Methods, apparatus, devices, and storage media for controlling the TRPM8 channel to be in the open state

Abstract

This invention provides a method, apparatus, device, and storage medium for controlling the TRPM8 channel to be in an open state. The method includes: irradiating the TRPM8 channel with a terahertz wave at a center frequency of 41–53 THz and an electric field strength of 0.2–2.0 V / nm; and extending the open time of the TRPM8 channel by enhancing the hydrogen bond stability at key locations, thus keeping the TRPM8 channel in an open state. The apparatus includes a terahertz wave emission module and a parameter control module, the latter adjusting the terahertz wave parameters to a preset range to achieve the aforementioned stabilization effect. This method solves the understability problem in TRPM8 structural analysis, provides an open conformation locking strategy for cryo-electron microscopy, can construct in vitro research tools for TRPM8 channel gating and desensitization mechanisms, and can also build a related drug screening platform.

Description

Technical Field

[0001] This invention relates to the field of biophysics, specifically to ion channel regulation and structural analysis techniques, and more particularly to a method, apparatus, device, and storage medium for controlling TRPM8 channels to be in an open state. Background Technology

[0002] The transient receptor potential melastatin 8 (TRPM8) channel, a member of the M subfamily, is a receptor for cold and menthol in the human body. As a typical multimodal ion channel, it can respond to a variety of physical and chemical stimuli, including osmotic pressure, protons, membrane depolarization, and phosphatidylinositol (4,5) bisphosphate (PIP2). This channel is a non-selective cation channel for calcium ion permeation and is highly expressed in nociceptive neurons. It plays a key role in diseases such as oxaliplatin-induced neuropathic pain, nerve injury-related cold hypersensitivity, and migraines, making it an important target for precision drug development.

[0003] Determining the high-resolution structure of TRPM8 is a prerequisite for elucidating its physiological and pathological mechanisms. Atomic-level channel structures can reveal ligand-binding pockets and conformational changes in gated helices, revealing the molecular mechanisms by which temperature and ligands drive channel opening and closing. This also provides direct guidance for rational drug design, avoiding the blind spots of traditional screening methods. However, the structural determination of TRPM8 has long faced significant challenges: its inherent instability on the natural cell membrane is substantial, and its crystal structure was not obtained until 2018. Furthermore, early determinations were all from avian species, and the ion-selective filtering region was not visible under an electron microscope. The successful determination of the open-state structure of mammalian TRPM8 in 2022 also confirmed its significant structural differences from avian TRPM8.

[0004] Both crystallography and single-particle cryo-electron microscopy require proteins to remain in a specific conformation for a sufficient period of time to meet the stringent requirements of conformational stability and uniformity for data acquisition. However, TRPM8 has a complex and dispersed gating mechanism, lacking independent, strongly coupled ligand-binding domains. Its open states are a collection of conformations composed of multiple metastable states, resulting in a flat energy landscape and insufficient native stability. To address this issue, current techniques require the simultaneous use of two agonists to provide additional allosteric energy through synergistic effects, thereby increasing the proportion of open-state populations and locking the conformation to meet the experimental requirements for structural resolution. Summary of the Invention

[0005] This invention provides a method for controlling a TRPM8 channel to be in an open state, comprising: irradiating the TRPM8 channel with a terahertz wave, wherein the center frequency of the terahertz wave is 41–53 THz and the electric field strength is 0.2–2.0 V / nm; and extending the open time of the TRPM8 channel by enhancing the hydrogen bond stability at key positions of the TRPM8 channel, thereby keeping the TRPM8 channel in an open state.

[0006] According to one embodiment of the present invention, the center frequency range of the terahertz wave is 42 to 43 THz.

[0007] According to one embodiment of the present invention, before irradiating the TRPM8 channel with terahertz waves, the method further includes: activating the TRPM8 channel with an agonist; the irradiation timing of the terahertz waves is: irradiation is initiated with a preset irradiation pre-time before applying the agonist to the TRPM8 channel, and the continuous irradiation duration is a preset continuous irradiation duration.

[0008] According to one embodiment of the present invention, during irradiation, the electric field polarization direction of the terahertz wave is perpendicular to the cell membrane plane where the TRPM8 channel is located.

[0009] According to one embodiment of the present invention, the key location of the TRPM8 channel is the S6 transmembrane chain and the TRP structural domain near the channel aperture, and the -COO in the hydrogen bond between the terahertz wave and the S6 transmembrane chain and the TRP structural domain. - Group stretching vibration resonance.

[0010] According to one embodiment of the present invention, before irradiating the TRPM8 with the terahertz wave, the TRPM8 channel is activated by one or more of the following stimulation combinations: low temperature, menthol, proton or membrane depolarization. The multimodal response characteristics of the TRPM8 channel provide an activation basis for stabilizing its open state under terahertz wave irradiation.

[0011] According to one embodiment of the present invention, the method further includes cryo-electron microscopy structural analysis, specifically including: after locking the open conformation of the TRPM8 channel by terahertz wave irradiation, rapidly freezing and fixing the sample containing the TRPM8 channel, and then performing cryo-electron microscopy data acquisition and structural analysis; the method further includes an open state verification method, specifically including: detecting the minimum radius of a preset site of the TRPM8 channel by preset software, wherein the statistical average value of the radius is greater than the preset channel radius.

[0012] The present invention also provides a device for controlling a TRPM8 channel to be in an open state, comprising: a terahertz wave transmitting module for transmitting terahertz waves into the TRPM8 channel; and a parameter adjustment module for adjusting the center frequency of the terahertz wave to a preset frequency range and the electric field strength to a preset electric field strength range; wherein the terahertz wave extends the open time of the TRPM8 channel by enhancing the hydrogen bond stability at key positions of the TRPM8 channel, thereby keeping the TRPM8 channel in an open state.

[0013] The present invention also provides an electronic device, including a memory, a processor, and a computer program stored in the memory and running on the processor, wherein the processor executes the computer program to implement the method of controlling the TRPM8 channel to be in an open state as described in the above embodiments.

[0014] The present invention also provides a non-transitory computer-readable storage medium having a computer program stored thereon, wherein the computer program, when executed by a processor, implements the method of controlling the TRPM8 channel to be in an open state as described in the above embodiments.

[0015] This invention achieves multi-dimensional technological breakthroughs and enhances practical value by irradiating the TRPM8 channel with terahertz waves of specific parameters. The terahertz waves resonate with the stretching vibrations of specific groups at key positions in the TRPM8 channel, enhancing the hydrogen bond stability between the transmembrane chain and structural domains, thereby stabilizing the channel's physiologically open conformation. This method eliminates the need for synergistic effects of two agonists, fundamentally avoiding the risk of overstabilization and additional conformational perturbations. It allows the locked open state to more closely resemble the physiological state, meeting the stringent requirements of conformational uniformity and stability for structural analysis.

[0016] Terahertz waves within a suitable parameter range can significantly prolong the open time of the TRPM8 channel. Irradiation within the optimal frequency range can maintain the minimum lower gate radius of the channel at a stable level for a long period, ensuring a continuous and stable open state. Simultaneously, terahertz waves can also act on relevant functional groups at the channel entrance, promoting calcium ion accumulation and accelerating inward penetration. Combined with the extended open time, the duration and amplitude of the calcium ion current are significantly enhanced, resulting in a substantial increase in calcium flux compared to the unirradiated state. This effectively induces the channel to enter a desensitized state, and this effect exhibits a clear frequency dependence.

