasmid combination according to the sixth aspect of the present application; the mammalian cells are, for example, 293T cells.

In a ninth aspect, the present application provides a method for preparing the TRPM3 truncations according to the fourth aspect of the present application, comprising infecting mammalian cells with the recombinant baculovirus according to the seventh aspect of the present application or transfecting mammalian cells with the recombinant lentivirus according to the eighth aspect of the present application.

Preferably, the mammalian cell is a 293S or 293T cell.

In a tenth aspect, the application provides a method of preparing a cell line according to the third aspect of the application, comprising infecting mammalian cells with a recombinant lentivirus according to the eighth aspect of the application.

Preferably, the mammalian cells are 293T cells.

On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the application.

The reagents and materials used in the present application are commercially available.

The application has the positive progress effects that:

the application creatively combines with the structure prediction of the protein, truncates and reforms the wild type human TRPM3 protein sequence which is originally expressed in vitro and has no function, and designs the MBP protein tag sequence at the N end of the protein, thereby being convenient for purifying the protein which is expressed in a heterogeneous way, and improving the stability and the expression quantity of the protein. Secondly, the application designs an enzyme cutting site (HRV 3C) of the protein sequence, and the modified sequence can be applied to a small molecular drug library, a biological molecular interaction experiment, a patch clamp drug screening experiment and the like of nucleic acid coding. In addition, the TRPM3 truncate provided by the application does not change a conserved functional region, compared with wild type human TRPM3, the stability and the yield of the TRPM3 truncate are obviously improved, and the whole-cell patch clamp can record larger continuous and stable specific channel current.

Drawings

FIG. 1 is a schematic diagram showing the construction of a TRPM3 truncate nucleotide sequence, a6 XHis tag nucleotide sequence, an MBP protein nucleotide sequence and an HRV3C protease cleavage site nucleotide sequence on a pEGBacMam vector by a seamless cloning technique.

FIG. 2 is a schematic diagram showing the construction of a nucleotide sequence of TRPM3 truncations on a PCDH vector by means of a seamless cloning technique.

Fig. 3 is a SEC diagram of wild-type human TRPM3 protein.

FIG. 4 is a SDS-PAGE diagram of wild-type human TRPM3 protein.

Fig. 5 is a SEC graph of TRPM3 truncations.

FIG. 6 is a SDS-PAGE map of TRPM3 truncations.

Fig. 7 is a graph of patch clamp recording voltage ramp stimulus delivered by channel current.

FIG. 8 is a plot of the change in amplitude versus time of the current of wild-type human TRPM3 channel at +100mV under 10 μM CIM0216 stimulation.

FIG. 9 is a typical graph of wild-type human TRPM3 channel currents under 10. Mu.M CIM0216 stimulation.

FIG. 10 is a plot of TRPM3 truncate channel current versus time at an amplitude of +100mV under 10. Mu.M CIM0216 stimulation.

FIG. 11 is a typical graph of TRPM3 truncate channel currents under 10. Mu.M CIM0216 stimulation.

FIG. 12 is a plot of TRPM3 truncate channel current over time at an amplitude of +100mV under 1 μM CIM0216 and 50 μM pregS stimulation.

FIG. 13 is a typical graph of TRPM3 truncate channel currents under 1. Mu.M CIM0216 and 50. Mu.M pregS stimulation.

FIG. 14 is a plot of TRPM3 truncate channel current versus time at +100mV amplitude for different concentrations of pregS stimulation.

FIG. 15 is a graph showing typical TRPM3 truncate channel currents at different concentrations of pregS stimulation.

FIG. 16 is the EC50 (semi-activation concentration) of pregS on TRPM3 truncate channel activation.

FIG. 17 is a plot of the magnitude versus time of various concentrations of Oonetin (Ono for short) inhibiting TRPM3 truncate channel currents (activated using 50uM pregS) at +100 mV.

FIG. 18 is a typical graph of Ononetin inhibition TRPM3 truncate channel currents (activated using 50uM pregS) at different concentrations.

FIG. 19 is an IC50 (half inhibitory concentration) of Oonetin inhibition of TRPM3 truncate channel (using 50uM pregS activation).