[0017] At the application level, this method provides an innovative strategy for cryo-electron microscopy structural analysis, enabling the locking of open conformations with only a single agonist, simplifying the experimental procedure and providing crucial support for high-resolution physiological structural analysis of TRPM8 in mammals. Furthermore, this method can precisely regulate the influx of calcium ions into cells expressing TRPM8, facilitating in-depth research into channel gating and desensitization mechanisms and providing a novel technical means for basic ion channel research. Based on its regulatory role in the desensitization process, it can also be used to construct targeted drug screening platforms to screen compounds that can alleviate TRPM8 desensitization, laying the foundation for precision drug development for related diseases, and possessing significant scientific value and clinical translational potential. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 This is a schematic diagram of the process for promoting TRPM8 channel opening and desensitization by terahertz wave irradiation provided in an embodiment of the present invention.

[0020] Figure 2 This is a simplified structure of the TRPM8 channel and a schematic diagram showing the changes in the channel radius provided in an embodiment of the present invention.

[0021] Figure 3 This is a statistical diagram illustrating the minimum radius of the lower gate of the TRPM8 channel under terahertz wave irradiation at different frequencies, provided in an embodiment of the present invention.

[0022] Figure 4 This is a schematic diagram showing the change of the minimum aperture of the lower gate of the TRPM8 channel over time under three conditions: no irradiation, 42.5THz irradiation, and 34.5THz irradiation, as provided in the embodiments of the present invention.

[0023] Figure 5 This is a schematic diagram of the menthol-induced TRPM8 inward current signal waveform under four environments provided in this embodiment of the invention: no terahertz irradiation, 34.5THz irradiation, 42.5THz irradiation, and 42.5THz irradiation without calcium ions.

[0024] Figure 6 This is a schematic diagram showing the statistical results of TRPM8 current amplitude, full width at half maximum (FWHM), and AUC under four environments: no terahertz irradiation, 34.5 THz irradiation, 42.5 THz irradiation, and 42.5 THz irradiation without calcium ions, provided in the embodiments of the present invention.

[0025] Figure 7This is a schematic diagram comparing the inward current signals triggered by two menthol stimulations of neurons under conditions of 42.5THz irradiation and without, provided by an embodiment of the present invention.

[0026] Figure 8 This is a statistical diagram illustrating the desensitization status of neuronal cells to secondary menthol stimulation under conditions of 42.5THz irradiation and without, as provided in an embodiment of the present invention.

[0027] Figure 9 This is a schematic diagram of the carboxyl group distribution at key positions in the TRPM8 channel provided in an embodiment of the present invention.

[0028] Figure 10 This is a schematic diagram of the device provided by the present invention for controlling the TRPM8 channel to be in the open state.

[0029] Figure 11 This is a schematic diagram of the structure of the electronic device provided by the present invention.

[0030] Figure label: 101: TRPM8 channel; 102: Terahertz wave; 103: Menthol; 104: Calcium ion; 105: Desensitization state of TRPM8 channel; 201: Pore region helix; 202: S5 transmembrane chain; 203: S6 transmembrane chain; 204: Glycine 913 site; 205: Valine 976 site; 301: Frequency axis; 302: Minimum radius of the lower gate corresponding to the frequency; 303: Critical line of the open state; 401: Time axis; 402: Minimum radius of the lower gate corresponding to the time; 501: Time axis; 502: Current axis; 503: No terahertz irradiation group; 504: No calcium ion experimental group; 601, 802: Current amplitude; 6 02, 803: Full width at half maximum (FWHM); 603, 804: AUC; 701: Neuron; 702: TRPM8 inward current signal after two menthol stimulations; 801: Percentage of desensitized neurons; 901: S4 transmembrane chain; 902: S5 transmembrane chain; 903: S6 transmembrane chain; 904: TRP domain; 905: Carboxyl group; 906: Aspartate 991 site; 907: Arginine 851 site; 1000: Device for controlling the TRPM8 channel to be in the open state; 1010: Terahertz wave emission module; 1020: Parameter control module; 1110: Processor; 1120: Communication interface; 1130: Memory; 1140: Communication bus. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0032] The following is combined with Figures 1 to 11 This invention describes a method for controlling a TRPM8 channel to be in an open state. The method includes: irradiating the TRPM8 channel with a terahertz wave, the center frequency of which is 41–53 THz, and the electric field strength of which is 0.2–2.0 V / nm; and extending the open time of the TRPM8 channel by enhancing the hydrogen bond stability at key positions, thereby keeping the TRPM8 channel in an open state.

[0033] Specifically, the method for controlling the TRPM8 channel 101 to be in an open state provided by this invention achieves the stabilization of the open state of channel 101 through terahertz wave 102 irradiation with specific parameters. All preset parameters have been verified through extensive molecular dynamics simulations and electrophysiological experiments, possessing both scientific rigor and operability, and can meet the application needs of various scenarios such as structural analysis and mechanism research. The complete execution logic of this method can be obtained through... Figure 1 The presentation clearly shows the sequence and relationship between the key steps, from the initiation of terahertz wave 102 irradiation, the activation of channel 101 by menthol 103, to the stabilization of the open state of channel 101 and the formation of the desensitized state 105, providing a visual guide for the implementation of the technical solution.

[0034] The method involves irradiating TRPM8 channel 101 with terahertz wave 102, the center frequency of which is limited to 41–53 THz, and the electric field strength to 0.2–2.0 V / nm. These parameters comprehensively consider factors such as the interaction efficiency between terahertz wave 102 and the key structure of channel 101, and the tolerance of biological samples. This ensures effective excitation of the target effect that enhances hydrogen bond stability, while avoiding damage to the biological sample due to excessively high parameters or failure due to excessively low parameters, thus ensuring the practicality and safety of the technical solution. Through terahertz wave 102 irradiation within this parameter range, energy can be precisely applied to the key structure of TRPM8 channel 101, directly enhancing hydrogen bond stability and extending the opening time of channel 101. This keeps TRPM8 channel 101 stably in an open state, meeting the stringent requirements for conformational uniformity in structural analysis.

[0035] To further optimize the control effect, the center frequency of the terahertz wave 102 is preferably 42–43 THz. This optimal range was determined by comparing the open-state stability of the TRPM8 channel 101 at different frequencies. Within this range, the minimum lower gate radius 302 of the channel can be maintained at a high level for a long period, and the uniformity and duration of the open state are significantly better than other frequency ranges, maximizing the control efficiency of the technical solution. Parameter selection must actively exclude the water absorption frequency ranges of 0–30 THz and 44–50 THz. Water, which is widely present in the biological environment, strongly absorbs the terahertz wave 102 in these two ranges, causing severe energy loss during transmission and preventing effective delivery of the irradiation energy to the TRPM8 channel 101. Therefore, excluding these ranges is a crucial prerequisite for ensuring irradiation effectiveness and an important reflection of the scientific nature of parameter setting.

[0036] Before irradiation, TRPM8 channel 101 needs to be activated with an agonist. A commonly used specific agonist is menthol 103, which can rapidly initiate the opening program of TRPM8 channel 101, creating the foundation for the subsequent stable open state of terahertz wave 102. The timing of terahertz wave 102 irradiation is clearly defined: a 5-second pre-irradiation time, meaning that terahertz wave 102 irradiation is initiated 5 seconds before applying menthol 103 to TRPM8 channel 101, with a continuous irradiation duration of 1 minute. This duration ensures that the key structures of TRPM8 channel 101 fully respond to irradiation, allowing the hydrogen bonding enhancement effect to fully manifest and be stably maintained, thus providing long-term support for the open state of TRPM8 channel 101. Simultaneously, this duration avoids energy accumulation due to prolonged irradiation, preventing non-specific damage to the native structure of the TRPM8 channel 101 protein or host cell activity, achieving an optimal balance between regulatory efficacy and biosafety.

[0037] The electric field polarization direction of terahertz wave 102 must be strictly controlled to be perpendicular to the cell membrane plane where TRPM8 channel 101 is located. This orientation is compatible with the natural arrangement of the S5 transmembrane chain 202, S6 transmembrane chain 203, and TRP domain 904 of TRPM8 channel 101, maximizing the interaction efficiency between terahertz wave 102 and key functional groups in channel 101, making the resonance effect more concentrated and the hydrogen bond enhancement more precise, thereby further improving the reliability of open-state stability. If the polarization direction is parallel to the cell membrane plane, the electric field energy will be more dispersed on the membrane surface, making it difficult to effectively act on the target hydrogen bonds inside channel 101, resulting in a weakened resonance effect and failing to achieve the ideal open-state stability effect.

[0038] The key components of TRPM8 channel 101 are the S6 transmembrane chain 203 and the TRP domain 904 near the channel pore. These two structures directly participate in the regulation of channel 101 opening and closing, and the hydrogen bonds between them are crucial for maintaining the open conformation. The structural layout of TRPM8 channel 101 and the difference in channel radius between the open and closed states can be determined by... Figure 2 The relative positions of key structures such as the pore region helix 201, S5 transmembrane chain 202, S6 transmembrane chain 203, and valine 976 site 205 are clearly labeled, demonstrating the dynamic changes in the radius of channel 101 under different states. This provides visual support for understanding the conformational regulation mechanism. When irradiated with terahertz wave 102, it resonates with the stretching vibration of the carboxyl group 905 in the hydrogen bond between the S6 transmembrane chain 203 and the TRP domain 904. This resonance enhances the hydrogen bond strength and reduces the probability of hydrogen bond breakage, thus firmly locking the open conformation of TRPM8 channel 101. This avoids the problem of easy fluctuation in the open state under natural conditions, providing a stable conformational basis for structural analysis.

[0039] In addition to the stable open state, this method can also regulate the desensitization state 105 of TRPM8 channel 101, further expanding the application scenarios of the technology. During irradiation, the terahertz wave 102 simultaneously acts on the widely distributed carboxyl groups 905 at the entrance of TRPM8 channel 101. These groups change the local charge distribution under irradiation, promoting the rapid accumulation of calcium ions 104 at the entrance of channel 101 and accelerating the inward penetration of calcium ions 104. Combined with the extended opening time of channel 101 due to the terahertz wave 102, the duration and amplitude of the calcium ion 104 current are enhanced, ultimately leading to a large accumulation of intracellular calcium ions 104, forming calcium overload, which in turn triggers TRPM8 channel 101 to spontaneously enter the desensitization state 105, providing a controllable regulatory means for in-depth research on the desensitization mechanism of TRPM8 channel 101.

[0040] This method can also be directly applied to cryo-electron microscopy (cryo-EM) structural analysis, with a clearly defined implementation procedure. First, the open conformation of TRPM8 channel 101 is locked by terahertz wave 102 irradiation, ensuring the homogeneity and stability of the channel 101 population in the open state. Then, samples containing TRPM8 channel 101 are rapidly cryo-fixed to permanently preserve the open conformation. Finally, cryo-EM data acquisition and structural analysis are performed. This procedure eliminates the need for two agonists, fundamentally avoiding the overstabilization risks and local conformational perturbations inherent in traditional methods, resulting in structures that more closely resemble physiological states.

[0041] Validation of the open state must adhere to clear standards. The validation software used is HOLE, specifically designed for detecting the pore size and radius of biomolecular channels, achieving atomic-level precision. The detection site is near valine 976 at position 205 in the lower gate of TRPM8 channel 101. This site is a critical region for the opening and closing of channel 101, and its radius change directly reflects the channel 101 state. The detection time is 200 nanoseconds, comprehensively reflecting the long-term stability of the channel 101 conformation and avoiding misjudgments caused by short-term fluctuations. The judgment threshold is 1.6 Å, corresponding to the open state threshold 303. The validation standard is that the average value of the minimum radius 302 at this site is greater than 1.6 Å within the 200 nanosecond monitoring time. This value is based on extensive ion permeation experiments and structural comparisons. When the radius is greater than this value, TRPM8 channel 101 allows calcium ions (104 ions) to pass through smoothly, conforming to the functional definition of the open state.

[0042] The structural characteristics of TRPM8 channel 101, the parameter setting of terahertz wave 102, the timing and method of irradiation, and the verification standards are all coordinated to form a complete technical system. This method does not require the introduction of additional chemical substances and achieves the stabilization of the open state and the regulation of the desensitized state 105 of TRPM8 channel 101 through purely physical means. It not only solves the shortcomings of existing dual-agonist methods, but also provides reliable technical support for the structural analysis, mechanism research, and application transformation of TRPM8 channel 101, and has significant scientific significance and practical value.

[0043] According to an embodiment of the present invention, the center frequency range of the terahertz wave is 42 to 43 THz.

[0044] Specifically, the optimal frequency range of terahertz wave 102 is 42 to 43 terahertz. This range is the optimal range for modulation effect obtained through systematic experimental screening within the effective frequency range. In molecular dynamics simulations and neurocellular electrophysiological experiments, terahertz wave 102 within this range can form an efficient resonance with the target group carboxyl group 905 at the key position of TRPM8 channel 101, keeping the minimum lower gate radius 302 of the channel stable at more than 2.0 angstroms for a long time, which is much higher than the 1.6 angstrom open state determination threshold corresponding to the open state threshold 303. At the same time, the current amplitude 601, full width at half maximum 602, and AUC603 (the area integral under the inward current trace) all reach their peak values, fully ensuring the stability and uniformity of the open state of TRPM8 channel 101.

[0045] The water absorption frequency ranges are specifically 0 to 30 terahertz and 44 to 50 terahertz. Water is widely present in biological experimental systems and physiological environments, and this water strongly absorbs the terahertz waves 102 within these two ranges, causing severe energy loss during transmission and preventing the irradiation energy from effectively reaching the TRPM8 channel 101 and triggering the expected resonance effect. Experimental data show that when the terahertz wave 102 frequency is within these water absorption ranges, the minimum lower gate radius 302 of the channel is mostly below the 1.6 Å open-state critical line 303, making it impossible to maintain an effective open state. The current signal is not significantly different from the non-terahertz irradiation group 503. Therefore, these frequency ranges are explicitly excluded from the effective parameter range to ensure that the irradiation energy can accurately act on the TRPM8 channel 101, guaranteeing the reliability and repeatability of the open-state stability effect.

[0046] According to an embodiment of the present invention, before irradiating the TRPM8 channel with terahertz waves, the method further includes: activating the TRPM8 channel with an agonist; the irradiation timing of the terahertz waves is: irradiation is initiated with a preset irradiation pre-time before applying the agonist to the TRPM8 channel, and the continuous irradiation duration is a preset continuous irradiation duration.