Detailed Description

The application is further illustrated by means of the following examples, which are not intended to limit the scope of the application. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

Example 1 Gene synthesis and plasmid construction

The nucleotide sequence of wild-type human TRPM3 (SEQ ID NO: 6), the nucleotide sequence of MBP protein (SEQ ID NO: 10), the nucleotide sequence of TRPM3 truncations (SEQ ID NO: 8) were synthesized by Nanjing, kyowa Biotechnology Co., ltd, carrying EcoRI and NotI cleavage sites, respectively, and the above MBP protein sequence and TRPM3 truncate sequence, and the 6 XHis tag sequence and HRV3C protease cleavage site sequence were ligated and recombined to pEGBacMam plasmid (FIG. 1).

EXAMPLE 2 preparation of recombinant baculovirus

1. Extraction of recombinant baculovirus plasmid

A recombinant pEGBacMam plasmid (FIG. 1) containing a nucleotide sequence (SEQ ID NO: 7) of a TRPM3 truncate to which MBP protein was linked was introduced into E.coli DH10Bac competent cells (Shanghai only) by heat shock transformation, and cultured at 37℃for 48-72 hours in LB solid medium containing 50. Mu.g/mL kanamycin (aladin), 7. Mu.g/mL gentamicin (aladin), 10. Mu.g/mL tetracyclomycin (aladin), 100. Mu.g/mL Bluo-gal (Thermofish), 40. Mu.g/mL IPTG (aladin). The recombinant baculovirus plasmid was extracted by selecting the homogeneous white spots to 5mL of LB liquid medium containing three antibiotics (50. Mu.g/mL kanamycin, 7. Mu.g/mL gentamicin, 10. Mu.g/mL tetracyclomycin) and culturing at 37℃for 12-16 hours at 200 rpm.

2. Recombinant baculovirus production

Cell culture 6 well plates (Nest) were taken, 1X 10 per well ⁶ 2mL sf9 Insect cells were subjected to wall culture in a constant temperature and humidity incubator at 27℃for 30min, and 100. Mu.L of select Medium (Sf-900) ^TM III SFM) 10. Mu.L of transfection reagent (Cellfectin) and 100. Mu.L of instrument Medium are added with 5. Mu.g of recombinant baculovirus plasmid, and after mixing, the mixture is incubated for 20 minutes at room temperature, the transfection complex is evenly dripped into a 6-well plate, the culture is continued for 72 hours at 27 ℃, centrifugation is carried out at 6000rpm for 15 minutes at 4 ℃, 2% FBS is added into the supernatant, and the P1-generation recombinant baculovirus is obtained after 4-degree preservation in the dark.

The P1 generation recombinant baculovirus is taken to be infected with 2mL of cell density of 5 multiplied by 10 according to the proportion of 1:100 ⁵ And (3) culturing the sf9 insect cells/mL for 72 hours at 27 ℃ in an adherence way, centrifuging at 6000rpm at 4 ℃ for 15 minutes, adding 2% FBS into the supernatant, and preserving at 4 ℃ in a dark place to obtain the P2 generation recombinant baculovirus.

The separation density is 1X 10 ⁶ 40mL sf9 insect cells per mL are cultured for 24 hours, the P2 generation recombinant baculovirus is taken to be infected according to the proportion of 1:100, the culture is carried out for 96 hours under the condition of 27 ℃ and 120rpm, the centrifugation is carried out for 15 minutes at 6000rpm at 4 ℃, after the supernatant is filtered by a 0.22 mu m filter device, 2 percent FBS is added, and the P3 generation recombinant baculovirus is obtained after the preservation at 4 ℃ in the dark.

The separation density is 1X 10 ⁶ 200mL sf9 insect cells of/mL are cultured for 24 hours, and the P3 generation recombinant baculovirus is taken to feel according to the proportion of 1:100Dyeing, culturing at 27deg.C and 120rpm for 96 hr, centrifuging at 4deg.C and 6000rpm for 15 min, filtering supernatant with 0.22 μm filter, adding 2% FBS, and storing at 4 deg.C in dark place to obtain P4 generation recombinant baculovirus.