[0047] Specifically, menthol 103 was selected as the agonist to activate TRPM8 channel 101. It possesses high specificity and efficient activation capabilities, accurately identifying the ligand binding site of TRPM8 channel 101 and rapidly initiating the channel 101 opening process, creating the necessary foundation for the subsequent stable open state of terahertz wave 102. As a classic agonist of TRPM8 channel 101, menthol 103 can effectively activate channel 101 at physiological concentrations without causing additional perturbation to the TRPM8 channel 101 structure. This synergistic effect with the physical regulation of terahertz wave 102 ensures that the open state of TRPM8 channel 101 is closer to its natural physiological state.

[0048] The timing of terahertz wave 102 irradiation was determined through systematic experimental screening. The preset irradiation pre-time was 5 seconds, meaning that terahertz wave 102 irradiation needed to be initiated 5 seconds before the application of menthol 103 to TRPM8 channel 101. This pre-time setting allows the key structures of TRPM8 channel 101 to respond to terahertz wave 102 irradiation in advance before the agonist takes effect, initially adjusting the vibrational state of the relevant groups in TRPM8 channel 101 to a state conducive to resonance. After the application of menthol 103, a synergistic effect from activation to stability can be rapidly formed, significantly improving the stability and uniformity of the open state of TRPM8 channel 101.

[0049] The preset continuous irradiation duration was 1 minute. This duration was determined by comprehensively considering the structural response efficiency of TRPM8 channel 101 and the tolerance of biological samples. One minute of continuous irradiation ensures sufficient resonance between the terahertz wave 102 and the hydrogen bonds at key positions of TRPM8 channel 101, allowing the hydrogen bond enhancement effect to be fully manifested and stably maintained, thus providing long-term support for the open state of TRPM8 channel 101. Simultaneously, this duration avoids energy accumulation due to prolonged irradiation, preventing non-specific damage to the native structure of the TRPM8 channel 101 protein or the activity of host cells, achieving an optimal balance between regulatory effect and biological sample safety. Experimental data show that under these irradiation timing and duration parameters, the open time of TRPM8 channel 101 is significantly prolonged, and the stability and persistence of current signal indicators such as current amplitude 601, full width at half maximum 602, and AUC603 are superior to other irradiation parameter combinations, further validating the scientific validity and rationality of this setting.

[0050] According to an embodiment of the present invention, during irradiation, the electric field polarization direction of the terahertz wave is perpendicular to the cell membrane plane where the TRPM8 channel is located.

[0051] Specifically, during irradiation, the electric field polarization direction of the terahertz wave 102 must be strictly controlled to be perpendicular to the cell membrane plane where the TRPM8 channel 101 is located. As a transmembrane protein, the TRPM8 channel 101's structure, including the S5 transmembrane chain 202, S6 transmembrane chain 203, and TRP domain 904, is arranged perpendicular to the cell membrane plane, and the channel's extension direction is also perpendicular to the cell membrane plane. This polarization direction setting precisely matches the arrangement direction of the channel's key structures, allowing the electric field energy to penetrate the cell membrane more concentratedly and act on the target sites within the channel.

[0052] The advantage of this orientation is that it maximizes the interaction efficiency between the terahertz wave 102 and the key groups in the channel. In the hydrogen bonds at the key positions of channel 101 in TRPM8, the stretching vibration direction of the carboxyl group 905 is more aligned with the transmembrane chain arrangement, i.e., closer to perpendicular to the cell membrane plane, rather than strictly perpendicular. This orientation characteristic may be one of the key factors in improving interaction efficiency: when the electric field polarization direction of the terahertz wave 102 is in the same direction or nearly in the same direction, photon energy can be transferred to these groups more efficiently, triggering a resonance effect, and thus significantly enhancing the binding strength of hydrogen bonds. If the polarization direction is parallel to the cell membrane plane, the electric field energy will be more dispersed on the membrane surface, making it difficult to effectively act on the target hydrogen bonds inside the channel, resulting in a weakened resonance effect, a less significant hydrogen bond enhancement effect, and a significant decrease in the stability of the open state of channel 101 in TRPM8. It should be noted that the specific mechanism by which a more perpendicular polarization direction yields better results is not yet fully understood; the above-mentioned correlation of carboxyl group vibration orientation is only one possible explanation.

[0053] Experimental data further validated the scientific validity of this directional setting. Under all frequency conditions, the polarization direction perpendicular to the cell membrane plane was superior to the parallel direction. Only when the terahertz wave frequency was in the range of 41 to 53 terahertz could the advantage of the vertical direction be fully manifested through the resonance effect, with 42 to 43 terahertz being the optimal range. The minimum lower gate radius of the channel in the vertical direction group could be stably maintained above 2.0 angstroms, directly corresponding to... Figure 3 The frequency range of 42-43 terahertz corresponds to the minimum gate radius curve of the channel at 302. In this range, due to its efficient resonance with carboxyl group 905, the radius value is significantly higher than the open-state critical line 303 and remains stable at a high level for a long period. Simultaneously, key indicators such as current amplitude 601, full width at half maximum (FWHM) 602, and AUC 603 are all at their peak values, directly corresponding to… Figure 6 The statistical results of the 42.5 terahertz irradiation group show that all current indicators are better than those of other groups.

[0054] Regardless of whether the frequency is within the 41-53 terahertz range, effective control of the parallel polarization direction group is impossible. Its channel radius fluctuates significantly, mostly falling below the open-state critical value of 1.6 angstroms, directly corresponding to... Figure 3 The frequencies in the 0-30 terahertz and 44-50 terahertz water absorption ranges correspond to the minimum gate radius of the channel, curve 302. These ranges lack resonance effects, making it difficult to maintain a stable open state even with vertical polarization. The parallel direction further weakens energy transfer efficiency, resulting in an effect consistent with the non-resonant frequency ranges. The current signal of the parallel direction group is not significantly different from that of the non-terahertz irradiation group 503, directly corresponding to... Figure 6 The statistical results of the non-terahertz irradiation group 503 show that all current indicators are significantly lower than those of the 42.5 terahertz irradiation group.

[0055] Therefore, limiting the electric field polarization direction of the terahertz wave 102 to be perpendicular to the cell membrane plane where the TRPM8 channel 101 is located during irradiation is a key technical feature to ensure irradiation effect and improve the stability and accuracy of the open state, providing an important guarantee for the efficient implementation of the entire method.

[0056] According to an embodiment of the present invention, the key location of the TRPM8 channel is the S6 transmembrane chain and the TRP structural domain near the channel aperture, and the -COO in the hydrogen bonds between the terahertz wave and the S6 transmembrane chain and the TRP structural domain. - Group stretching vibration resonance.

[0057] Specifically, the key functional regions of TRPM8 channel 101 are concentrated in the S6 transmembrane chain 203 and the TRP structural domain 904 near the channel pore. The spatial arrangement and interaction of these two structures directly determine the opening and closing state of TRPM8 channel 101. As an important component of the channel pore, the conformational changes of the S6 transmembrane chain 203 can directly change the width of the pore; the TRP structural domain 904, through hydrogen bonding with the S6 transmembrane chain 203, plays a stabilizing role in the pore conformation. Together, they constitute the structural basis for maintaining the open state of TRPM8 channel 101. Figure 9 The distribution of carboxyl groups at key positions in the TRPM8 channel is shown. Numerous carboxyl groups (905) are present in the hydrogen bonds between the S6 transmembrane chain 203 and the TRP domain 904. These carboxyl groups carry -COO... - Groups are the targets of resonance.