EXAMPLE 3 protein purification bench scale

HEK-293S cells were passaged at a concentration of 1.5X10 ⁶ Culturing at 37deg.C, 8% CO2, 60% humidity and 120rpm for 24 hr, infecting mammal cells with P4 generation recombinant baculovirus at a ratio of 1:10, shaking culture for 12-18 hr, and adding sodium butyrate to final concentration of 10mM.

After 72 hours, cells were collected by centrifugation at 6000rpm at 4℃for 15 minutes, and cell pellet was resuspended in Lysis buffer (50mM HEPES,pH7.4, 150mM NaCl,1%Protease Inhibitor Cocktail,EDTA-Free,1% GDN,1mM TCEP) and the membrane was turned over at 4℃for 3 hours. After the completion of the membrane dissolution, the supernatant was centrifuged at 4℃and 40000rpm for 45 minutes, the supernatant was combined with MBP affinity chromatography packing for 2.5 hours at 4℃in a reverse manner, the combined supernatant was passed through a gravity column, the impurity protein was eluted by Wash buffer (25mM HEPES,pH7.4, 150mM NaCl,0.02%GDN,1mM TCEP), the target protein was concentrated to 500. Mu.L by using a ultrafiltration tube for eluting the target protein of 100kDa by Elute buffer (25mM HEPES,pH7.4, 150mM NaCl,0.02%GDN,1mM TCEP,40mM Maltose), the gel filtration chromatography was performed using a gel column model Superlose 6Increase 10/300GL (cytova) and the buffer was SEC buffer (25mM HEPES,pH7.4, 150mM NaCl,0.02%GDN,1mM TCEP), the protein sample was collected, the concentration of A280 was tested, and SDS PAGE gel electrophoresis was performed to detect the size and purity of the target protein. As a result, TRPM3 truncations of high purity and high yield can be expressed as shown in FIGS. 5 and 6.

The wild-type TRPM3 protein was expressed by the same experimental procedure, and as a result, as shown in fig. 3 and 4, it was impossible to express the wild-type TRPM3 protein in high purity and high yield.

EXAMPLE 4 construction of stable cell lines

The nucleotide sequence (SEQ ID NO: 8) of TRPM3 truncated form is synthesized by Nanjing department biotechnology limited company, and is connected and recombined to a lentiviral vector (figure 2) to transform and extract plasmids.

1. Preparation of lentiviruses

The biological safety cabinet is irradiated by ultraviolet lamp for 30 minutes, and the culture medium and the reagent are placed at normal temperature. 293T cells in good condition were removed from the incubator, the original medium was aspirated, washed with PBS, then digested with pancreatin, the dish was gently tapped, and the cells in the dish were transferred to a 15ml centrifuge tube for centrifugation at 1000rpm for 5min. The cell sediment is resuspended in a 10cm cell culture dish, 10mL of cell suspension is added into each hole, the cells are taken out from the incubator after the overnight, the transfection can be started after the observation density of the inverted microscope reaches about 90%, the original culture medium is sucked out, 10mL of opti-MEM is slowly added along the dish wall, and the mixture is placed in the incubator for the transfection. HEK293T cells were transfected with the lentiviral vectors and helper plasmids described above. The helper plasmids are, in particular, the psPAX2 vector containing the gag gene, the pol gene and the rev gene and the pMD2G vector containing the vsvg gene. At 6 hours after transfection, opti-MEM was discarded and replaced with fresh DMEM medium. The supernatant was aspirated from the dishes 48 hours after transfection, ultracentrifuged, filtered through a 0.22 μm pinhole filter and stored.

2. Transfecting cells

Cells were seeded in 6-well plates at about 30% confluency, and the following day the collected lentiviruses were infected to 293T cells with polybrene (5 mg/mL) added for additional transfection, and medium was removed 12 hours after transfection and replaced with fresh DMEM medium. After further culturing for 24 hours, 1. Mu.g/mL puromycin was added to screen positive cells, fresh puromycin-containing selection medium was changed daily until cells no longer continue to die, low concentration puromycin was changed to maintain cell growth, and target protein expression was detected by FACS after cell expansion was completed.