[0058] These -COO - The stretching vibrations of the radical exhibit specific frequency characteristics, precisely matching the frequency range of terahertz waves 102. When terahertz waves 102 are irradiated, their photon energy is similar to that of -COO. - The stretching vibration frequencies of the functional groups resonate, and this resonance effect significantly enhances the hydrogen bond strength between the S6 transmembrane chain 203 and the TRP domain 904, reducing the probability of hydrogen bond breakage and thus firmly locking their relative positions, avoiding conformational fluctuations in the open state. Simultaneously, the concerted conformation formed by the S4 transmembrane chain 901, S5 transmembrane chain 902, and S6 transmembrane chain 903 provides structural support for the distribution of the carboxyl group 905. The aspartic acid 991 site 906 and the arginine 851 site 907 further stabilize this hydrogen bond structure through charge interactions, making the resonance enhancement effect more concentrated and persistent, ultimately extending the opening time of the TRPM8 channel 101, providing a stable conformational basis for structural analysis and mechanistic studies.

[0059] According to an embodiment of the present invention, before irradiating TRPM8 with terahertz waves, the TRPM8 channel is activated by one or more of the following stimulation combinations: low temperature, menthol, protons or membrane depolarization. The multimodal response characteristics of the TRPM8 channel are utilized to provide an activation basis for stabilizing its open state under terahertz wave irradiation.

[0060] Specifically, before irradiating TRPM8 channel 101 with terahertz waves, the channel needs to be activated by cryotherapy or menthol 103. Alternatively, protons or membrane depolarization can be combined to form a stimulation combination depending on experimental requirements. TRPM8 channel 101, as a typical multimodal channel, can initiate its opening program without relying on a single stimulus signal and can produce a synergistic response to multiple physical and chemical stimuli. This characteristic provides a key foundation for the implementation of this method. Cryotherapy, as a physiological activation signal, can directly trigger a conformational change in the channel, initiating ion permeability. Menthol 103, as a classic chemical agonist, can precisely bind to ligand binding sites, rapidly initiating the opening program, synergistically with the physical regulation of terahertz waves 102. Protons indirectly assist opening by altering the pH of the microenvironment surrounding the channel, affecting the charge distribution and conformational stability of the channel protein. Membrane depolarization further facilitates gating initiation by changing the cell membrane potential and acting on the voltage-sensitive region of the channel. The use of these stimuli, individually or in combination, ensures that TRPM8 channel 101 efficiently enters the initial open state, providing a reliable activation basis for subsequent terahertz wave irradiation to enhance hydrogen bond stability and lock the open conformation. This multi-stimulus selective activation approach allows the method to be flexibly adapted to different experimental designs and application scenarios, expanding its applicability to in vitro mechanism studies, drug screening, and other scenarios, and further demonstrating the broad practical value of the technical solution.

[0061] According to an embodiment of the present invention, the method further includes cryo-electron microscopy structural analysis, specifically including: after locking the open conformation of the TRPM8 channel by terahertz wave irradiation, rapidly freezing and fixing the sample containing the TRPM8 channel, and then performing cryo-electron microscopy data acquisition and structural analysis; the method further includes an open state verification method, specifically including: detecting the minimum radius of a preset site of the TRPM8 channel by preset software, wherein the statistical average value of the radius is greater than the preset channel radius.

[0062] Specifically, the application procedure of this method in cryo-electron microscopy structural analysis is clear and operable. First, terahertz wave irradiation (THz 102) locks the open conformation of TRPM8 channel 101, ensuring the homogeneity and stability of the channel 101 population in the open state. This fundamentally avoids the overstabilization risks and local conformational perturbations associated with traditional dual-agonist methods. Subsequently, samples containing TRPM8 channel 101 are rapidly cryo-fixed to permanently preserve the stable open conformation. Finally, cryo-electron microscopy data acquisition and structural analysis are performed, resulting in structures that more closely resemble physiological states. This provides crucial support for high-resolution physiological structural analysis of TRPM8 in mammals.

[0063] The validation of the open state was achieved through a standardized detection process. All key parameters and judgment criteria were experimentally verified to ensure accurate and reliable results. The validation software used was HOLE, specifically designed for the detection of pore size and radius of biomolecular channels. This software offers atomic-level precision and can accurately capture subtle changes in the pore size of TRPM8 channel 101, meeting the stringent requirements of structural analysis for data accuracy. The validation site was selected near valine 976 at position 205 in the lower gate of TRPM8 channel 101. This site is the regulatory region for the opening and closing of channel 101; its radius change directly relates to the permeability of the channel pore. In the open state, the radius of this site expands significantly, while in the closed state, it contracts noticeably. Detecting this site allows for a direct and accurate determination of the channel conformation. The validation time was set to 200 nanoseconds. This duration is determined based on the conformational fluctuations of TRPM8 channel 101, comprehensively covering the dynamic changes in the conformation of channel 101, avoiding misjudgments caused by short-term fluctuations, and ensuring that the validation results represent the true stable state of channel 101. The critical distance for verification was 1.6 Å, corresponding to the open state critical line 303. This open state determination criterion was summarized through numerous ion permeation experiments and structural comparisons. Specifically, within a 200 nanosecond monitoring period, if the average minimum radius near site 205 of valine 976 was greater than 1.6 Å, then TRPM8 channel 101 was determined to be in a stable open state. At this point, the channel pore size was sufficient to allow calcium ions (104 ions) to pass through smoothly, meeting the functional definition of an open state. If the average value was less than 1.6 Å, it indicated that channel 101 had not reached an effective open state or that the open state stability was insufficient, failing to meet the requirements of applications such as structural analysis. This verification method, through precise site selection, long-term monitoring, and quantitative standard setting, achieved objective and repeatable determination of the open state, providing a clear detection basis for the effectiveness of the technical solution.

[0064] The method and technical characteristics for controlling the TRPM8 channel 101 in the open state are based on rigorous experimental research, and its key parameters and operating standards have been determined. To further clarify the scientific validity and reproducibility of the technical solution, the following detailed explanation reveals the experimental acquisition process, verification logic, and data support for key parameters such as the preset frequency range, electric field intensity, and irradiation timing, presenting the selection criteria for each technical feature and providing solid experimental evidence for the effectiveness of the entire method. This section will be explained in conjunction with relevant figures, all of which are clearly labeled in the figure descriptions. The figures are as follows: Figure 3 The statistical results of the minimum gate radius of the TRPM8 channel under terahertz wave irradiation at different frequencies are presented. Figure 4 The minimum aperture of the lower gate of the TRPM8 channel changes over time under three conditions: no irradiation, 42.5 THz irradiation, and 34.5 THz irradiation. Figure 5The waveforms of menthol-induced TRPM8 inward current signals are shown under four environments: no terahertz irradiation, 34.5 THz irradiation, 42.5 THz irradiation, and 42.5 THz irradiation without calcium ions. Figure 6 These are the statistical results of TRPM8 current amplitude, full width at half maximum (FWHM), and AUC under the above four environments; Figure 7 Compare the inward current signals triggered by two menthol stimulations of neurons under conditions of 42.5 THz irradiation and without. Figure 8 The study investigated the desensitization state of neurons to secondary menthol stimulation under 42.5 THz irradiation conditions.