Example 5 patch clamp electrophysiological detection

The current of the TRPM3 channel in the cell line can be recorded by utilizing the whole cell patch clamp technology, and in order to test the effect of the TRPM3 truncations, the application makes current comparison of the wild type human TRPM3 protein before and after truncations, namely the wild type human TRPM3 protein and the TRPM3 truncations. Collecting HEK293T cells transfected with wild-type human TRPM3 protein or TRPM3 truncate for about 24 hours, discarding culture solution, adding artificially prepared extracellular bufferAnd (3) continuously pouring an extracellular fluid at the temperature of 22 ℃ into a recording bath, controlling the flow rate of the perfusion solution by using a peristaltic pump perfusion system, controlling the flow rate to be 2mL/min, finding out proper target cells under a microscope, generally selecting single full cells with clear edges and no concave cells, pouring artificially prepared electrode inner liquid into glass microelectrodes with the liquid inlet impedance of 2-2.5MΩ which are drawn in advance, moving the glass electrode to the cells by micro operation, controlling the air pressure in the glass electrode by using an injector, giving negative pressure, observing gradual reduction of a tested current square wave by recording software, continuously giving negative pressure, enabling an opening of the glass electrode to establish high-resistance (G omega) sealing with a cell membrane, continuously giving the negative pressure to the glass electrode to enable the cell membrane to be broken so as to be communicated with the glass electrode to form a whole cell recording mode, controlling the voltage in the cells by using an amplifier (HEKA 10 amplifier matched with a patch master recording software) connected with the glass electrode, and setting different voltages so as to record the channel currents of the whole cells. The extracellular fluid formulation used when recording TRPM3 was as follows: 138mM NaCl,5.4mM KCl,1mM MgCl ₂ ，2mM CaCl ₂ 10mM HEPES,10mM D-glucose, deionized water was dissolved, pH was adjusted to 7.4 using NaOH, and the osmolality was measured to be about 315mOsm; the formulation of the electrode inner liquid is as follows: 130mM CsMES (cesium methanesulfonate), 2mM MgCl ₂ ，10mM CsCl ₂ 10mM EGTA,10mM HEPES, deionized water was dissolved and the pH was adjusted to 7.4 using CsOH with an osmotic pressure of about 315mOsm.

Cells were given a voltage Ramp (Ramp) stimulus of-100 mV to +100mV for a period of 400ms, once every 5s, with the stimulus maintaining the cell voltage at-60 mV (FIG. 7).

The channel is activated by using 50 mu M pregnenolone sulfate (pregnenolone sulfate, hereinafter referred to as PregS) and the channel current is continuously recorded, the recorded sampling frequency is 50KHz, and the low-pass filtering is used for filtering high-frequency interference noise by using 2 KHz. When the influence of the drug taking TRPM3 as a target point on the channel current of the drug is detected, different drug concentration gradients are set, extracellular fluid is used for dissolving, if water-insoluble drugs are used, after DMSO is used for assisting in dissolving, the ratio of the final concentration of the drug to the DMSO is not more than 0.1%, so that the damage of the DMSO to the recorded cells is avoided. And continuously feeding medicines with different concentrations through the medicine feeding device while recording the current, observing the influence of the different concentrations on the cell current, calculating the current amplitude of the channel under +100mV stimulation voltage, and making a scatter diagram of the amplitude versus time. In order to calculate the half-inhibitory concentration (IC 50) of a drug on TRPM3, usually 5-6 concentration gradients are set, the inhibition ratios of different concentrations on current are calculated, and the half-inhibitory concentration is calculated by fitting a Hill evaluation to a concentration inhibition curve in a software graphpad. The inhibition rate per test of a new compound typically requires an overall test of the system with a positive inhibitor of TRPM3 and as a positive control to ensure that the patch clamp recording system is free of problems.

1. Channel current comparison of wild-type human TRPM3 protein before and after truncation

It is difficult to activate channel currents of wild-type human TRPM3 proteins using 10 μm CIM 0216. FIG. 8 shows the amplitude of wild-type human TRPM3 protein channel currents on HEK293T cell membranes recorded by whole-cell patch clamp, with protocol recorded at a voltage Ramp (Ramp) of-100 mV to +100mV for a stimulation period of 400ms, once every 5s (patch clamp method is shown in FIG. 7), each point in FIG. 8 representing the current amplitude at +100mV for Ramp stimulation. The control (control) is an artificial extracellular fluid without any agonist or inhibitor and unifies the concentration of DMSO with the small molecules added later (no more than 0.1%). From fig. 8, it can be seen that the current amplitude of the wild-type human TRPM3 protein at +100mV stimulation is not increased by the administration of the agonist.