[0065] The preset frequency range, preset optimal frequency range, and preset water absorption frequency interval of terahertz wave 102 were determined through a combined screening of molecular dynamics simulations and neuronal electrophysiological experiments. In the molecular dynamics simulation stage, a membrane-protein simulation system containing TRPM8 channel 101 was constructed, and a 200-nanosecond long-term dynamic simulation was performed using GROMACS software. A gradient frequency of 1 to 60 terahertz was set, and terahertz waves 102 of different frequencies were applied to the system. Simultaneously, the minimum radius change of the lower gate of TRPM8 channel 101 was monitored in real time using HOLE software. Simulation results showed that when the frequency was in the 41-53 terahertz range, the average value of the minimum radius of the lower gate was consistently greater than 1.6 Å, maintaining an effective open state. Within the 42-43 terahertz range, the average radius remained consistently above 2.0 Å, exhibiting optimal conformational stability and uniformity. Therefore, this range was determined as the preset optimal frequency range. This result is consistent with... Figure 3 The statistical trend of the minimum gate radius of the corresponding channel at medium frequency is consistent, with the radius value in the 42 to 43 terahertz range being significantly higher than other ranges and remaining stable at a high level. Figure 3 The mid-frequency coordinate axis 301 marks the gradient range from 1 to 60 terahertz, which corresponds perfectly to the frequency range selected in the experiment, showing the correlation between frequency and channel radius. However, when the frequency is in the 0-30 terahertz and 44-50 terahertz ranges, the moisture in the simulated system strongly absorbs the terahertz wave 102, causing the irradiation energy to be unable to be effectively transferred to the key structure of TRPM8 channel 101. The minimum lower gate radius of the channel is mostly below 1.6 Å, showing no significant difference from the detection results of the non-terahertz irradiation group 503. This phenomenon... Figure 4 This is also reflected in the fact that the minimum aperture of the lower gate of the channel in the 34.5THz irradiation group (which is near the water absorption frequency range) fluctuates greatly over time and is generally low, with no significant difference from the non-irradiation group. Figure 4The time axis 401 covers the entire duration of the kinetic simulation, demonstrating the dynamic changes in channel aperture at different time points, providing a temporal dimension to support the determination of frequency validity. Therefore, these two intervals were identified as the preset water absorption frequency ranges and excluded. Neuronal electrophysiological experiments further validated the validity of the above frequency ranges. Neuronal cells expressing TRPM8 channel 101 were selected, and after irradiation with terahertz waves 102 at different frequencies, the channels were activated with menthol 103, and current signals were recorded. The results showed that in the 41-53 terahertz frequency range, the current amplitude 601, full width at half maximum (FWHM) 602, and AUC 603 were significantly higher than in other frequency ranges, with all current parameters reaching their peak values ​​in the 42-43 terahertz range. Figure 5 and Figure 6 The experimental data also confirms this, showing that the current signal waveform of the 42.5THz irradiation group is more stable and has a larger amplitude. Figure 5 The time axis 501 and the current axis 502 clearly define the time range of signal recording and the current intensity scale, making it easier to compare waveform differences (such as peak value and duration) between different groups, and providing a basis for the quantitative analysis of current indicators. The corresponding current amplitude, full width at half maximum (FWHM), and AUC statistics are all better than those of the 34.5 THz irradiated group and the non-irradiated group, further confirming that this frequency range can effectively enhance the open-state stability of the TRPM8 channel 101.

[0066] The preset electric field strength range for the terahertz wave 102 is 0.2 to 2.0 V / nm, determined based on experience from molecular dynamics simulations combined with experimental verification. During the simulation, it was found that the choice of electric field strength needs to balance the stability of the open state with the integrity of the protein structure; too small a field strength makes it difficult to achieve the ideal regulatory effect, while too large a field strength can easily disturb the overall protein structure, leading to deformation. In the experimental phase, an appropriate electric field strength within this range was selected, and under the uniform condition of 42.5 terahertz irradiation for 1 minute, samples of TRPM8-expressing neurons were processed. Simultaneously, the activity of neuronal cells 701 was assessed using a cell viability assay kit, and current signals were recorded in conjunction with electrophysiological experiments.

[0067] Simulation results show that at an electric field strength of 0.1 V / nm, the average minimum gate radius of the channel is only 1.4 Å, failing to reach the critical value for the open state, and the current signal is not significantly different from the unirradiated group, indicating insufficient hydrogen bond enhancement to maintain a sufficiently large channel radius or a long open state. When the electric field strength is increased to 0.2 V / nm, the average channel radius reaches 1.7 Å, the current amplitude is 60% higher than the 0.1 V / nm group, and cell activity is maintained above 95%. Within the electric field strength range of 0.2 to 2.0 V / nm, the channel radius stabilizes between 1.7 and 2.2 Å, cell activity remains above 90%, and the stability and persistence of the current signal are good, which is consistent with... Figure 6The current parameters of the 42.5 THz irradiation group showed excellent performance. When the electric field strength reached 2.5 V / nm, although the channel radius could still be maintained at 1.8 Å, the activity of neuron 701 dropped to below 70%. Electron microscopy showed conformational distortion of the S6 transmembrane chain 203 in some TRPM8 channels 101, indicating that excessively high electric field strength can cause structural damage to channel proteins and cytotoxicity.

[0068] It should be noted that the HOLE software is primarily used to calculate the channel radius in a simulated environment, or to further calculate the channel radius to verify the effect after obtaining atomic data of the channel structure through cryo-electron microscopy in an experimental environment. Based on the patterns summarized from simulations, the results of experimental verification, and the balance between open-state stability and the tolerance of biological samples, a preset electric field strength range of 0.2 to 2.0 volts per nanometer was ultimately determined.

[0069] The preset pre-irradiation time and preset continuous irradiation duration of the terahertz wave 102 were determined based on the activation characteristics of the TRPM8 channel and actual experimental operations, adapting to the rapid activation characteristics of the channel and ensuring the full realization of the irradiation effect. The pre-irradiation time is set to about 5 seconds before the agonist is applied because menthol can rapidly activate the TRPM8 channel. If irradiation is started only when menthol is applied, the timing will be delayed and the regulatory effect will not be able to be exerted in time. The duration of 5 seconds mainly corresponds to the operation interval from starting the irradiation switch to applying menthol. In practice, it can be flexibly adjusted to 2 seconds, 3 seconds, or even shorter times according to the operation speed. The key is to shorten the interval as much as possible to avoid affecting the natural state of the channel before activation.

[0070] The preset duration of continuous irradiation is set to 1 minute, a setting that is consistent with... Figure 7 The signal characteristics presented are directly related. Figure 7 The current signal width of the TRPM8 channel can only be maintained for a maximum of 60 seconds, after which the channel will automatically close, and further irradiation at this point will no longer produce an effective modulation effect. Meanwhile, a 1-minute irradiation duration ensures that the hydrogen bond enhancement effect is fully realized, allowing the channel to remain stably open, with the corresponding current signal... Figure 5 The stable waveforms of the 42.5THz irradiation group were consistent. Considering operational practicality, the channel's own shut-off behavior, and the control effect, a preset continuous irradiation duration of 1 minute was ultimately determined.

[0071] The selection of the electric field polarization direction of terahertz wave 102 was determined based on molecular dynamics simulations combined with experimental verification. The simulations clearly showed that the optimal modulation effect was achieved when the polarization direction aligned with the inherent arrangement of the TRPM8 channels, a direction perpendicular to the tangent of the cell membrane where the channels are located. Since cells are spherical and TRPM8 channels are uniformly distributed across different regions of the cell surface, precise control of the polarization direction of each channel was not possible in the experiments; the overall modulation effect could only be verified by fixing the irradiation direction.