Fig. 9 shows a typical graph of TRPM3 channel currents on HEK293T cell membranes recorded by whole cell patch clamp, showing a graph of complete currents for one ramp stimulus under different conditions. From fig. 9, it can be seen that the wild-type human TRPM3 protein hardly records the activation current on 293T cells using patch clamp, and the current curve for the agonist CIM0216 of 10uM is almost the same as that of the blank, and the current amplitude at +100mV is not increased.

After the wild-type human TRPM3 protein is truncated, not only 10uM CIM0216 can activate TRPM3, but also 1 mu M CIM0216 and 50 mu M pregS can activate current. FIG. 12 shows the amplitude of TRPM3 (post-truncate) channel current on HEK293T cell membranes recorded by whole cell patch clamp, protocol recorded by patch clamp was voltage Ramp (Ramp) stimulation of-100 mV to +100mV for a period of 400ms, once every 5s of stimulation (patch clamp method is shown in FIG. 7), each point in FIG. 10 representing the current amplitude of Ramp stimulation at +100 mV. The control (control) is an artificial extracellular fluid without any agonist or inhibitor and unifies the concentration of DMSO with the small molecules added later (no more than 0.1%).

FIG. 11 shows a typical graph of TRPM3 truncate channel currents recorded by whole cell patch clamp on HEK293T cell membranes, FIG. 11 shows a graph of the complete current of one ramp stimulus under different conditions, after sequence truncation, the change in current can be recorded under the action of 10uM agonist CIM 0216.

Fig. 12 shows the change in TRPM3 truncations current amplitude for different agonists, and it can be seen that both agonists can record changes in current amplitude after sequence truncation, suggesting that channel opening to TRPM3 can be recorded on 293T cell membranes after truncation.

FIG. 13 shows a typical graph of TRPM3 truncate channel currents recorded by whole cell patch clamp on HEK293T cell membranes, FIG. 13 shows a graph of the complete current of one ramp stimulus under different conditions, after sequence truncation, the change in current can be recorded under the action of 1. Mu.M agonists CIM0216 and 50. Mu.M agonist pregS.

2. Testing EC50 (half maximal effector concentration) of TRPM3 agonist PregS on TRPM3 truncate channel activation on HEK293T cell membrane

FIG. 14 shows the change in TRPM3 truncations current amplitude at different concentrations of pregS agonist, and it can be seen that after sequence truncation, each concentration of pregS agonist can record changes in current amplitude, suggesting that channel opening of TRPM3 can be recorded on 293T cell membranes after truncation.

Fig. 15 shows a typical graph of TRPM3 truncate channel currents recorded by whole cell patch clamp on HEK293T cell membranes, fig. 15 shows a graph of the complete currents of one ramp stimulus under different conditions, after sequence truncation, changes in currents can be recorded under different concentrations of agonist PregS.

As shown in fig. 16, EC50 = 11.13 μm was calculated by fitting a plot of activation rate versus log concentration.

3. Testing IC50 (half inhibition concentration) of TRPM3 inhibitor Oonetin (formononetin) on TRPM3 truncate channel inhibition on HEK293T cell membrane

FIG. 17 shows the change in TRPM3 truncations current amplitude at different concentrations of Onetin inhibitor following activation with 50uM pregS, and it can be seen that each concentration of Onetin inhibitor registers a change in current amplitude following sequence truncation, suggesting that channel opening of TRPM3 can be registered on 293T cell membranes following truncation.

FIG. 18 shows a typical graph of TRPM3 truncate channel currents recorded by whole cell patch clamp on HEK293T cell membranes, FIG. 18 shows a graph of the complete current of one ramp stimulus under different conditions, after sequence truncation, changes in current can be recorded under the action of different concentrations of the inhibitor Oonetin after activation with 50. Mu.M pregS.

As shown in fig. 19, IC50 = 126nM was calculated by fitting a curve of inhibition versus log concentration.