[0072] The experiment used TRPM8-expressing neuronal cell samples and applied terahertz irradiation in a single direction under uniform conditions of 42.5 terahertz frequency, 1.0 V / nm electric field strength, and 1 minute continuous irradiation. At this time, the channels at different locations on the cell surface formed different angles with the irradiation polarization direction. Some channels achieved polarization in the same or nearly the same direction as their own orientation, while others deviated or even became parallel. The minimum lower gate radius of the channels under simulated conditions was calculated using HOLE software, and current signals and cell activity were recorded simultaneously during the experiment.

[0073] The results showed that when irradiated along this direction, the average minimum lower gate radius of the channel population matching the polarization direction remained stable at 2.1 Å, and the current amplitude 601, full width at half maximum (FWHM) 602, and AUC 603 were all at high levels, with cell viability maintained above 93%. This is consistent with the current performance of the optimal parameter group in previous experiments, and the corresponding current waveforms and statistical indicators are also consistent with... Figure 5 , Figure 6 The results are consistent with the excellent data from the 42.5 THz irradiation group. However, for channel populations deviating from or parallel to the polarization direction, the average minimum gate radius was only 1.5 Å, mostly below the open-state critical value. The current signal was not significantly different from the unirradiated group, and although cell activity did not decrease significantly, the open-state stabilization effect was extremely poor. This result corroborates the simulation conclusions, fully demonstrating that maximizing the interaction efficiency between terahertz wave 102 and key channel groups when the polarization direction is along the channel's own arrangement direction. Furthermore, the overall regulatory effect formed by irradiation along a single direction in the experiment further verifies the rationality of this polarization direction setting, which is a key setting to ensure the regulatory effect.

[0074] The regulatory effect of terahertz wave 102 on the desensitization state 105 of TRPM8 channel 101 was also verified by experimental data and corresponding figures. In the experiment, terahertz wave 102 acted on the carboxyl group 905 at the entrance of TRPM8 channel 101, promoting the accumulation and influx of calcium ions 104, ultimately inducing intracellular calcium overload and triggering desensitization. Figure 7 The difference in current signals 702 between neurons stimulated twice with and without 42.5 THz irradiation was shown. After irradiation with terahertz wave 102, the current signal of neuron 701 under secondary menthol stimulation (the signal on the right side of 702) was significantly reduced. Figure 8 Further analysis of the proportion of desensitized neurons showed that the proportion of desensitized neurons in the terahertz wave 102 irradiation group (801) was significantly higher than that in the non-irradiation group. Moreover, the current amplitude (601), full width at half maximum (FWHM) (602), and AUC (603) corresponding to the secondary stimulation all decreased significantly, fully demonstrating the effectiveness of this desensitization regulation mechanism.

[0075] The scientific validity of the open-state verification method is also supported by experimental data and accompanying figures. During the verification process, HOLE software was used to detect the minimum radius near position 205 of valine 976 in channel 101 of TRPM8. A value greater than 1.6 Å over a 200-nanosecond monitoring period was considered a stable open state. This standard is consistent with... Figure 3 The critical line 303 in the open state corresponds to 1.6 angstroms. The minimum radius of the lower gate of the channel for all effective parameter groups is higher than this critical line, while the radius values ​​of most ineffective parameter groups or no-irradiation groups are lower than this value, further confirming the rationality and practicality of the verification standard.

[0076] The present invention also provides a device 1000 for controlling the TRPM8 channel 101 to be in an open state, comprising: a terahertz wave transmitting module 1010 for transmitting terahertz waves to the TRPM8 channel; and a parameter adjustment module 1020 for adjusting the center frequency of the terahertz wave to a preset frequency range and the electric field strength to a preset electric field strength range; the terahertz wave enhances the hydrogen bond stability at key positions of the TRPM8 channel, prolonging the open time of the TRPM8 channel, thereby keeping the TRPM8 channel in an open state.

[0077] Specifically, Figure 10 This is a schematic diagram of the device for controlling the TRPM8 channel 101 to be in the open state provided by the present invention. The device 1000 for controlling the TRPM8 channel 101 to be in the open state is a dedicated hardware carrier for implementing the aforementioned control method. Its overall structure and functional design are closely matched to the terahertz wave 102 control mechanism. Through inter-module collaborative work, it ensures accurate parameter output and efficient operation. Its composition and working logic are as follows: Figure 10 As shown.

[0078] The terahertz wave emission module 1010 serves as the energy output, precisely emitting terahertz waves 102 towards the target TRPM8 channel 101. This module employs a solid-state terahertz source design, featuring stable output and strong directionality, ensuring that the terahertz wave 102 energy is concentrated on the sample area, avoiding energy diffusion that could reduce modulation efficiency. The module is equipped with a dedicated emission fiber probe, which can adjust the emission angle and focusing range according to the sample type (HEK293T cells, neurons, or acute brain slices in the recombinant TRPM8 channel), ensuring uniform wavefield coverage of the TRPM8 channel 101 area while minimizing non-specific impacts on the surrounding biological environment. Furthermore, the module integrates an energy monitoring unit to provide real-time feedback on the energy intensity of the emitted wave, ensuring consistent energy output during irradiation and providing hardware support for stable modulation effects.

[0079] The parameter control module 1020 is the control center of the device 1000, responsible for precisely adjusting the key parameters of the terahertz wave 102 to the preset range. Its adjustment capability directly matches the effective parameter range determined in the aforementioned experiments. Regarding center frequency adjustment, the module supports continuous adjustment from 41 to 53 terahertz and can precisely lock the optimal frequency range of 42 to 43 terahertz, while automatically avoiding the water absorption frequency ranges of 0 to 30 terahertz and 44 to 50 terahertz, achieving intelligent frequency selection without manual intervention. Regarding electric field strength adjustment, the module covers an adjustment range of 0.2 to 2.0 volts per nanometer, with an adjustment accuracy of 0.01 volts per nanometer. It can precisely set the electric field strength value according to experimental requirements, ensuring hydrogen bond enhancement while avoiding damage to the sample from excessively high electric field strength. Furthermore, the parameter control module 1020 also supports the adjustment function of the electric field polarization direction. Given the randomness of the distribution and arrangement of TRPM8 channels on the cell surface, it is currently impossible to directly and precisely match the natural arrangement of each channel through preset. This adjustment function can flexibly adjust the polarization direction of the terahertz wave according to experimental needs, so as to apply irradiation in an orientation as close as possible to the orientation perpendicular to the cell membrane plane where the channel is located, thereby maximizing the interaction efficiency between the terahertz wave and the key groups of the channel.

[0080] The two modules of device 1000 are coordinated and controlled through a data transmission line, with a clear and orderly workflow. Before the experiment begins, the user can input information such as sample type and experimental purpose through the operation interface provided with the module. The parameter control module 1020 will automatically match the corresponding optimal parameter combination. After startup, the parameter control module 1020 first completes the setting and calibration of frequency and electric field strength, and then sends a start signal to the terahertz wave emission module 1010 to ensure that the emission wave parameters are consistent with the preset values. During irradiation, the parameter control module 1020 receives the energy monitoring data of the terahertz wave emission module 1010 in real time. If parameter drift occurs, it will immediately and automatically calibrate to ensure the parameter stability of the entire irradiation process.

[0081] The device 1000 also boasts excellent compatibility and practicality, seamlessly integrating with experimental equipment such as cryo-electron microscopes and cell electrophysiology recorders, adapting to various application scenarios including structural analysis and mechanism research. Its integrated design simplifies experimental procedures, eliminating the need for complex external equipment to achieve stable control of the open state of the TRPM8 channel 101. This provides reliable hardware support for the standardized conduct of related experiments and also makes the practical application of the aforementioned purely physical control methods more feasible.