The sequences used in the present application are shown in the following table:

reference to the literature

[1]Goldberg,D.S.,&McGee,S.J.(2011).Pain as a global public health priority.BMC Public Health,11,770 10.1186/1471-2458-11-770.

[2]Yaksh,T.L.,Woller,S.A.,Ramachandran,R.,&Sorkin,L.S.(2015).The search for novel analgesics:Targets and mechanisms.F1000Prime Rep,7,56 10.12703/P7-56.

[3]Yekkirala,A.S.,Roberson,D.P.,Bean,B.P.,&Woolf,C.J.(2017).Breaking barriers to novel analgesic drug development.Nature Reviews.Drug Discovery,16,545–564.10.1038/nrd.2017.202.

[4]Held,K.,Gruss,F.,Aloi,V.D.,Janssens,A.,Ulens,C.,Voets,T.,Vriens,J.Mutations in the voltage-sensing domain affect the alternative ion permeation pathway in the TRPM3 channel.J.Physiol.2018,596,2413–2432.

[5]Hofmann,T.,Chubanov,V.,Gudermann,T.,Montell,C.TRPM5is a voltage-modulated and Ca2+-activated monovalent selective cation channel.Curr.Biol.2003,13,1153–1158.

[6]Voets,T.,Owsianik,G.,Janssens,A.,Talavera,K.,Nilius,B.TRPM8voltage sensor mutants reveal a mechanism for integrating thermal and chemical stimuli.Nat.Chem.Biol.2007,3,174–182.

[7]Chang,Y.,Schlenstedt,G.,Flockerzi,V.,Beck,A.Properties of the intracellular transient receptor potential(TRP)channel in yeast,Yvc1.FEBS Lett.2010,584,2028–2032.

[8]Moqrich,A.,Hwang,S.W.,Earley,T.J.,Petrus,M.J.,Murray,A.N.,Spencer,K.S.R.,Andahazy,M.,Story,G.M.,Patapoutian,A.Impaired thermosensation in mice lacking TRPV3,a heat and camphor sensor in the skin.Science 2005,307,1468–1472.

[9]Cenac,N.,Altier,C.,Chapman,K.,Liedtke,W.,Zamponi,G.,Vergnolle,N.Transientreceptor potential vanilloid-4 has a major role in visceral hypersensitivity symptoms.Gastroenterology 2008,135,937–946.

[10]Thilo,F.,Scholze,A.,Liu,D.Y.,Zidek,W.,Tepel,M.Association of transient receptorpotential canonical type3(TRPC3)channel transcripts with proinflammatory cytokines.Arch.Biochem.Biophys.2008,471,57–62.

[11]Kim,S.R.,Lee,D.Y.,Chung,E.S.,Oh,U.T.,Kim,S.U.,Jin,B.K.Transient receptorpotential vanilloid subtype1 mediates cell death of mesencephalic dopaminergic neurons in vivoand in vitro.J.Neurosci.2005,25,662–671.

[12]Hutter,M.M.,Wick,E.C.,Day,A.L.,Maa,J.,Zerega,E.C.,Richmond,A.C.,Jordan,T.H.,Grady,E.F.,Mulvihill,S.J.,Bunnett,N.W.,et al.Transient receptor potential vanilloid(TRPV-1)promotes neurogenic inflammation in the pancreas via activation of the neurokinin-1receptor(NK-1R).Pancreas 2005,30,260–265.

[13]Reiser,J.,Polu,K.R.,C.C.,Kenlan,P.,Altintas,M.M.,Wei,C.,Faul,C.,Herbert,S.,Villegas,I.,Avila-Casado,C.,et al.TRPC6 is a glomerular slit diaphragm-associatedchannel required for normal renal function.Nat.Genet.2005,37,739–744.

[14]Winn,M.P.,Conlon,P.J.,Lynn,K.L.,Farrington,M.K.,Creazzo,T.,Hawkins,A.F.,Daskalakis,N.,Kwan,S.Y.,Ebersviller,S.,Burchette,J.L.,et al.Medicine:A mutation in theTRPC6 cation channel causes familial focal segmental glomerulosclerosis.Science 2005,308,1801–1804.

[15]Thebault,S.,Flourakis,M.,Vanoverberghe,K.,Vandermoere,F.,Roudbaraki,M.,Lehen’kyi,V.,Slomianny,C.,Beck,B.,Mariot,P.,Bonnal,J.L.,et al.Differential role of transientreceptor potential channels in Ca2+entry and proliferation of prostate cancer epithelial cells.Cancer Res.2006,66,2038–2047.

[16]Humphray,S.J.,Oliver,K.,Hunt,A.R.,Plumb,R.W.,Loveland,J.E.,Howe,K.L.,Andrews,T.D.,Searle,S.,Hunt,S.E.,Scott,C.E.,et al.DNA sequence and analysis of humanchromosome 9.Nature 2004,429,369–374.

[17]Shiels,A.TRPM3_miR-204:A complex locus for eye development and disease.Hum.Genom.2020,14,7.

[18]Behrendt,M.Transient receptor potential channels in the context of nociception andpain—Recent insights into TRPM3 properties and function.Biol.Chem.2019,400,917–926.

[19]Grimm,C.,Kraft,R.,Sauerbruch,S.,Schultz,G.,Harteneck,C.Molecular andfunctional characterization of the melastatin-related cation channel TRPM3.J.Biol.Chem.2003,278,21493–21501.

[20]Hoffmann,A.,Grimm,C.,Kraft,R.,Goldbaum,O.,Wrede,A.,Nolte,C.,Hanisch,U.-K.,Richter-Landsberg,C.,Brück,W.,Kettenmann,H.,et al.TRPM3 is expressed in sphingosine-responsive myelinating oligodendrocytes:TRPM3 in oligodendrocytes.J.Neurochem.2010,114,654–665.

[21]Kunert-Keil,C.,Bisping,F.,Krüger,J.,Brinkmeier,H.Tissue-specific expression ofTRP channel genes in the mouse and its variation in three different mouse strains.BMC Genom.2006,7,159.

[22]Samuel,W.,Jaworski,C.,Postnikova,O.A.,Kutty,R.K.,Duncan,T.,Tan,L.X.,Poliakov,E.,Lakkaraju,A.,Redmond,T.M.Appropriately differentiated ARPE-19 cells regainphenotype and gene expression profiles similar to those of native RPE cells.Mol.Vis.2017,23,60–89.

[23]Son,A.R.,Yang,Y.M.,Hong,J.H.,Lee,S.I.,Shibukawa,Y.,Shin,D.M.OdontoblastTRP channels and thermo/mechanical transmission.J.Dent.Res.2009,88,1014–1019.

[24]Wang,H.-P.,Pu,X.-Y.,Wang,X.-H.Distribution profiles of transient receptor potentialmelastatin-related and vanilloid-related channels in prostatic tissue in rat.Asian J.Androl.2007,9,634–640.

[25]Held,K.,Voets,T.,Vriens,J.TRPM3 in temperature sensing and beyond.Temperature2015,2,201–213.

[26]Held,K.,Kichko,T.,De Clercq,K.,Klaassen,H.,Van Bree,R.,Vanherck,J.-C.,Marchand,A.,Reeh,P.W.,Chaltin,P.,Voets,T.,et al.Activation of TRPM3 by a potent syntheticligand reveals a role in peptide release.Proc.Natl.Acad.Sci.USA2015,112,E1363–E1372.

[27]Behrendt,M.(2019).Transient receptor potential channels in the context of nociceptionand pain—Recent insights into TRPM3 properties and function.Biological Chemistry,400(7),917–926.10.1515/hsz-2018-0455.

[28]Vriens,J.,Owsianik,G.,Hofmann,T.,Philipp,S.E.,Stab,J.,Chen,X.,…Voets,T.(2011).TRPM3 is a nociceptor channel involved in the detection of noxious heat.Neuron,70(3),482–494.10.1016/j.neuron.2011.02.051.

[29]Alkhatib,O.,da Costa,R.,Gentry,C.,Quallo,T.,Bevan,S.,Andersson,D.A.Promiscuous G-protein-coupled receptor inhibition of transient receptor potential melastatin 3ion channels by Gβγsubunits.J.Neurosci.2019,39,7840–7852.