[0082] The functional design of the device 1000 is completely consistent with and mutually referential to the aforementioned method of controlling the TRPM8 channel 101 to be in an open state, forming a complete technical system of hardware and software synergy. The irradiation operation of the TRPM8 channel 101 using terahertz waves 102 directly corresponds to the energy output function of the terahertz wave emission module 1010. The module's focusing design and energy monitoring capabilities are precisely to ensure the accuracy and stability of the irradiation in the method. The method's specified preset frequency range of 41 to 53 terahertz, preset electric field strength range of 0.2 to 2.0 volts per nanometer, optimal frequency range of 42 to 43 terahertz, and exclusion of the water absorption frequency range are all achieved through the precise adjustment function of the parameter control module 1020. The module's adjustment range and precision perfectly match the experimentally determined values ​​of the method. The setting of the electric field polarization direction perpendicular to the cell membrane plane is solidified through pre-configured settings before the device leaves the factory, ensuring consistency between the hardware and the method's technical characteristics. Furthermore, the device's design for adjusting the emission angle for different sample types is compatible with the multi-source application scenarios of the TRPM8 channel 101 in the method.

[0083] The method provides a clear technical basis for the structural design and parameter configuration of the device, while the device provides standardized and repeatable hardware support for the implementation of the method. The high degree of functional and logical compatibility between the two allows the device settings to be calibrated by referring to the parameter requirements of the method during the experiment, and the scientific validity of the method can be verified by the operation effect of the device. This forms a mutually supportive and mutually verifying technical closed loop, further enhancing the practicality and reliability of the entire invention.

[0084] Figure 11 An example is a schematic diagram of the physical structure of an electronic device, such as... Figure 11 As shown, the electronic device may include a processor 1110, a communications interface 1120, a memory 1130, and a communication bus 1140. The processor 1110, communications interface 1120, and memory 1130 communicate with each other via the communication bus 1140. The processor 1110 can call logic instructions in the memory 1130 to execute a method for controlling the TRPM8 channel to be in an open state. This method includes: irradiating the TRPM8 channel with a terahertz wave, the center frequency of which is 41–53 THz, and the electric field strength which is 0.2–2.0 V / nm; and extending the open time of the TRPM8 channel by enhancing the hydrogen bond stability at key positions, thereby keeping the TRPM8 channel in an open state.

[0085] Furthermore, the logical instructions in the aforementioned memory 1130 can be implemented as software functional units and, when sold or used as independent products, can be stored in a computer-readable storage medium. Based on this understanding, the technical solution of the present invention, essentially, or the part that contributes to the prior art, or a part of the technical solution, can be embodied in the form of a software product. This computer software product is stored in a storage medium and includes several instructions to cause a computer device (which may be a personal computer, server, or network device, etc.) to execute all or part of the steps of the methods described in the various embodiments of the present invention. The aforementioned storage medium includes various media capable of storing program code, such as USB flash drives, portable hard drives, read-only memory (ROM), random access memory (RAM), magnetic disks, or optical disks.

[0086] In another aspect, the present invention also provides a non-transitory computer-readable storage medium storing a computer program thereon, which, when executed by a processor, implements a method for controlling the TRPM8 channel to be in an open state provided by the above methods, the method comprising: irradiating the TRPM8 channel with a terahertz wave having a center frequency of 41–53 THz and an electric field strength of 0.2–2.0 V / nm; and extending the open time of the TRPM8 channel by enhancing the hydrogen bond stability at key positions of the TRPM8 channel, thereby keeping the TRPM8 channel in an open state.

[0087] The device embodiments described above are merely illustrative. The units described as separate components may or may not be physically separate. The components shown as units may or may not be physical units; that is, they may be located in one place or distributed across multiple network units. Some or all of the modules can be selected to achieve the purpose of this embodiment according to actual needs. Those skilled in the art can understand and implement this without any creative effort.

[0088] Through the above description of the embodiments, those skilled in the art can clearly understand that each embodiment can be implemented by means of software plus necessary general-purpose hardware platforms, and of course, it can also be implemented by hardware. Based on this understanding, the above technical solutions, in essence or the part that contributes to the prior art, can be embodied in the form of a software product. This computer software product can be stored in a computer-readable storage medium, such as ROM / RAM, magnetic disk, optical disk, etc., and includes several instructions to cause a computer device (which may be a personal computer, server, or network device, etc.) to execute the methods described in the various embodiments or some parts of the embodiments.

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for controlling a TRPM8 channel to be in an open state, characterized in that, include: The TRPM8 channel was irradiated with terahertz waves, the center frequency of which was 41–53 THz and the electric field strength was 0.2–2.0 V / nm. By enhancing the hydrogen bond stability at key positions of the TRPM8 channel, the open time of the TRPM8 channel is extended, thus keeping the TRPM8 channel in an open state.

2. The method according to claim 1, characterized in that, The center frequency range of the terahertz wave is 42–43 THz.

3. The method according to claim 1, characterized in that, Prior to irradiating the TRPM8 channel with terahertz waves, the method further includes: activating the TRPM8 channel with an agonist; The terahertz wave irradiation timing is as follows: irradiation is initiated after a preset irradiation pre-time before the agonist is applied to the TRPM8 channel, and the continuous irradiation duration is the preset continuous irradiation duration.

4. The method according to claim 1, characterized in that, During irradiation, the electric field polarization direction of the terahertz wave is perpendicular to the cell membrane plane where the TRPM8 channel is located.

5. The method according to claim 1, characterized in that, The key location of the TRPM8 channel is the S6 transmembrane chain and TRP structural domain near the channel aperture. The -COO atoms in the hydrogen bonds between the terahertz wave and the S6 transmembrane chain and TRP structural domain are also key components. - Group stretching vibration resonance.

6. The method according to claim 1, characterized in that, Before irradiating the TRPM8 channel with the terahertz wave, the TRPM8 channel is activated by one or more of the following stimulations: low temperature, menthol, proton or membrane depolarization. The multimodal response characteristics of the TRPM8 channel provide an activation basis for stabilizing its open state under terahertz wave irradiation.

7. The method according to claim 1, characterized in that, The method also includes cryo-electron microscopy structure analysis, specifically including: after locking the open conformation of the TRPM8 channel by terahertz wave irradiation, the sample containing the TRPM8 channel is rapidly frozen and fixed, and then cryo-electron microscopy data acquisition and structure analysis are performed; The method also includes an open-state verification method, specifically including: detecting the minimum radius of the preset site of the TRPM8 channel using preset software, wherein the statistical average value of the radius is greater than the preset channel radius.

8. A device for controlling a TRPM8 channel to be in an open state, characterized in that, include: Terahertz wave transmitting module, used to transmit terahertz waves into the TRPM8 channel; The parameter control module is used to adjust the center frequency of the terahertz wave to a preset frequency range and the electric field strength to a preset electric field strength range. The terahertz wave enhances the hydrogen bond stability at key locations in the TRPM8 channel, extending the open time of the TRPM8 channel and keeping it in an open state.

9. An electronic device comprising a memory, a processor, and a computer program stored in the memory and running on the processor, characterized in that, When the processor executes the computer program, it implements the method for controlling the TRPM8 channel to be in the open state as described in any one of claims 1 to 7.

10. A non-transitory computer-readable storage medium having a computer program stored thereon, characterized in that, When the computer program is executed by the processor, it implements the method for controlling the TRPM8 channel to be in the open state as described in any one of claims 1 to 7.

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