[30]Badheka,D.,Yudin,Y.,Borbiro,I.,Hartle,C.M.,Yazici,A.,Mirshahi,T.,Rohacs,T.Inhibition of transient receptor potential melastatin 3 ion channels by G-proteinβγsubunits.eLife 2017,6,e26147.

[31]Quallo,T.,Alkhatib,O.,Gentry,C.,Andersson,D.A.,Bevan,S.G proteinβγsubunitsinhibit TRPM3 ion channels in sensory neurons.eLife 2017,6,e26138.

[32]Dembla,S.,Behrendt,M.,Mohr,F.,Goecke,C.,Sondermann,J.,Schneider,F.M.,Schmidt,M.,Stab,J.,Enzeroth,R.,Leitner,M.G.,et al.Anti-nociceptive action of peripheralmu-opioid receptors by G-beta-gammaprotein-mediated inhibition of TRPM3 channels.eLife2017,6,e26280.

[33]Jia,S.,Zhang,Y.,&Yu,J.(2017).Antinociceptive effects of isosakuranetin in a ratmodel of peripheral neuropathy.Pharmacology,100(3–4),201–207.10.1159/000478986.

[34]Krugel,U.,Straub,I.,Beckmann,H.,&Schaefer,M.(2017).Primidone inhibitsTRPM3 and attenuates thermal nociception in vivo.Pain,158(5),856–867.10.1097/j.pain.

[35]Straub,I.,Krugel,U.,Mohr,F.,Teichert,J.,Rizun,O.,Konrad,M.,…Schaefer,M.(2013).Flavanones that selectively inhibit TRPM3 attenuate thermal nociception in vivo.Molecular Pharmacology,84(5),736–750.10.1124/mol.113.086843.

Claims

1. A TRPM3 truncate, characterized in that the TRPM3 truncate comprises an amino acid sequence as shown in SEQ ID No. 5.

2. The TRPM3 truncate of claim 1, wherein said TRPM3 truncate is further linked to a6 xhis tag, MBP protein and/or HRV3C protease cleavage site;

preferably, said ligation occurs at the N-terminus of said TRPM3 truncations and/or between said 6 xhis tag, said MBP protein and said HRV3C protease cleavage site by a linker;

3. A cell line comprising a TRPM3 truncate according to claim 1 or 2;

preferably, the cell line is selected from 293T cells.

4. A method of screening for a compound that interacts with TRPM3, comprising

i) Expressing the TRPM3 truncations of claim 1 or 2 on a cell membrane, contacting the compound with the TRPM3 truncations, recording channel currents using whole cell patch clamp; or (b)

ii) contacting the compound with the cell line of claim 3, recording channel current using whole cell patch clamp;

preferably, the compounds are TRPM3 agonists and/or inhibitors.

5. An isolated nucleic acid encoding the TRPM3 truncations of claim 1 or 2;

6. A recombinant expression vector comprising a promoter and the nucleic acid of claim 5;

7. A plasmid combination comprising the recombinant expression vector of claim 6;

preferably, the plasmid combination further comprises a second plasmid encoding a gag gene, a pol gene, a rev gene, and a third plasmid encoding a vsvg gene;

more preferably, the backbone plasmid of the second plasmid is a psPAX2 vector and the backbone plasmid of the third plasmid is a pMD2G vector.

8. A recombinant baculovirus comprising the nucleic acid of claim 5; preferably, the recombinant baculovirus is produced by infecting insect cells with the recombinant expression vector of claim 6.

9. A recombinant lentivirus comprising the nucleic acid of claim 5; preferably, the recombinant lentivirus is produced by transfecting mammalian cells with the plasmid combination of claim 7; the mammalian cells are, for example, 293T cells.

10. A method for preparing the TRPM3 truncations according to claim 1 or 2, comprising infecting mammalian cells with the recombinant baculovirus of claim 8 or transfecting mammalian cells with the recombinant lentivirus of claim 9;

preferably, the mammalian cell is a 293S or 293T cell.

11. A method of preparing the cell line of claim 3, comprising transfecting a mammalian cell with the recombinant lentivirus of claim 9;

preferably, the mammalian cells are 293T cells.